Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex

Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Assembly and phospho-regulatory mechanisms of the budding yeast outer kinetochore KMN complex Noah N. Turner , View ORCID Profile Ziguo Zhang , Jing Yang , View ORCID Profile Kyle W. Muir , View ORCID Profile Stephen H. McLaughlin , Tomos Morgan , View ORCID Profile David Barford doi: https://doi.org/10.1101/2025.06.03.657598 Noah N. Turner 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK 2 Present address: Noah N. Turner: John Innes Centre, Norwich, UK; Kyle W. Muir: Institute of Cell Biology, University of Edinburgh , Edinburgh, EH9 3BF, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: noah.turner{at}jic.ac.uk dbarford{at}mrc-lmb.cam.ac.uk Ziguo Zhang 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ziguo Zhang Jing Yang 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kyle W. Muir 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK 2 Present address: Noah N. Turner: John Innes Centre, Norwich, UK; Kyle W. Muir: Institute of Cell Biology, University of Edinburgh , Edinburgh, EH9 3BF, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kyle W. Muir Stephen H. McLaughlin 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stephen H. McLaughlin Tomos Morgan 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site David Barford 1 MRC Laboratory of Molecular Biology, Francis Crick Avenue , Cambridge, CB2 0QH, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David Barford For correspondence: noah.turner{at}jic.ac.uk dbarford{at}mrc-lmb.cam.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract During mitosis and meiosis kinetochores mediate interactions between chromosomes and spindle microtubules. Kinetochores are multi-megadalton protein complexes essential for chromosome segregation in all eukaryotes, however recent structural, functional, and evolutionary studies have revealed divergent mechanisms of kinetochore assembly. In this study, we use cryo-EM to understand the structural mechanisms by which the budding yeast microtubule-binding outer kinetochore KMN complex assembles, and how its interactions with the centromere-binding inner kinetochore are regulated. The ten-subunit KMN complex comprises three subcomplexes: Knl1c, Mis12c Mtw1c and Ndc80c. We show that α-helical motifs in the C-termini of the Mis12c Mtw1c subunits Dsn1, Mis12 Mtw1 and Nnf1 bind Knl1c and Ndc80c. At the opposite end of the Mis12c Mtw1c stalk, an N-terminal auto-inhibitory segment of Dsn1 (Dsn1 AI ) folds into two α-helices that engage the Mis12c Mtw1c head 1 domain, thereby occluding binding sites for the inner kinetochore subunits CENP-C Mif2 and CENP-U Ame1 , reducing their affinity for Mis12c Mtw1 . Our structure reveals how Aurora B Ipl1 phosphorylation of Dsn1 AI would release this auto-inhibition to substantially strengthen pre-existing connections between the inner and outer kinetochore. Summary Kinetochore-localised KMN complexes mediate chromosome segregation by attaching chromosomes to spindle microtubules. Cryo-EM, biochemical and genetic experiments reveal conserved underlying mechanisms of complex assembly, auto-inhibition and phospho-regulation that ensure precise control of KMN assembly onto centromeres to enable genome division. Introduction Kinetochores are large nucleoprotein complexes that mediate faithful chromosome segregation during cell division ( Ariyoshi and Fukagawa, 2023 ; McAinsh and Marston, 2022 ; Musacchio and Desai, 2017 ). Kinetochores assemble at centromeres to create an essential load-bearing linkage between chromosomes and microtubules of the mitotic spindle ( Akiyoshi et al., 2010 ; Lang et al., 2018 ). The point centromeres of budding yeast Saccharomyces cerevisiae comprise genetically defined ∼118 bp CEN sequences with three centromere DNA elements (CDEI to III) ( Clarke and Carbon, 1980 ; Clarke and Carbon, 1983 ; Fitzgerald-Hayes et al., 1982 ; Hyman and Sorger, 1995 ). The CEN sequence and CENP-A Cse4 nucleosome provide a foundation upon which the rest of the kinetochore assembles ( Lang et al., 2018 ). The CENP-C Mif2 protein mediates specific interactions between the CENP-A nucleosome and the inner kinetochore constitutive centromere associated network (CCAN) complex, and also interacts with the outer kinetochore KMN complex ( Ariyoshi et al., 2021 ; Dimitrova et al., 2016 ; Hornung et al., 2014 ; Kato et al., 2013 ; Przewloka et al., 2011 ; Screpanti et al., 2011 ; Xiao et al., 2017 ; Yan et al., 2019 ). CCAN complexes entrap centromeric DNA within topological chambers ( Dendooven et al., 2023 ; Yatskevich et al., 2022 ). The KMN complex bridges the inner kinetochore with the microtubule ( Cheeseman et al., 2006 ; Deluca et al., 2006 ). Mis12c/Mis12c Mtw1c (human/budding yeast) interacts with Knl1c and Ndc80c to assemble the KMN complex ( Figures 1A and S1A) and mediates recruitment of the entire complex to kinetochores ( Dimitrova et al., 2016 ; Ghodgaonkar-Steger et al., 2020 ; Petrovic et al., 2014 ; Petrovic et al., 2010 ; Polley et al., 2024 ; Yatskevich et al., 2024 ). Ndc80c also associates with CENP-T independently of Mis12c Mtw1c and Knl1c ( Malvezzi et al., 2013 ; Nishino et al., 2013 ). Mis12c engages Knl1c and Ndc80c at the apex of a central α-helical stalk ( Petrovic et al., 2014 ). The base of the stalk bifurcates to form the head 1 and head 2 domains that mediate interactions with the inner kinetochore ( Akiyoshi et al., 2013 ; Dimitrova et al., 2016 ; Emanuele et al., 2008 ; Kim and Yu, 2015 ; Petrovic et al., 2016 ; Rago et al., 2015 ; Walstein et al., 2021 ). Head 1 interacts with N-terminal motifs of CENP-C Mif2 and CENP-U Ame1 in budding yeast, and CENP-C Mif2 and CENP-T in vertebrates ( Dimitrova et al., 2016 ; Hornung et al., 2014 ; Huis In’T Veld et al., 2016; Killinger et al., 2020 ; Petrovic et al., 2016 ). The mutually exclusive binding of inner kinetochore subunits to Mis12c Mtw1c (Huis In’T Veld et al., 2016; Killinger et al., 2020 ) recruits multiple copies of KMN to the inner kinetochore ( Cieslinski et al., 2023 ; Dhatchinamoorthy et al., 2017 ; Joglekar et al., 2006 ; Johnston et al., 2010 ). These interactions are auto-inhibited by a region within the intrinsically disordered N-terminus of the Mis12c Mtw1c subunit Dsn1 in a manner that is relieved by Aurora B Ipl1 phosphorylation at mitotic kinetochores ( Dimitrova et al., 2016 ; Petrovic et al., 2016 ; Polley et al., 2024 ; Takenoshita et al., 2024 ; Walstein et al., 2021 ; Yatskevich et al., 2024 ). Download figure Open in new tab Figure 1: Overall organisation of the S. cerevisiae KMN junction complex A) Subcomplex and subunit composition of the S. cerevisiae KMN complex. B) Composite cryo-EM density map of the S. cerevisiae KMN junction complex derived from docking the apex and base bodies (Figure S2Diii) into the consensus density map. C) Model of the KMN junction complex, highlighting the motifs that mediate the interactions between Mis12c Mtw1c and Knl1c or Ndc80c. D ) and E) Comparison of cryo-EM structures of the inactive S. cerevisiae KMN junction complex with head 2 and Dsn1 AI defined (this study) (D) with the inactive human KMN junction complex (PDB: 8PPR) from ( Yatskevich et al., 2024 ) (E). Structures shown in D and E were superimposed on the stalk segment (CC2, CC3) of the Mis12/Mis12 Mtw1 chain. F) Cartoon schematic of the S. cerevisiae KMN junction complex, based on the structures determined in this study. The interactions between Mis12 Mtw1_Cterm-helix and Knl1 Spc105_RWD-N ; Nnf1 Cterm_helix and Knl1 Spc105_RWD-C ; Dsn1 Cterm-helix and Mtw1 Spc24-helix and the Spc24 and 25 RWD domains, and Mis12c Mtw1c and the two auto-inhibitory α-helices in Dsn AI , and CENP-C Mif2 and CENP-Q Ame1 binding sites are highlighted. The four interfaces discussed in the text are numbered in parenthesis. G) Cartoon schematic of the human KMN junction complex, based on the structures determined in Yatskevich et al ., 2024 (PDB: 8PPR). Interactions between Nsl1 Cterm and Knl1c; Mis12c stalk CC3 region and Knl1 RWD-C ; Dsn1 Cterm-helix and Nsl1 Spc24-helix and the Spc24 and 25 RWD domains, and Mis12c head 1 and Dsn1 AI , CENP-C binding site are highlighted. Ndc80c interacts with microtubules through N-terminal domains of its constituent Ndc80 and Nuf2 subunits ( Alushin et al., 2012 ; Alushin et al., 2010 ; Ciferri et al., 2008 ; Lampert et al., 2013 ; Muir et al., 2023 ; Wei et al., 2007 ), augmented through interactions with the unrelated Astrin/SKAP and Ska complexes in human and the Dam1 complex in budding yeast ( Helgeson et al., 2018 ; Huis In’T Veld et al., 2019 ; Kern et al., 2017 ; Lampert et al., 2010 ; Muir et al., 2023 ; Tien et al., 2010 ; Van Hooff et al., 2017 ). The Ndc80 and Nuf2 microtubule-binding calponin homology (CH) domains are at the opposite end of the 570 Å-long Ndc80 complex to RWD domains in the C-termini of the Spc24 and Spc25 subunits (Spc24 RWD :Spc25 RWD ) that interact with Mis12c Mtw1c ( Wang et al., 2008 ; Wei et al., 2006 ; Wei et al., 2005 ). The long Ndc80c is formed by the end on interactions of the Ndc80:Nuf2 and Spc24:Spc25 coiled-coils ( Ciferri et al., 2005 ; Valverde et al., 2016 ). Kinetochore-associated Knl1c contributes to spindle assembly checkpoint (SAC) and error correction (EC) signalling, reviewed in ( Fischer, 2023 ; McAinsh and Kops, 2023 ). Although the general mechanisms of KMN complex assembly and regulation are conserved from budding yeast to human, specific details of how Mis12c Mtw1c interacts with both Ndc80c and Knl1c differ ( Figure 1F , G). The Spc24:Spc25 RWD domains of Ndc80c interact with α-helical motifs in C-terminal extensions of the rod-like stalk domain of human Mis12c and budding yeast Mis12c Mtw1c . These include a conserved C-terminal α-helix in the Dsn1 subunit ( Dimitrova et al., 2016 ; Polley et al., 2024 ; Yatskevich et al., 2024 ), and, additionally in humans, an α-helix in the Mis12c subunit Nsl1 ( Polley et al., 2024 ; Yatskevich et al., 2024 ). No equivalent motif exists in budding yeast Nsl1 ( Dimitrova et al., 2016 ). Instead, an α-helix in the budding yeast Mis12 Mtw1 binds to the Spc24 and Spc25 RWD domains cooperatively with the Dsn1 α-helix ( Ghodgaonkar-Steger et al., 2020 ). In humans, motifs at the C-terminus of Nsl1 mediate binding of Mis12c to the tandem RWD domains of the human Knl1c subunit Knl1 ( Kiyomitsu et al., 2007 ; Petrovic et al., 2016 ; Petrovic et al., 2014 ; Polley et al., 2024 ; Yatskevich et al., 2024 ). The orthologous budding yeast protein Knl1 Spc105 also uses tandem RWD domains to interact with Mis12c Mtw1c ( Maskell et al., 2010 ), although the regions of Mis12c Mtw1c that interact with yeast Knl1c are largely uncharacterised. Likewise, due to sequence differences between human and budding yeast Dsn1, the structural basis of Mis12c Mtw1c auto-inhibition by an N-terminal region of Dsn1, and its relief by Aurora B Ipl1 phosphorylation ( Akiyoshi et al., 2013 ; Kim and Yu, 2015 ; Yang et al., 2008 ) is unclear. To understand these differences, we determined cryo-EM structures of the S. cerevisiae KMN complex. The structure reveals that interactions between the three KMN sub-complexes are organised by α-helical motifs in the C-termini of the Mis12c Mtw1c subunits Dsn1, Mis12 Mtw1 and Nnf1, which engage interfaces on Knl1c and Ndc80c. Our work shows that a combination of conserved and plastic interfaces, divergent at the sequence and subunit level, underpin a comparable KMN complex architecture between point and regional centromeres. Furthermore, we show that Aurora B Ipl1 overcomes auto-inhibition of human Mis12c and budding yeast Mis12c Mtw1c through similar structural mechanisms. Results Structural features of the budding yeast KMN complex To determine the cryo-EM structure of the S. cerevisiae KMN complex, we purified Ndc80c and a Knl1c:Mis12c Mtw1c complex (K HB-RWD M) from insect cells (Figure S1A, B). K HB-RWD M comprised a truncated Knl1 Spc105 subunit (Knl1 Spc105_HB-RWD ) that incorporated its helical bundle (HB) and tandem RWD domains but had a deleted N-terminal disordered region (retaining residues 445-917). Protein band intensities on SDS-PAGE gels (Figure S1B) indicated that the purified K HB-RWD M contained one subunit of each of Knl1 Spc105_HB-RWD and ZWINT Kre28 , a stoichiometry consistent with the number of Knl1 Spc105 and ZWINT Kre28 proteins at kinetochores in cells defined using fluorescence microscopy ( Roy et al., 2022 ). We pursued numerous strategies to reconstitute and stabilise KMN on cryo-EM grids, with the best results obtained using the GraFix methodology ( Kastner et al., 2008 ). The KMN complex ( Figure 1A ) was reconstituted by incubating K HB-RWD M and Ndc80c (Figure S1C, D) at an equimolar ratio. K HB-RWD MN complex formation was indicated by the co-migration in a glycerol gradient of its ten subunits, separate from K HB-RWD M and Ndc80c (Figure S1E). The reconstituted K HB-RWD MN (henceforth KMN) was crosslinked and purified using a 10-30% glycerol gradient with 0.0-0.2% glutaraldehyde (Figure S1F). We collected a large cryo-EM dataset on this sample. Cryo-electron micrographs and 2D class averages revealed elongated particles (Figure S2A, B) that corresponded closely with the previously observed structures of isolated Mis12c Mtw1c , Mis12c and Ndc80c, the entire human KMN complex, and negative-staining EM of the budding yeast KMN complex ( Ciferri et al., 2008 ; Dudziak et al., 2025; Hornung et al., 2011 ; Maskell et al., 2010 ; Petrovic et al., 2014 ; Petrovic et al., 2010 ; Polley et al., 2024 ; Wang et al., 2008 ; Wei et al., 2005 ; Yatskevich et al., 2024 ). We determined medium-resolution cryo-EM maps of a KMN junction complex that contained the stalk and head 1 domains of Mis12c Mtw1c , and the RWD domains of Knl1c and Ndc80c (Figures S2D-F and Table S1). Overall, the S. cerevisiae KMN junction complex architecture ( Figure 1B , C and Video S1) (schematic in Figure 1F ) resembles the human KMN complex ( Polley et al., 2024 ; Yatskevich et al., 2024 ) ( Figure 1E , G). The Mis12c Mtw1c structure is dominated by an α-helical bundle assembled from its four subunits that form a central stalk. This stalk domain comprises three coiled-coil (CC) regions (CC1, CC2 and CC3) ( Dimitrova et al., 2016 ). Similar to human KMN, the Knl1c and Spc24:Spc25 RWD domains are bound at the apex of the Mis12c Mtw1c stalk, and although in close proximity, are not in direct contact. The Mis12c Mtw1c head 1 domain, comprising a short four α-helical bundle formed from the Mis12 Mtw1 and Nnf1 subunits, is attached to the base of the stalk through loops, docking against Dsn1 and Nsl1 of the CC1 region ( Figure 1B , C). In the composite cryo-EM map of the S. cerevisiae KMN junction complex, no density was observed for portions of Ndc80c comprising the Spc24:Spc25 coiled-coils and the entire Ndc80:Nuf2 subcomplex. Likewise, for Knl1c, all regions preceding the C-terminal RWD domains of Knl1 Spc105 and ZWINT Kre28 were not visible. Disorder of both the Ndc80c and Knl1c subcomplexes in S. cerevisiae KMN, indicative of their conformational flexibility, was also observed in the human KMN structure ( Polley et al., 2024 ; Yatskevich et al., 2024 ). Our structure of S. cerevisiae KMN also revealed important differences with the human KMN complex, specifically in how Mis12c Mtw1c interacts with both Knl1c and Ndc80c, and a C-terminal RWD domain in ZWINT Kre28 (ZWINT Kre28_RWD ) that is absent from the human ortholog ( Figure 1C-G ). An RWD domain in S. cerevisiae ZWINT Kre28 had previously been predicted through phylogenetic analyses and AF2 models ( Polley et al., 2024 ; Tromer et al., 2019 ). In S. cerevisiae KMN, ZWINT Kre28_RWD interacts with the N-terminal RWD domain of Knl1 Spc105 , a role played by the ZWINT C-terminal α-helix in human Knl1 ( Yatskevich et al., 2024 ). Lastly, in this reconstruction of the S. cerevisiae KMN junction complex, cryo-EM density for the Mis12c Mtw1c head 2 domain is not visible due to the subtraction of head 2 cryo-EM density for cryo-EM data processing. Interactions of Mis12c Mtw1c with Knl1c and Ndc80c and comparison with human KMN Mis12c Mtw1c contacts Knl1c through the Dsn1:Nnf1 CC3 coiled-coil and a peptidic extension of Mis12 Mtw1 The Dsn1, Mis12 Mtw1 and Nnf1 subunits mediate Mis12c Mtw1c interactions at two contact sites on Knl1c. First, conserved aliphatic residues in Dsn1 and the C-terminal α-helix of Nnf1 (Nnf1 Cterm-helix ) ( Figure 2C ), part of Mis12c Mtw1c CC3, engage a hydrophobic patch on the C-terminal RWD domain of Knl1 Spc105 (Knl1 Spc105_RWD-C ) (interface (1)) ( Figures 1C , F and 2A-C and 3A, C and Video S1). In general, this S. cerevisiae Mis12c Mtw1c – Knl1c interface resembles its counterpart in human KMN ( Figure 2E , F) ( Polley et al., 2024 ; Yatskevich et al., 2024 ). Download figure Open in new tab Figure 2: Structural details of the interactions between Mis12c Mtw1c and Knl1c Cryo-EM density map (Figure S2Diii) highlighting the interactions between Mis12c Mtw1c and Knl1c. [C] and [D] highlight regions of the map that are shown close up in panels C and D , for interface 1 and interface 2, respectively. B) Model of Knl1c engaging in interactions with motifs from Mis12c Mtw1c , highlighting the Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix motifs from Mis12c Mtw1c that interact with the Knl1 Spc105_ RWD-N and Knl1 Spc105_RWD-C domains, respectively. The molecule is rotated 90° anti-clockwise related to Fig. 1C . C) Structural details of the interaction between the Nnf1 Cterm-helix helix, the hydrophobic patch on the Knl1 Spc105_RWD-C domain, and the Dsn1 CC3_α-helix at interface (1). D) Structural details of the interaction between the Mis12 Mtw1_Cterm-helix and the interface formed by the ZWINT Kre28_RWD and Knl1 Spc105_ RWD-N domains at interface (2). E) and F) Comparison of S. cerevisiae (E) and human (F) KMN junction complexes at the Mis12c interface with Knl1:Zwint. Human structure PDB: 8PPR from ( Yatskevich et al., 2024 ). Structures were superimposed on the Knl1 RWD domains. The comparison highlights the interchangeable roles played by the C-termini of Mis12 Mtw1 in S. cerevisia e and Nsl1 in human in contacting the Knl1 RWD-C domain. Views in (E) and (F) similar to (B). At the second Mis12c Mtw1c – Knl1c contact site, which differs from human KMN ( Polley et al., 2024 ; Yatskevich et al., 2024 ), the extreme C-terminal α-helix of Mis12 Mtw1 (residues 282-289: Mis12 Mtw1_Cterm-helix ) engages a channel formed at the interface of the Knl1 Spc105 RWD-N domain (Knl1 Spc105_RWD-N ) and an α-helix of ZWINT Kre28 (ZWINT Kre28_α1-helix ) (interface (2)) ( Figures 1C , F and 2A, B, D-F and 3B, C). The ZWINT Kre28_α1-helix combines with Knl1 Spc105_RWD-N to create a hydrophobic channel that is ideally suited to bind the mixed hydrophobic and acidic Mis12 Mtw1_Cterm-helix ( Figure 2D ). Immediately preceding Mis12 Mtw1_Cterm-helix , a β-strand from Mis12 Mtw1 augments the β-sheet of Knl1 Spc105_RWD-N ( Figure 2B ). Consistent with the observation that the ZWINT Kre28_RWD domain is not essential for S. cerevisiae viability (Dudziak et al., 2025), our structure shows that although ZWINT Kre28_RWD may indirectly stabilise ZWINT Kre28_α1-helix through its contact with Knl1 Spc105 ( Figure 2B ), ZWINT Kre28_RWD does not contribute directly to contacting Mis12c Mtw1c subunits. In human KMN, ZWINT lacks an RWD domain, and Mis12 does not contact Knl1c. Instead, the C-terminus of Nsl1 contacts the Knl1 RWD-N :ZWINT interface and a shallow groove on Knl1 RWD-N ( Petrovic et al., 2014 ; Polley et al., 2024 ; Yatskevich et al., 2024 ) ( Figures 1G , 2F). Our experimental structure of the Mis12 Mtw1 – Knl1c interface agrees with AF2 predictions of the Knl1c:Mis12c Mtw1c complex ( Figure 3C-E ), supporting our assignment of cryo-EM densities to Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix ( Figure 3A , B). Download figure Open in new tab Figure 3: Modelling of interactions between Mis12c Mtw1c and Knl1c A) Molecular model of the interaction between Nnf1 Cterm-helix and Knl1c docked into the cryo-EM density map, showing density for amino acid side chains relevant to the interaction. View similar to Fig. 3C . B) Molecular model of the interaction between Mis12 Mtw1_Cterm-helix at interface (2) and Knl1c docked into the cryo-EM density map, showing density for amino acid side chains relevant to the interaction. View similar to Fig. 3D . C) AF2 structure prediction of the Knl1c:Mis12c Mtw1c interaction. The prediction was performed using full-length protein sequences apart from Knl1 Spc105 , for which residues 1-444 were excluded. The structure prediction is coloured by chain. D) AF2 structure prediction presented in panel C with the model coloured by residue pLDDT score and with the predicted alignment error (PAE) score shown as an inset in the top right corner of the panel. E) Structural alignment of the Knl1c:Mis12c Mtw1c AF2 model with the Mis12c Mtw1c and Knl1c experimental molecular model generated in this study. The predicted and experimentally modelled Knl1 Spc105 and ZWINT Kre28 subunits are coloured identically, whereas the Nnf1 Cterm-helix and Mis12 Mtw1_Cterm-helix are coloured distinctly according to whether they derive from the AF2 model or the experimental molecular model. F) Pulldown experiment to investigate the role of the Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix helical motifs in mediating the interaction between Knl1c and Mis12c Mtw1c . Knl1c composed of Knl1 Spc105_RWD and ZWINT Kre28_RWD containing a C-terminal twin-strep-tag II (K RWD ) was coexpressed with Mis12c Mtw1c in insect cells. I = input, E = eluate, K = K RWD complex present, - = K RWD complex absent, Mis12 Mtw1_ΔC = Mis12 Mtw1_Δ272-C , Nnf1 ΔC = Nnf1 Δ180-C . Experimental details are provided in Materials and methods. E, F Structures superimposed on Knl1 RWD domains. Consistent with our structure, deletion of residues comprising and surrounding Mis12 Mtw1_Cterm-helix mildly reduced levels of Knl1 Spc105 and ZWINT Kre28 that co-purified with Mis12c Mtw1c isolated from yeast ( Ghodgaonkar-Steger et al., 2020 ). Indeed, we found that deletion of either Mis12 Mtw1_Cterm-helix or Nnf1 Cterm-helix reduced the co-purification of Mis12c Mtw1c with Knl1c from insect cells ( Figure 3F ). These deletions did not affect the stability nor assembly of Mis12c Mtw1c (Figure S3A, B) nor its interaction with Ndc80c (Figure S3C, D), suggesting that the specific functions of Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix are to mediate the interaction between Mis12c Mtw1c and Knl1c. To investigate the function of Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix in vivo we used the auxin inducible degron (AID) system ( Tanaka et al., 2015 ) to deplete the endogenous MTW1 and NNF1 gene products and tested whether mutant mtw1 and nnf1 proteins with their C-terminal α-helices deleted ( mtw1 ΔC and nnf1 ΔC , respectively) rescued depletion of the endogenous gene product. Western blotting confirmed that the mAID 3 -FLAG 5 tagged gene products of the endogenous MTW1 and NNF1 loci were depleted after addition of IAA (Figure S1G, H), and that the rescue alleles were expressed from the exogenous loci (Figure S1I, J). Consistent with the essentiality of Mis12 Mtw1 and Nnf1 ( Euskirchen, 2002 ), strains with an empty cassette incorporated at the exogenous locus were unable to grow on plates containing IAA (Figure S3E). The expression of wild type MTW1 and NNF1 rescue alleles rescued this lethality (Figure S3E). The mutant mtw1 ΔC and nnf1 ΔC alleles also rescued lethality caused by depletion of endogenous MTW1 and NNF1 at 30°C (Figure S3E). These data are consistent with our in vitro results that deletion of either Mis12 Mtw1_Cterm-helix or Nnf1 Cterm-helix helices alone reduces, but does not abolish, co-purification of Mis12c Mtw1c with Knl1c ( Figure 3F ). We were unable to test the consequence to cell growth of deleting Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix simultaneously because strains combining both mtw1 ΔC and nnf1 ΔC alleles with AID-tagged MTW1 and NNF1 could not be isolated, suggestive of lethality. Mis12c Mtw1c contacts Ndc80c through the Dsn1:Nsl1 CC3 coiled-coil and peptidic extensions of Dsn1 and Mis12 Mtw1 In the S. cerevisiae KMN junction complex, Mis12c Mtw1c engages Ndc80c through multiple contacts involving the RWD domains of Spc24 and Spc25 (Spc24 RWD and Spc25 RWD ). These interactions comprise the CC3 region of the Mis12c Mtw1c stalk, and peptidic C-terminal extensions of Dsn1 and Mis12 Mtw1 ( Figures 1C , F, 4 and 5A, B and Video S1). First, as similarly observed in human KMN ( Polley et al., 2024 ; Yatskevich et al., 2024 ), the hydrophobic patch of the Spc24 RWD β-sheet interacts with hydrophobic residues of Dsn1 and Nsl1 in CC3 (interface 3) ( Figures 1C , F and 4B, C, E, F and 5A). Second, differing from human KMN, an α-helical segment of Mis12 Mtw1 (Mis12 Mtw1_Spc24-helix ) buttresses Spc24 RWD and the Spc24:Spc25 coiled-coil (interface 4) ( Figures 1C , F and 4B, D-F and 5B), before Mis12 Mtw1 crosses to contact Knl1c through Mis12 Mtw1_Cterm-helix ( Figure 1C , F). The interaction of Mis12 Mtw1_Spc24-helix residues Tyr236 and Arg240 with Spc24 RWD and Spc25 RWD is supported by well-resolved cryo-EM density ( Figure 5B ). These interactions likely rationalise why Asp substitution of Mis12 Mtw1 residues Arg233 and Tyr236 reduced levels of Spc24:Spc25 associated with Mis12c Mtw1c in an in vitro assay, and sensitises yeast to benomyl ( Ghodgaonkar-Steger et al., 2020 ). In human KMN, S. cerevisiae Mis12 Mtw1_Spc24-helix is functionally replaced by a short α-helix of Nsl1 (Nsl1 Spc24-helix ) that buttresses Spc24 RWD and the Spc24:Spc25 coiled-coil, before Nsl1 also crosses to contact Knl1c (Nsl1 Cterm ) ( Polley et al., 2024 ; Yatskevich et al., 2024 ) ( Figure 1F , G). Thirdly, in another interaction that is conserved with human KMN ( Polley et al., 2024 ; Yatskevich et al., 2024 ) the C-terminal Dsn1 α-helix (residues 560-574: Dsn1 Cterm-helix ) docks into a deep channel at the interface of Spc24 RWD and Spc25 RWD (interface 4: Figures 1C , F and 4B, D-F and 5B). However, the budding yeast KMN complex differs from human in that the Mis12 Mtw1_Spc24-helix stabilises this interaction. The four-turn Mis12 Mtw1_Spc24-helix bridges both Dsn1 Cterm-helix and Spc24 RWD through interactions with Val244 and Leu245 of Mis12 Mtw1_Spc24-helix ( Figure 4D and 5B ). The shorter human Nsl1 Spc24-helix does not extend to contact the Dsn1 Cterm-helix ( Polley et al., 2024 ; Yatskevich et al., 2024 ). This interaction of budding yeast Dsn1 Cterm-helix with Mis12 Mtw1_Spc24-helix might be the basis by which Dsn1 Cterm-helix and Mis12 Mtw1_Spc24-helix bind to Spc24:Spc25 cooperatively. This interface could also explain how Asp substitutions of Mis12 Mtw1 Val244, together with Leu248, a residue not resolved in our structure, strongly impaired binding of Spc24:Spc25 and Mis12c Mtw1c in vitro , and confers benomyl sensitivity in vivo ( Ghodgaonkar-Steger et al., 2020 ). Download figure Open in new tab Figure 4: Structural details of interactions between Ndc80c and Mis12c Mtw1c A) Cryo-EM density map (Figure S2Diii) highlighting the interactions between Mis12c Mtw1c and Spc24 and Spc25 RWD domains. [C] and [D] highlight regions of the map that are shown close-up in panels C and D for interface 3 and interface 4, respectively. B) Model of Spc24 and Spc25 RWD domains interacting with motifs from Mis12c Mtw1c , highlighting the Mis12 Mtw1_Spc24-helix and Dsn1 Cterm-helix helical motifs from Mis12c Mtw1c that interact with Spc24 and Spc25 RWD domains at interface (3). The interaction between the CC3 region of the Mis12c Mtw1c stalk domain and the Spc24 RWD domain is also featured. C) Structural details of the interactions between the CC3 region of the Mis12c Mtw1c stalk domain and the solvent exposed hydrophobic patch on the Spc24 RWD domain at interface (4). D) Structural details of the interactions between Dsn1 Cterm-helix and Mis12 Mtw1_Spc24-helix motifs and the Spc24 and Spc25 RWD domains. E) and F) Comparison of S. cerevisiae (E) and human (F) KMN junction complexes at the Mis12c interface with Spc24:Spc25 RWD domains. In both S. cerevisiae and human KMN junction complexes the Dsn1 Cterm-helix contacts Spc24:Spc25 RWD interface. The comparison highlights the interchangeable roles played by the C-termini of Mis12 Mtw1 in S. cerevisiae (Mis12 Mtw1_Spc24 helix ) and Nsl1 in human in contacting the Spc24 RWD . In the S. cerevisiae KMN junction complex, the longer Mis12 Mtw1_Spc24 helix contacts Dsn1 Cterm-helix . Structures superimposed on Spc24 RWD domain. Human KMN junction (PDB: 8PPR) from ( Yatskevich et al., 2024 ). Views in (E) and (F) similar to (B). Download figure Open in new tab Figure 5: Modelling of interactions between Mis12c Mtw1c and Ndc80c A) Molecular model of the interactions between the CC3 region of the Mis12c Mtw1c stalk domain and the Spc24 and Spc25 RWD domains at interface (3) docked into the cryo-EM density map, showing density for select amino acid side chains relevant to the interaction. View similar to Fig. 4C . B) Molecular model of the interactions between Mis12 Mtw1_Spc24-helix and Dsn1 Cterm-helix motifs with the Spc24 and Spc25 RWD domains at interface (4) docked into the cryo-EM density map, showing density for select amino acid side chains relevant to the interaction. View similar to Fig. 4D . C) Structural alignment of the crystal structure of Spc24 and Spc25 RWD domains bound to Dsn1 560-576 peptide (PDB: 5T6J) ( Dimitrova et al., 2016 ) onto the Mis12c Mtw1c and Spc24 and Spc25 experimental molecular models (this study). The Spc24 and Spc25 chains derived from 5T6J and this study are coloured identically, whilst Dsn1 Cterm-helix is coloured distinctly according to whether it derives from 5T6J or this study. D) AF2 model of the interaction between Spc24, Spc25, and Mis12c Mtw1c (coloured in red), structurally aligned against the Spc24, Spc25, and Mis12c Mtw1c molecular models produced in this study (coloured by chain). The prediction was performed using full-length and wild-type protein sequences for Spc24, Spc25, and Mis12c Mtw1c subunits. E) AF2 structure prediction presented in panel D coloured by residue pLDDT score, with the predicted alignment error (PAE) score plot shown in inset in the top right corner. Lastly, our structure of Mis12 Mtw1_Spc24-helix and Dsn1 Cterm-helix bound to Spc24 RWD :Spc25 RWD agrees with a crystal structure of the Spc24 RWD :Spc25 RWD :Dsn1 Cterm-helix complex ( Figure 5C ) ( Dimitrova et al., 2016 ), and an AF2 prediction of Mis12c Mtw1c with Spc24 and Spc25 ( Figure 5D , E). Mechanism of Mis12c Mtw1c auto-inhibition and activation by Aurora B Ipl1 kinase An auto-inhibitory segment of Dsn1 engages the head 1 domain of Mis12c Mtw1c The Dsn1 N-terminal intrinsically disordered region (N-IDR), situated immediately N-terminal of head 2, auto-inhibits the interaction of Mis12c Mtw1c with the inner kinetochore ( Dimitrova et al., 2016 ; Petrovic et al., 2016 ; Walstein et al., 2021 ; Yatskevich et al., 2024 ), whereas Aurora B Ipl1 phosphorylation of this region strengthens inner and outer kinetochore interactions ( Akiyoshi et al., 2013 ; Kim and Yu, 2015 ; Yang et al., 2008 ). Due to signal subtraction of the Mis12c Mtw1c head 2 domain during cryo-EM processing, our model of the KMN junction complex did not reveal the structural basis of Mis12c Mtw1c auto-inhibition. However, we observed cryo-EM density on the surface of head 1 not accounted for in our model (Figure S2Dii). We reasoned this density might correspond to either a region of head 2, and/or a region of Dsn1 N-IDR involved in auto-inhibition ( Dimitrova et al., 2016 ). To investigate these possibilities, we performed focussed 3D classification of the non-signal subtracted KMN particles using a mask encompassing the base of the Mis12c Mtw1c stalk, head 1 and putative head 2 domain (Figure S4A). Using this approach, we obtained a subset of particles that resulted in 3D reconstructions with defined cryo-EM density for the Mis12c Mtw1c head 1 and head 2 domains (Figure S4 and Table S1). The small number of particles (18,160) exhibiting head 2 associated with head 1 limited the global resolution of the reconstruction to 6.5 Å (Figure S4B), with a local resolution of 6.0 Å in regions of the map closest to the head 1 central region (Figure S4C). The overall conformations of the head 1 and Mis12c Mtw1c stalk domains are unchanged compared to the composite cryo-EM map of the KMN junction complex (Figure S5A, B). To interpret the putative head 2 density (Figure S5A), we used an AF2 prediction of S. cerevisiae Spc24:Spc25:Mis12c Mtw1c (Figure S5C, D). This accounted for the helical bundle of head 2 docked onto head 1, but not an additional α-helical-like density contacting head 1, nor continuous density between CC1 of the Mis12c Mtw1c stalk and the head 2 domain (Figure S5A, B). Using crosslinking mass spectrometry (CL-MS) on the K HB-RWD M complex, we identified crosslinks between residues in head 1 and head 2, in agreement with the docked configuration of head 2 ( Figure 6A ). Download figure Open in new tab Figure 6: Interactions between the Mis12c Mtw1c head 1 and head 2 domains A) Observed CL-MS crosslinks on the K HB-RWD M complex, visualised using the molecular model from Figure S5B. Inter-subunit crosslinks between residues in the Mis12c Mtw1c head 1 and head 2 domains are highlighted, and relevant Cα atoms of crosslinked residues are shown as spheres. Crosslinks shown are between residues that satisfy the physical distance constraint of the chemical crosslinker. Crosslinks between two residues are only displayed if both residues are visible in the panel. B) Cryo-EM density map with improved occupancy for the Mis12c Mtw1c head 2 domain (Figure S4), interpreted and colour-coded according to experimental model of Sc Mis12c Mtw1c . C) Experimental cryo-EM model of Sc Mis12c Mtw1c head 1 and head 2 domains with Dsn1 auto-inhibitory region (Dsn1 AI ) at the base of the Sc Mis12c Mtw1c stalk domain. D) CL-MS cross-links from the Sc Mis12c Mtw1 dataset that satisfy the physical distance constraint of the chemical crosslinker, indicated in green, mapped onto Sc Mis12c Mtw1c head 1, head 2 and stalk domains and Dsn1 AI . E) Molecular model showing the interaction between the Sc Mis12c Mtw1c head 1 domain and Dsn1 AI (residues 229-255). Sc Mis12c Mtw1c head 2 domain is hidden. F) Multiple sequence alignment of Dsn1 protein from distantly related budding yeast demonstrates that the Kl Dsn1 201-230 segment of the Dsn1 N-terminal intrinsically disordered region is conserved. G) Isothermal titration calorimetry experiments to determine the identity of the unassigned α-helical-like density. K D and n values are an average from i) three experiments, or ii) from two experiments. i) Titration of Dsn1 228-254 peptide to Knl1c:Mis12c Mtw1c complex (K HB-RWD M Dsn1Δ257 ) with Knl1 Spc105 residues 1-444 and Dsn1 residues 1-257 deleted. ii) Titration of Dsn1 228-254 peptide to a head 1 deleted Knl1c:Mis12c Mtw1c complex (K HB-RWD M Dsn1Δ257_βhead1 ): Knl1 Spc105 residues 1-444, Dsn1 residues 1-257, Mis12 Mtw1 residues 1-106, and Nnf1 residues 1-104 deleted. iii) Titration of Dsn1 228-254 peptide to K HB-RWD M Dsn1Δ257 complex (K HB-RWD M Dsn1Δ257_Mis12-3A ) containing mutant Mis12 Mtw1_E69A,E73A,E77A . iv) Isothermal titration calorimetry data for phosphomimetic mutations at Aurora B sites in Dsn1 228-254 . K D and n values are an average from two experiments. Titration of Dsn1 228-254 peptide with S240E and S250E phosphomimetic mutations (Dsn1 228-254,S240E,S250E ) to Knl1c:Mis12c Mtw1c complex (K HB-RWD M Dsn1Δ257 ) with Knl1 Spc105 residues 1-444 and Dsn1 residues 1-257 deleted. The AF2 model of S. cerevisiae Mis12c Mtw1c ( Sc Mis12c Mtw1c ) was not consistent with the conformation of head 2 in the Sc Mis12c Mtw1c cryo-EM map with improved head 2 density (Figure S5E, F). By contrast, an AF2 prediction of Mis12c Mtw1c from the related budding yeast Kluyveromyces lactis ( Kl Mis12c Mtw1c ) better recapitulated the contacts between head 1 and 2 observed in the density map. In the Kl Mis12c Mtw1c model the head 2 domain is confidently predicted to contact head 1 (Figure S5G, H) and matched our docking of S . cerevisiae head 2 into cryo-EM density (Figure S5B, G). With this prediction of Kl Mis12c Mtw1c , we built a model of Sc Mis12c Mtw1c which yielded an excellent fit into the density map ( Figure 6B , C). This model rationalises all cryo-EM density including that which could not be assigned using AF2 predictions of Sc Mis12c Mtw1c head 2 (grey density in Figure S5A). A linker within Nsl1 connecting CC1 and head 2 docks into the continuous density between CC1 and head 2 ( Figure 6B , C), consistent with observed CL-MS crosslinks between the Nsl1 linker and CC1 residues Dsn1 K436 , Nsl1 K105 and Nsl1 Y106 ( Figure 6D ). Within the N-IDR of K. lactis Dsn1, residues 201-230 ( Kl Dsn1 201-230 ) are predicted to fold as two α-helices joined by a short linker (Figure S5G, H). Indeed, this region of Kl Dsn1 mediates association of the Kl Mis12c Mtw1c head 1 and head 2 domains in vitro and auto-inhibits the interaction of Kl Mis12c Mtw1c with Kl CENP-C Mif2 and Kl CENP-U Ame1 ( Dimitrova et al., 2016 ). This region of Dsn1 is referred to as the Dsn1 auto-inhibitory segment (Dsn1 AI ). Based on the sequence similarity of Sc Dsn1 228-257 and Kl Dsn1 201-230 ( Figure 6F ), we built Dsn1 AI of Sc Mis12c Mtw1c into the α-helical-like density as Sc Dsn1 229-255 ( Figure 6B , C) using the Kl Mis12 Mtw1c AF2 model. This assignment is supported by a CL-MS crosslink between Sc Mis12 Mtw1 Glu84 in head 1 and Sc Dsn1 229-255 Ser240 ( Figure 6E ). Importantly, Sc Dsn1 229-255 incorporates the Aurora B Ipl1 consensus sites Ser240 and Ser250 whose replacement with Asp enhanced the association of Sc Dsn1 with inner kinetochore proteins ( Akiyoshi et al., 2013 ). In agreement with this, phospho-mimetic mutants of the equivalent residues in Kl Dsn1 201-230 (Ser213 and Ser223) substantially increased the affinity of Kl Mis12c Mtw1c for N-terminal fragments of CENP-C Mif2 and CENP-U Ame1 ( Dimitrova et al., 2016 ). Consistent with our structure, an isothermal titration calorimetry (ITC) experiment showed that a peptide of Dsn1 residues 228-254 (Dsn1 228-254 ) bound to K HB-RWD M with the entire Dsn1 N-IDR (residues 1-257) deleted (K HB-RWD M Dsn1Δ257 ) with a K D of 1.3 μM ( Figure 6Gi ). By contrast, the Dsn1 228-254 peptide did not bind to K HB-RWD M with both the Dsn1 N-IDR and head 1 deleted (K HB-RWD M Dsn11′257_1′head1 ) ( Figure 6Gii ). Dsn1 229-255 is basic and projects Lys238, Arg245 and Arg248 towards a cluster of negatively-charged residues in head 1 ( Figure 6E ). That the Dsn1 228-254 peptide-binding site corresponds to the site on head 1 associated with the α-helical-like density was also consistent with a threefold-increased K D for its association with the K HB-RWD M Dsn1Δ257_Mis12-3A mutant, which has a disrupted negatively-charged patch on head 1 (Ala substitutions of Mis12 Mtw1 Glu69, Glu73 and Asp77) ( Figure 6E , Giii). Phosphoregulation of CENP-C Mif2 and CENP-U Ame1 binding to Mis12c Mtw1c K. lactis Dsn1 residues, equivalent to those of S. cerevisiae Dsn1 that form the negatively-charged patch of head 1 ( Figure 6E ), were implicated in binding the N-termini of CENP-C Mif2 and CENP-U Ame1 ( Dimitrova et al., 2016 ; Killinger et al., 2020 ). To understand the mechanism of Mis12c Mtw1c auto-inhibition, we superimposed the crystal structure of the Kl Mis12c Mtw1c head 1 bound to CENP-C Mif2 ( Dimitrova et al., 2016 ) onto our model of the KMN junction complex ( Figure 7A ). This indicated that Dsn1 AI (Dsn1 229-255 ) overlaps with CENP-C Mif2 bound to head 1, supporting a mechanism for Mis12c Mtw1c auto-inhibition in which Dsn1 AI partially occludes the binding site of CENP-C Mif2 . Download figure Open in new tab Figure 7: Regulation of Mis12c Mtw1c auto-inhibition by Aurora B kinase and its effect on the interaction with CENP-C Mif2 A) Structural alignment of the crystal structure of Kluyveromyces lactis CENP-C Mif2 residues 4-36 ( Kl CENP-C Mif2_4-36 ) as bound to the Kl Mis12c Mtw1c head 1 domain (PDB 5T59) ( Dimitrova et al., 2016 ) to the experimental model of Sc Mis12c Mtw1c (this study). The models are coloured by chain; Mis12 Mtw1 and Nnf1 are coloured identically in 5T59 and our experimentally determined model. Kl CENP-C Mif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1 AI . Only Kl CENP-C Mif2_4-36 is shown. B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12c Mtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-C Mif2 residues 1-63 (CENP-C Mif2_1-63 ) indicated within red boxes C) Coomassie brilliant blue stained SDS-PAGE gels of the chromatograms shown in panel B . B-C) K HB-RWD M = Full-length Dsn1. K HB-RWD M 2E = Full-length Dsn1 containing Dsn1 S240E and Dsn1 S250E phosphomimetic mutations. K HB-RWD M Dsn1Δ257 = Dsn1 residues 1-257 are deleted (Dsn1 Δ1-257 ). K HB-RWD M Dsn1Δ226 = Dsn1 residues 1-226 are deleted (Dsn1 Δ1-226 ). K HB-RWD M Dsn1Δ226-2E = Dsn1 residues 1-226 are deleted and Dsn1 S240E and Dsn1 S250E phosphomimetic mutations (Dsn1 β1-226,S240E,S250E ). CENP-C Mif2_1-63 is indicated within a red box. D) Isothermal titration calorimetry (ITC) data for ( i ) CENP-C Mif2_1-38 interactions with KM, and ( ii ) CENP-C Mif2_1-38 interactions with KM 2E . Phosphomimetic mutants of Aurora B Ipl1 consensus sites in Sc Dsn1 229-255 and Kl Dsn1 201-230 relieve Mis12c Mtw1c auto-inhibition and increased CENP-C Mif2 and CENP-U Ame1 affinity for the complex ( Dimitrova et al., 2016 ; Lang et al., 2018 ). Sc Dsn1 S250 , an Aurora B Ipl1 kinase substrate ( Maskell et al., 2010 ; Westermann et al., 2003 ), projects towards the negatively-charged patch of Mis12c Mtw1c head 1 ( Figure 6E ). Phosphorylation of Sc Dsn1 S250 , and the equivalent residue in Kl Dsn1 (Ser223), would cause electrostatic repulsion between the phosphoserine and this negatively-charged patch, and likely clash with Dsn1 residues of head 2. This might relieve Mis12c Mtw1c auto-inhibition by reducing the binding affinity of Dsn1 AI for head 1, releasing it from the CENP-C Mif2 binding site. Sc Dsn1 S240 and its equivalent residue, Kl Dsn1 S213 , conform to the Aurora B Ipl1 substrate consensus sequence. Although not reported to be phosphorylated by Aurora B Ipl1 , Sc Dsn1 S240 is phosphorylated in vivo ( Lanz et al., 2021 ). Sc Dsn1 S240 projects away from head 1 ( Figure 6E ), however phosphorylation of Sc Dsn1 S240 and Kl Dsn1 S213 could establish intramolecular interactions with positively charged residues Sc Dsn1 R237 and Sc Dsn1 R247 (conserved in K. lactis , Figure 6F ) to relieve Mis12c Mtw1c auto-inhibition by destabilising the Dsn1 AI α-helices. Consistent with these proposals, an ITC experiment demonstrated that an S. cerevisiae Dsn1 228-254 peptide with phosphomimetic mutations at the Dsn1 S240 and Dsn1 S250 Aurora B Ipl1 sites bound to K HB-RWD M Dsn1Δ257 with a K D of 6.0 μM ( Figure 6Giv ), a five-fold lower affinity than the wildtype peptide ( Figure 6Gi ). To test the model that Dsn1 AI auto-inhibits Sc Mis12c Mtw1c , we used analytical SEC to assess binding of an N-terminal fragment of CENP-C Mif2 comprising residues 1-63 (CENP-C Mif2_1-63 ) to K HB-RWD M complexes containing Dsn1 N-terminal truncations. CENP-C Mif2_1-63 did not bind K HB-RWD M containing full length Dsn1 ( Figure 7B , Cii) but did interact with K HB-RWD M Dsn1Δ257 ( Figure 7B , Ciii), similar to previous reports for Kl Mis12c Mtw1c ( Dimitrova et al., 2016 ). This indicated that the region mediating Mis12c Mtw1c auto-inhibition is within Dsn1 N-IDR . CENP-C Mif2_1-63 did not bind K HB-RWD M Dsn1Δ226 which lacked the N-terminal 226 amino acids of Dsn1 ( Figure 7B , Civ), demonstrating that residues 227-257 of Dsn1, corresponding to Dsn1 AI , are sufficient to confer Mis12c Mtw1c auto-inhibition. Aurora B Ipl1 Dsn1 S240E and Dsn1 S250E phosphomimetic mutants within Dsn1 AI in either full-length Dsn1 ( Figure 7B , Cv) or Dsn1 Δ1-226 ( Figure 7B , Cvi) were sufficient to abolish Mis12c Mtw1c auto-inhibition. Together, these results indicate that Dsn1 AI auto-inhibits Mis12c Mtw1c by binding to head 1, and that either Aurora B Ipl1 phosphorylation of Ser240 and Ser250 or deletion of the N-terminal 257 amino acids of Dsn1 relieves Mis12c Mtw1c auto-inhibition. We further investigated the effect of Dsn1 AI on the interaction between K HB-RWD M (which has wild-type unphosphorylated Dsn1 AI ) and a peptide modelled on the N-terminal 38 residues of CENP-C Mif2 (CENP-C Mif2_1-38 ). Using ITC, we did not detect an interaction between CENP-C Mif2_1-38 and K HB-RWD M ( Figure 7Di ). In contrast, CENP-C Mif2_1-38 associated with K HB-RWD M 2E (incorporating Dsn1 S240E and Dsn1 S250E phosphomimetic mutants) with a K D of 0.98 μM ( Figure 7Dii ), an affinity similar to CENP-C Mif2_1-41 binding to Kl Mis12c Mtw1c with phosphomimetic mutations of Dsn1 AI ( K D of 1.75 μM) ( Dimitrova et al., 2016 ). These data are consistent with Aurora B Ipl1 phosphorylation of Dsn1 overcoming auto-inhibition of Mis12c Mtw1c stimulating its association with CENP-C Mif2 . Budding yeast Mis12c Mtw1c binds an N-terminal segment of CENP-U Ame1 , however there are conflicting data concerning whether Mis12c Mtw1c auto-inhibition affects this interaction ( Dimitrova et al., 2016 ; Hornung et al., 2014 ). A complex of S. cerevisiae CENP-U Ame1 and CENP-Q Okp1 (CENP-QU) binds to Sc Mis12c Mtw1c with wildtype Dsn1 AI when both are at micromolar concentrations, as assessed by analytic SEC ( Hornung et al., 2014 ). However, another study using fluorescence polarisation showed that, at lower concentrations (50 nM), the N-terminal 25 amino acids of K. lactis CENP-U Ame1 did not interact with Kl Mis12c Mtw1c containing an intact Dsn1 AI ( Dimitrova et al., 2016 ). To investigate these apparent discrepancies and to understand how Dsn1 AI regulates Mis12c Mtw1c – CENP-U Ame1 interactions, we superimposed an AF2 structure prediction of S. cerevisiae Mis12c Mtw1c bound to CENP-U Ame1 ( Figures 8A and 9C , D) onto our model of the KMN junction complex ( Figure 8B ). The AF2-predicted structure of CENP-U Ame1 residues 1-33 (CENP-U Ame1_1-33 ) bound to head 1 clashed with Dsn1 AI ( Figure 8B ) more extensively than CENP-C Mif2 bound to the Mis12c Mtw1c head 1 domain ( Figure 7A ). The mode of CENP-U Ame1 binding to head 1, involving a basic α-helix lying along the acidic patch of head 1, is strikingly similar to how Dsn1 AI interacts with head 1 ( Figures 6C , 8B,C and 9C). This suggests that Dsn1 AI also inhibits the interaction between Mis12c Mtw1c and CENP-U Ame1 by occluding the CENP-U Ame1 -binding site on head 1. The AF2 model predicts that CENP-U Ame1 binds to a site on head 1 that is mainly distinct from the CENP-C Mif2 -binding site ( Dimitrova et al., 2016 ), but that both ligands nevertheless share a small common binding surface ( Figure 8D ). This may explain why CENP-C Mif2 and CENP-U Ame1 associate with Mis12c Mtw1c mutually exclusively ( Killinger et al., 2020 ). Download figure Open in new tab Figure 8: Effect of Mis12c Mtw1c auto-inhibition on the interaction with CENP-U Ame1 A) AF2 structure prediction of the interaction between S. cerevisiae Mis12c Mtw1c and CENP-U Ame1 coloured by chain, showing only the Mis12c Mtw1c head 1 domain and residues 1-33 of CENP-U Ame1 predicted to interact with Mis12c Mtw1c . The structure prediction was performed using full-length and wildtype proteins apart from Dsn1, from which Dsn1 residues 1-257 were excluded to avoid Mis12c Mtw1c auto-inhibition. Top panel: Overview of complex. Lower panel: details of CENP-U Ame1 residues Asp25 to Thr31 interactions with head 1. Val26, Val27, Ile28 and Phe29 of CENP-U Ame1 engage a hydrophobic pocket on head 1, augmented by hydrogen bonds from Asp25 and Thr31. B) Superimposition of the AF2 model presented in Figure 8A onto the experimental model of Sc Mis12c Mtw1c (this study). The subunits are coloured by chain; Mis12 Mtw1 and Nnf1 are coloured identically in both the AF2 model and in the experimentally determined model. The apparent steric clash between CENP-U Ame1 residues 1-24 and Dsn1 AI is highlighted. C) Comparison of the AF2 model of CENP-U Ame1 bound to Sc Mis12c Mtw1c (top panel) and Dsn1 AI of Sc Mis12c Mtw1c (bottom panel). Both CENP-U Ame1 and Dsn1 AI form basic α-helices that engage the acid patch of head 1. D) CENP-C MIF2 and CENP-U Ame1 -binding sites overlap. E) SEC elution chromatograms of CENP-QU alone, Knl1c:Mis12c Mtw1c complexes that lack residues 1-444 of Knl1 Spc105 alone (K HB-RWD M), or after reconstitution of the interaction between CENP-QU and K HB-RWD M. F) Coomassie brilliant blue stained SDS-PAGE gels of the experiments of the experiments in panel E . G) Isothermal titration calorimetry (ITC) data for ( i ) CENP-U Ame1_1-33 interactions with KM, ( ii ) CENP-U Ame1_1-33 interactions with KM 2E and ( iii ) the CENP-U Ame1_1-33 mutant with Ala substitutions of D25, V27, I28, F29, T31 predicted to interact with Mis12c Mtw1c ( Figure 8C , D) to KM 2E . Download figure Open in new tab Figure 9: Comparison of cryo-EM structure of Sc Mis12c Mtw1c with AF2 models of Sc Mis12c Mtw1c in complex with CENP-C Mif2 and CENP-U Ame1 and Kl Mis12c and human Mis12c. A) AF2 model of Sc Mis12c Mtw1c in complex with CENP-C Mif2 (residues of 1-55). B) Model coloured by residue pLDDT score. The predicted alignment error (PAE) score plot is shown to the right. C) AF2 model of Sc Mis12c Mtw1c in complex with CENP-U Ame1 (residues 1-34 of CENP-U Ame1 shown). D) Model coloured by residue pLDDT score. The predicted alignment error (PAE) score plot is shown to the right. In both AF2 models, head 2 is predicted to rotate by 180° about two orthogonal axes relative to head 2 of the inactive Sc Mis12c Mtw1c structure in Figure 6C . E) Crystal structure of the heads and stalk region of activated human Mis12c in complex with CENP-C (residues 1-48) (PDB: 5LSK) ( Petrovic et al., 2016 ). F) Crystal structure of the heads and stalk region of K. lactis Mis12c Mtw1c (PDB: 5T58) ( Dimitrova et al., 2016 ). Head 2 adopts a similar conformation to head 2 of the AF2 model of Sc Mis12c Mtw1c (Figure S5E). G) and H) Comparison of the cryo-EM structures of the heads and stalk region of human Mis12c in the inactive unphosphorylated conformation (PDB: 8PPR) ( Yatskevich et al., 2024 ) (G) with the equivalent regions of S. cerevisiae Mis12c Mtw1c (this study) (H). The structures shown in G and H were superimposed on the Mis12 chain of head 1. Although analytical SEC showed that CENP-QU bound to K HB-RWD M with full-length wild-type Dsn1 with partial dissociation ( Figure 8E , F), consistent with previous results ( Hornung et al., 2014 ), in an ITC experiment using 16 μM K HB-RWD M, we did not detect K HB-RWD M binding to 400 μM CENP-U Ame1_1-33 (residues 1-33 residues of CENP-U Ame1 ) ( Figure 8Gi ). Consistent with the model that Aurora B Ipl1 kinase phosphorylation of Dsn1 strengthens CENP-U binding to KMN, KM 2E with Dsn1 S240E and Dsn1 S250E phosphomimetic mutations, bound CENP-U Ame1_1-33 with a K D of 0.24 μM ( Figure 8Gii ). This binding affinity is six-fold higher than that observed for residues 1-25 of K. lactis CENP-U Ame1_1-25 to Kl Mis12c Mtw1c containing phosphomimetic mutations ( K D of 1.48 μM) ( Dimitrova et al., 2016 ), an increase likely explained by the predicted interactions of CENP-U Ame1 residues 27-33 with Mis12c Mtw1c head 1 ( Figure 8A ). In agreement with this, substituting Ala residues in CENP-U Ame1_1-33 to disrupt interactions of CENP-U Ame1 residues 27-33 with KM 2E reduced CENP-U Ame1_1-33-Mut binding affinity to a K D of 1.31 μM ( Figure 8Giii ). The increased affinity of CENP-U Ame1_1-33 for KM 2E relative to CENP-C Mif2_1-38 , may explain the binding of CENP-QU and not CENP-C Mif2_1-63 to K HB-RWD M as assessed by SEC ( Figures 7C and 8E , F). Human Mis12c binds CENP-C more tightly than its budding yeast counterpart, with non-phosphorylated Mis12c also capable of binding CENP-C ( Petrovic et al., 2016 ; Yatskevich et al., 2024 ). One explanation for this difference is an N-terminal 13-residue extension in human CENP-C that interacts with Mis12c head 1 ( Petrovic et al., 2016 ), reminiscent of how Sc CENP-U Ame1 is predicted to bind Sc Mis12c Mtw1 ( Figure 9C , E). Model for Aurora B Ipl1 -dependent activation mediated by conformational changes of head 2 Relative to our cryo-EM structure, head 2 in the AF2 Sc Mis12c Mtw1c model is rotated ∼90° and displaced from head 1, with Dsn1 AI adopting an extended conformation ( Figures 6C and S5E). The separated configuration of head 1 and head 2 in the AF2 Sc Mis12c Mtw1c model is reminiscent of crystal structures of human Mis12c bound to CENP-C ( Petrovic et al., 2016 ) and Kl Mis12c Mtw1c from which the auto-inhibitory segment was absent ( Dimitrova et al., 2016 ) ( Figure 9E , F). Additionally, the orientation of head 2 in the Kl Mis12c Mtw1c crystal structure closely matches that of the AF2 Sc Mis12c Mtw1c model ( Figures 9F and S5E, F). This suggests that the AF2 Sc Mis12c Mtw1c model represents a plausible prediction of the Aurora B Ipl1 -phosphorylated, activated state of Sc Mis12c Mtw1c . Consistent with this proposal, binding sites for CENP-C Mif2 and CENP-U Ame1 on head 1 are also not occluded by Dsn1 AI in this conformation ( Figure 9A , C and Video S2). Dsn1 AI -mediated auto-inhibition of Sc Mis12c Mtw1c resembles mechanisms proposed for human Mis12c in which Dsn1 AI bridges the two head domains, and together with head 2, occludes the inner kinetochore-binding sites on head 1 ( Figure 9G ) ( Yatskevich et al., 2024 ). Model of the entire S. cerevisiae KMN complex We were unable to identify particles in our cryo-EM datasets that contained signal for the predicted Knl1c helical bundle domain (Figures S1A, and 10A, C), nor for the Spc24:Spc25 coiled-coils and Ndc80:Nuf2 subunits (Figures S1A, and 10A, B). AF2 generated a structure prediction for the Knl1c helical bundle (residues 492 to 632 of Knl1 Spc105 and 7 to 194 of ZWINT Kre28 ) with high confidence ( Figure 10A , C). This helical bundle is connected to the Knl1c RWD domains by two segments in Knl1 Spc105 and ZWINT Kre28 (‘linker 1’ and ‘linker 2’) that were predicted to adopt extended conformations with lower pLDDT scores ( Figure 10A , C). Flexibility of linker 2 would explain why we were unable to resolve Knl1c domains beyond the RWD domains. An equivalent linker does not interrupt the coiled-coil of human Knl1c, possibly explaining why its cryo-EM density was visible ( Yatskevich et al., 2024 ). We also performed AF2 structure predictions of Ndc80c. We observed a bent conformation that brings the Ndc80c microtubule binding elements (Ndc80c CH domains and Ndc80 N-tail) into proximity with an intermediate section of the Ndc80:Nuf2 coiled-coil ( Figure 10A , B). Although isolated Ndc80c has been observed to exhibit modest bending by negative stain electron microscopy ( Wang et al., 2008 ), such a highly bent conformation is unlikely to be adopted in the presence of microtubules or Dam1c rings ( Aravamudhan et al., 2014 ; Muir et al., 2023 ). In the structure predicted by AF2, the Spc24:Spc25 coiled-coils are connected to their respective RWD domains by a linker (‘Spc24:Spc25 linker’) ( Figure 10B ). We expect that we were unable to resolve domains beyond the Spc24:Spc25 linker in our cryo-EM analysis because it flexibly tethers the Ndc80c coiled-coils and microtubule binding elements to the KMN junction. FRET measurements performed in the absence of both microtubules and Dam1c showed that Ndc80c bending is dynamic, and that more tightly bent Ndc80c conformations than those predicted by AF2 are possible ( Scarborough et al., 2019 ). However, these tightly bent conformations are adopted with low propensity in complex with Mis12c Mtw1c ( Scarborough et al., 2019 ). Download figure Open in new tab Figure 10: Hypothetical model of the entire S. cerevisiae KMN complex and comparison to humans A) Model of the entire S. cerevisiae KMN complex based on the structures determined experimentally in this study and AF2 models of Knl1c and Ndc80c. Flexibility of Ndc80c has been previously demonstrated ( Scarborough et al., 2019 ; Wang et al., 2008 ), and flexibility of the Knl1c helical bundle is inferred from AF2 models. CH: calponin homology. N-tail: N-terminal tail. B) AF2 structure prediction of Ndc80c, coloured by residue pLDDT score. The structure was predicted using full-length, wildtype protein sequences. The predicted alignment error (PAE) score plot of the AF2 Ndc80c model is shown on the left of the panel. C) AF2 structure prediction of Knl1c:Mis12c Mtw1c showing the Knl1c helical bundle domain, Knl1c RWD domains, and Mis12c Mtw1c stalk CC3 region. The structure was predicted using full-length, wildtype protein sequences coloured by residue pLDDT score with the predicted alignment error (PAE) score plot shown above. We generated a model of the S. cerevisiae KMN complex by superimposing the AF2 predictions of Knl1c and Ndc80c onto their corresponding RWD domains in the KMN junction complex structure ( Figure 10A ). The close proximity of the Knl1c helical bundle to the Spc24:Spc25 coiled-coils in the hypothetical model is consistent with previous CL-MS analysis of the budding yeast KMN complex ( Ghodgaonkar-Steger et al., 2020 ), although the exact positions of the domains are probably not correct. The Knl1c helical bundle is also close to the Ndc80c microtubule binding elements. Discussion Our cryo-EM structure and biochemical data of the S. cerevisiae KMN junction complex revealed both similarities and differences in how budding yeast Mis12c Mtw1c and human Mis12c interact with Knl1c and Ndc80c, and the mechanism of Aurora B Ipl1 -mediated regulation of KMN assembly onto the inner kinetochore. For both species, Mis12c Mtw1c /Mis12c interacts with Knl1c and Ndc80c through two distinct interaction modes. In a conserved mode of interaction, the coiled-coil (CC3) α-helices of the Mis12c Mtw1c /Mis12c stalk creates a rigid interface for docking with Knl1c and Ndc80c ( Figure 1F , G). Specifically, Dsn1:Nsl1 contact Ndc80c, whereas Dsn1:Nnf1 contact Knl1c. The other mode of interaction is mediated by C-terminal extensions of Mis12c Mtw1c /Mis12c subunits that interact with sites on Knl1c and Ndc80c. Conserved in both human and budding yeast KMN is the interaction of Dsn1 Cterm-helix at the interface of the Spc24:Spc25 RWD domains of Ndc80c ( Figure 1F , G), a site that also binds the Ndc80c-binding α-helix of CENP-T ( Malvezzi et al., 2013 ; Nishino et al., 2013 ). The most striking difference between budding yeast and human KMN is the contrasting functions of the Mis12 Mtw1 and Nsl1 subunits to contact Knl1c and Ndc80c. In human KMN, an α-helix of Nsl1 (Nsl1 Spc24-helix ) contacts the Ndc80c Spc24:Spc25 RWD domains ( Figure 1G ). Nsl1 then engages Knl1c at Knl1 RWD-N , and at the Knl1 RWD-N :ZWINT interface. In budding yeast these functions are substituted by Mis12 Mtw1 . Mis12 Mtw1_Spc24-helix contacts the Spc24:Spc25 RWD domains, which also stabilises the Dsn1 Cterm-helix :Spc24 RWD :Spc25 RWD interaction ( Figure 1F ). The Mis12 Mtw1 chain then interacts with the Knl1 Spc105_RWD-N :Kre28 ZWINT interface of Knl1c. Our hypothetical model of the entire S. cerevisiae KMN complex including the flexible tethering of other Knl1c and Ndc80c domains to the RWD domains of the KMN junction could have functional consequences for spindle assembly checkpoint (SAC) signalling ( Fig. 10 ). Experiments in humans and budding yeast are consistent with a model in which SAC signalling is activated at kinetochores that are not attached to microtubules ( Rieder et al., 1995 ; Wells and Murray, 1996 ), and that therefore do not incorporate Dam1c rings ( Li et al., 2002 ; Tanaka et al., 2007 ; Tanaka et al., 2005 ). Under these conditions, Ndc80c might adopt a conformation in which its microtubule binding elements localise near the Knl1c helical bundle domain and the N-terminal disordered region of Knl1 Spc105 . This may facilitate Mps1, bound proximal to the Ndc80c CH domains ( Parnell et al., 2024 ; Pleuger et al., 2024 ; Zahm and Harrison, 2024 ), in phosphorylating MELT motifs in the Knl1 Spc105 N-terminal disordered region to stimulate SAC signalling. We determined a lower-resolution cryo-EM reconstruction of the KMN junction complex in which head 2 of Mis12c Mtw1c was resolved. The auto-inhibitory region Dsn1 AI suppressed binding of Mis12c Mtw1c to the N-terminal segments of both CENP-C Mif2 and CENP-U Ame1 . Phosphomimetic mutations of Aurora B Ipl1 sites within a peptide modelled on Dsn1 AI reduced its affinity for the K HB-RWD M Dsn11′257 complex. Additionally, the same phosphomimetic mutations in Dsn1 AI activated K HB-RWD M binding of both CENP-C Mif2 and CENP-U Ame1 . These results suggest that the intra-molecular association of Dsn1 AI with Mis12c Mtw1c head 1 is responsible for Mis12c Mtw1c auto-inhibition, and that Aurora B Ipl1 phosphorylation of serine residues within this region relieves Mis12c Mtw1c auto-inhibition by reducing the binding affinity of Dsn1 AI for head 1. Cryo-EM structures and AF2 models suggest that this is because the Dsn1 AI and CENP-U Ame1 would sterically clash if both bound to head 1 ( Figure 8C ). Such a steric clash does not occur between CENP-C Mif2 and Dsn1 AI ; instead, the two elements compete for contacts with the same amino side chains within head 1 ( Figure 7A ). The higher affinity of CENP-U Ame1 for KMN relative to CENP-C Mif2 , as evidenced by our ITC data, and the binding of CENP-QU, but not CENP-C Mif2_1-63 , to unphosphorylated K HB-RWD M on SEC (this study and Hornung et al., 2014 ), has implications for understanding observations that CENP-U Ame1 is essential for yeast viability ( Hornung et al., 2014 ; Schmitzberger et al., 2017 ), whereas the N-terminus of CENP-C Mif2 is dispensable ( Hornung et al., 2014 ). Aurora B Ipl1 localisation to CCAN is necessary for the correction of erroneous kinetochore microtubule attachments ( Fischböck-Halwachs et al., 2019 ; García-Rodríguez et al., 2019 ; Li et al., 2023 ). Our findings therefore suggest a mechanism by which attachments of mitotic chromosomes to spindle microtubules, mediated by the connection of KMN complexes to centromere-bound CCAN through either CENP-C Mif2 or CENP-U Ame1 , are strengthened at kinetochores that are able to correct errors in these attachments. Our work has revealed that the mechanisms mediating assembly and auto-inhibition of the KMN complex are conserved at the structural level in humans and budding yeast. Furthermore, we showed that Aurora B Ipl1 -mediated phosphorylation overcomes auto-inhibition of human Mis12c and budding yeast Mis12c Mtw1c through similar structural mechanisms. In humans, Aurora B regulation of Mis12c auto-inhibition probably restricts KMN complex recruitment to mitotic kinetochores that are competent to correct erroneous kinetochore-microtubule attachments through error correction signalling ( Yatskevich et al., 2024 ). This and a previous study ( Hornung et al., 2014 ) revealed that in budding yeast the inner kinetochore subunit CENP-U Ame1 binds to unphosphorylated Mis12c Mtw1c . In contrast to humans, the KMN complex is attached to the centromere throughout almost the entire cell cycle in budding yeast, with only transient disassembly of kinetochores during S-phase ( Kitamura et al., 2007 ). Furthermore, Aurora B Ipl1 is recruited to kinetochores from G1 to metaphase, being depleted from centromeres under tension at metaphase and relocalising to the spindle midzone ( Buvelot et al., 2003 ; Campbell and Desai, 2013 ; Edgerton et al., 2023 ; Nerusheva et al., 2014 ; Shimogawa et al., 2009 ). Consequently, relief of Mis12c Mtw1c auto-inhibition from G1-phase to metaphase through phosphorylation by Aurora B Ipl1 most likely strengthens pre-existing connections between the inner and outer kinetochore, allowing bioriented kinetochores to withstand the pulling and pushing forces exerted by spindle microtubules. However, the diminished, but not abolished, affinity of both CENP-U Ame1 for unphosphorylated Mis12c Mtw1c is possibly still able to support kinetochore-microtubule attachment during anaphase after Aurora B Ipl1 has localised away from kinetochores. Materials and methods Cloning of S. cerevisiae CENP-C Mif2 and S. cerevisiae KMN complex proteins Genes encoding S. cerevisiae Ndc80, Nuf2, Spc24, Spc25, Dsn1, Mis12 Mtw1 , Nnf1, Nsl1, residues 1-444 of Knl1 Spc105 , and ZWINT Kre28 were each amplified by Polymerase chain reaction (PCR) from S. cerevisiae strain S288c genomic DNA ( Zhang et al., 2016 ). Genes encoding Ndc80 and Nuf2 were cloned into the same pU1 plasmid ( Zhang et al., 2016 ). Genes encoding Spc24 and Spc25 were cloned into the same pF1 plasmid ( Zhang et al., 2016 ). Genes encoding Knl1 Spc105 residues 445-917 (Knl1 Spc105_HB-RWD ) and ZWINT Kre28 were cloned into the same modified pF1 plasmid. Genes encoding Knl1 Spc105 residues 710–917 (Knl1 Spc105_RWD ) and ZWINT Kre28 residues 268-385 (ZWINT Kre28_RWD ) were cloned into the same modified pF1 plasmid. TEV and HRV-3C cleavable twin-strep-tag II (TS) tags were introduced into the C-termini of Nuf2 and ZWINT Kre28 , respectively, and a tandem tag comprising maltose binding protein (MBP) fused to monomeric enhanced green fluorescent protein (mEGFP) was introduced into the N-terminus of Knl1 Spc105_RWD . Genes encoding Dsn1, Mis12 Mtw1 , Nsl1, and Nnf1 were cloned into the same modified pF1 plasmid for coexpression with Knl1 Spc105 and ZWINT Kre28 . For expression of Mis12c Mtw1c and Mis12c Mtw1c mutants, Dsn1 and Nsl1 were cloned into the same modified pF1 plasmid and twin-strep-tag II was introduced into the N-terminus of Dsn1. This plasmid was used to generate a virus for coexpression with a second virus generated from a another modified pF1 plasmid containing a Mis12 Mtw1 -Nnf1 pair. Constructs for Dsn1 Δ1-226 , Dsn1 Δ1-226,S240E,S250E , Dsn1 Δ1-257 , Dsn1 S240E,S250E , Mis12 Mtw1_E69A,E73A,E77A , Mis12 Mtw1_Δ272-C , and Nnf1 Δ180-C were created using primers supplied from Merck and the KLD reaction kit supplied by New England Biolabs and cloned into plasmids containing all other Mtw1c subunits for coexpression with Knl1 Spc105_HB-RWD and ZWINT Kre28 . Constructs for Mis12 Mtw1_Δ272-C and Nnf1 Δ_180-C were additionally cloned into the plasmid containing the Mis12 Mtw1 -Nnf1 pair and expressed together with a virus made from the plasmid containing a Dsn1-Nsl1 pair. To express Knl1c:Mis12c Mtw1c with the Mis12c head 1 domain deleted, constructs for Mis12 Mtw1_1Δ1-106 and Nnf1 1Δ1-104 were cloned with Dsn1 1′1-257 and wildtype Nsl1 into the same modified pF1 plasmid and coexpressed with Knl1 Spc105 and ZWINT Kre28 . To express Knl1c:Mis12c Mtw1c with mutated Dsn1, constructs for Mis12 Mtw1 , Nnf1, Nsl1, and mutant Dsn1 were cloned into the same modified pF1 plasmid and coexpressed with Knl1 Spc105 and ZWINT Kre28 . The gene encoding S. cerevisiae CENP-C Mif2 residues 1-63 (CENP-C Mif2_1-63 ) was amplified from S. cerevisiae strain S288c genomic DNA and cloned into a pET28 plasmid carrying a Kanamycin selectable marker. A 6xHis-SUMO-tag was introduced into the N-terminus of the protein. A construct to express protein for surface plasmon resonance (SPR) was assembled by introducing a 6xHis-SUMO-tag was introduced into the N-terminus of the protein and a glycine-serine-glycine linker followed by a twin-strep-tag II (TS) tag into the C-terminus of the protein. To construct plasmids for yeast strain construction, the promoter, coding regions, and terminator regions defined previously for the MTW1 and NNF1 genes in the yeast genome ( Euskirchen, 2002 ) were amplified from S. cerevisiae strain S288c genomic DNA. The MTW1 fragments were cloned into a modified pRS304 plasmid ( Frigola et al., 2013 ) and a HA 3 tag was introduced into the N-terminus of the protein in a single step using NEBuilder HiFi DNA assembly reaction kit supplied by New England Biolabs. The NNF1 fragments were cloned into a modified pRS305 plasmid ( Frigola et al., 2013 ) and a V5 3 tag was introduced into the N-terminus of the protein in a single step using NEBuilder HiFi DNA assembly reaction kit. A pRS304 series plasmid and a pRS305 series plasmid encoding HA 3 -Mis12 Mtw1_Δ272-C and V5 3 -Nnf1 Δ180-C were constructed from the plasmids described above using PCR with primers supplied by Merck followed by processing with the KLD reaction kit supplied by New England Biolabs. Expression and purification of KMN subcomplexes, and CENP-C Mif2_1-63 and CENP-QU complexes Baculoviruses for expression of KMN subcomplexes were generated from the plasmids described above using methods described previously ( Zhang et al., 2016 ). Ndc80c was expressed from two baculoviruses encoding a Ndc80-Nuf2 gene pair and a Spc24-Spc25 gene pair in High-5 insect cells. Six-subunit Knl1c:Mis12c Mtw1c complexes were expressed from two baculoviruses encoding a Knl1 Spc105_HB-RWD -ZWINT Kre28 gene pair and a Dsn1-Nsl1-Mis12 Mtw1 -Nnf1 gene quartet in High-5 insect cells. Isolated Mis12c Mtw1c was expressed from two baculoviruses encoding Mis12 Mtw1 -Nnf1 and Dsn1-Nsl1 gene pairs. The Sf9 and High-5 insect cell lines used for baculovirus generation and protein complex expression were not tested for mycoplasma contamination or authenticated. 2 L roller bottles containing 500 mL suspension cultures of High-5 cells were typically infected with 2.5% vol/vol of P3 Sf9 suspension cell culture, and the High-5 expression cultures were subsequently harvested 42–70 h after infection when the cell viability had dropped to 75–85%. All purification steps were carried out at 4 ° C or on ice. Cells expressing Ndc80c, Knl1c:Mis12c Mtw1c , or Mis12c Mtw1c were lysed by sonication in lysis buffer (50 mM Tris.HCl pH 8.5, 8 mM benzamidine, 1 mM EDTA, 1 mM TCEP, 0.2 mM PMSF, cOmplete EDTA-free protease inhibitor tablets) containing benzonase and either 300 mM NaCl and 7.5% glycerol (Ndc80c), 250 mM NaCl and 7.5% glycerol (Mis12c Mtw1c ), or 200 mM NaCl and 5% glycerol (Knl1c:Mis12c Mtw1c ). Lysates were cleared by ultracentrifugation at 38,000 x g for one h and loaded onto Strep-Tactin superflow plus cartridges (Qiagen). Immobilised complexes were washed with lysis buffer after adjusting to pH 8.0. Immobilised Knl1c:Mis12c Mtw1c complexes were subjected to further washes with a buffer containing ATP (50 mM Tris.HCl pH 8.0, 175 mM KCl, 10 mM MgCl 2 , 2.5 mM ATP sodium salt, 1 mM TCEP, 2.5% glycerol) followed by a buffer containing 50 mM Tris.HCl pH 8.0, 200 mM NaCl, 2.5% glycerol. Bound complexes were then eluted using a buffer containing 50 mM Tris.HCl pH 8.0, 2.5 mM d-desthiobiotin, 1 mM TCEP, and either 100 mM NaCl (Ndc80c, Mis12c Mtw1c , and Knl1c:Mis12c Mtw1c used for SPR) or 200 mM NaCl (Knl1c:Mis12c Mtw1c for all applications other than SPR). Knl1c:Mis12c Mtw1c used for SPR was incubated overnight with TEV protease whilst rolling at 4 ° C to remove the C-terminal twin-strep-tag II sequences from Kre28. Knl1c:Mis12c Mtw1c used for SPR and Ndc80c were further purified by loading the TEV protease cleavage reaction and Strep-Tactin eluate, respectively, onto a Resource Q anion exchange column (Cytiva). Bound complexes were eluted with buffer containing 50 mM Tris.HCl pH 8.0, 1 mM TCEP, 2.5% glycerol and a gradient from 100 mM to 1000 mM NaCl over 15-20 column volumes. Peak fractions were pooled, and salt concentration in pooled Knl1c:Mtw1c protein was reduced to 150 mM prior to incubation for 30 min whilst rolling at 4 ° C. Pooled protein was further purified using a Superose 6 10/300 increase GL column (Cytiva) equilibrated in KMN buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP). Peak Ndc80c fractions were pooled, concentrated, and flash frozen using liquid nitrogen. Mis12c Mtw1c complexes and Knl1c:Mis12c Mtw1c complexes used for all applications other than SPR were further purified by concentrating the Strep-Tactin eluate and applying it to a Superose 6 increase 10/300 GL column equilibrated in KMN buffer. The peak protein complex fractions were concentrated and flash frozen using liquid nitrogen. CENP-C Mif2_1-63 with a N-terminal 6xHis-SUMO-tag was expressed in E. coli BL-21 CodonPlus RIL cells by induction with 0.5 mM IPTG at a cell culture OD 600nm = 0.8 followed by incubation of cells a further 18 h at 18 ° C with shaking. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (30 mM Tris.HCl pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM TCEP, 0.2 mM PMSF) containing benzonase and 10 mM imidazole. The lysate was cleared by ultracentrifugation at 38,000 x g in a JA25.50 rotor for 1 h, and the clarified lysate was applied to a HisTrap excel column (Cytiva). Immobilised proteins were washed with lysis buffer and were eluted by successive applications of lysis buffer containing 50 mM, 100 mM, 150 mM, 300 mM, and 500 mM imidazole. The eluates obtained after application of 150 mM and 300 mM imidazole were pooled and applied to HiPrep 26/10 desalting columns (Cytiva) that were equilibrated in buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM TCEP) to reduce the imidazole content. CENP-C Mif2_1-63 with a N-terminal 6xHis-SUMO-tag and a C-terminal twin-strep-tag II (TS) tag (CENP-C Mif2_1-63 -TS) was expressed in E. coli BL-21 CodonPlus RIL cells by induction with 0.5 mM IPTG at a cell culture OD 600nm = 0.7 followed by incubation of cells a further 14 h 30 min at 18 ° C with shaking. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (30 mM Tris.HCl pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM TCEP, 0.2 mM PMSF) containing benzonase. The lysate was cleared by ultracentrifugation at 48,000 x g in a JA25.50 rotor for 45 min, and the clarified lysate was applied to Strep-Tactin superflow plus cartridges (Qiagen). Immobilised proteins were washed with lysis buffer followed by wash buffer 2 (20 mM HEPES pH 7.0, 150 mM NaCl, 1 mM TCEP) and were eluted using wash buffer 2 containing 2.5 mM d-desthiobiotin. The 6xHis-SUMO tag was removed from the CENP-C Mif2_1-63 and CENP-C Mif2_1-63 -TS by overnight incubation with recombinant Ulp1 protease carrying a 6xHis tag at 4 ° C. Cleaved protein was isolated from the protease by a reverse IMAC purification step. After diluting the salt concentration to 100 mM, the reverse IMAC flow through was applied to a Resource S cation exchange column (Cytiva) for further purification. CENP-C Mif2_1-63 was eluted using buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP. CENP-C Mif2_1-63 -TS was eluted using buffer containing 20 mM HEPES pH 7.5, 1 mM TCEP, and a gradient from 100 mM to 1000 mM NaCl over 15 column volumes. CENP-C Mif2_1-63 and CENP-C Mif2_1-63 -TS were concentrated and further purified by application to a Superdex 75 16/60 column (Cytiva) equilibrated in KMN buffer. Peak CENP-C Mif2_1-63 fractions were pooled, concentrated, and flash frozen using liquid nitrogen. CENP-QU was expressed and purified according to the method described in ( Dendooven et al., 2023 ). Reconstitution and GraFix crosslinking the S. cerevisiae KMN complex for cryo-EM The S. cerevisiae KMN complex was reconstituted and crosslinked for cryo-EM using a GraFix protocol ( Stark, 2010 ). Two buffers were used to prepare 10–30% continuous glycerol gradients (4 mL total volume) with an additional 0.0–0.2% glutaraldehyde component using a gradient master: buffer A contained 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM TCEP, and buffer B contained 20 mM HEPES pH 7.5, 150 mM NaCl, 30% glycerol, 1 mM TCEP, 0.2% glutaraldehyde. Stocks of Ndc80c and a Knl1c:Mis12c Mtw1c complex that contained Knl1 Spc105_HB-RWD (K HB-RWD M) were thawed on ice, and diluted and mixed at a 4μM equimolar (1:1) ratio in KMN buffer. The mixture was incubated for 30 min at 4 ° C to reconstitute the KMN complex. 200 pmol (50 μL volume) of reconstituted K HB-RWD MN complex was applied to each glycerol gradient, and gradients were subsequently subjected to centrifugation at 50,000 rpm in a SW 60 Ti Swinging-Bucket Rotor (Beckman Coulter) for 16 h. Glycerol gradients were manually fractionated into 250 μL fractions, and unreacted glutaraldehyde was quenched by pipetting the fractions over a solution of 1 M Tris.HCl pH 7.5 such that the final concentration of Tris.HCl in each fraction was 20 mM. Fractions encompassing the gradient volume 2.00–2.75 mL were pooled, glycerol content was reduced by application to Zeba spin desalting columns equilibrated in KMN buffer, and the protein complex was concentrated to a final concentration of 0.8 mg/mL. Cryo-EM grid preparation Cryo-EM grids were glow discharged in an Edwards S150B glow discharger for 60 s at setting 6, 20–25 Ma, 1.2 kV and 0.2 mBar (0.15 Torr) immediately prior to application of sample. 3 μL of reconstituted and crosslinked K HB-RWD MN complex was applied to freshly glow discharged UltrAuFoil 300 mesh gold R1.2/1.3 grids (Quantifoil Micro Tools). Grids were mounted in a FEI Vitrobot mark III humidity chamber (maintained at 4 ° C, 100% humidity), blotted with Whatman filter paper I (2 s blotting time,-7 blotting force), and vitrified in liquid ethane. Cryo-EM data acquisition Cryo-EM movies were collected on a Thermo Fisher Scientific Titan Krios 1 microscope operating at 300 keV using a Gatan K3 camera equipped with a Gatan energy filter. Movies were collected at x105,000 magnification (pixel size 0.825 Å px -1 ) with a 1.75 s exposure and 15.6 e - /px/s fluence, providing a total dose of 40 e - /Å 2 , and were split into 40 frames. A series of movies were collected over a range of defoci from-1.4 μm to-3.0 μm, at an interval of 0.4 μm. Data acquisition was performed using aberration-free image shifts (AFIS) as employed in the Thermo Fisher Scientific ‘EPU’ automated data acquisition software. The limited resolution (6.5 Å) of the consensus structure of the KMN junction complex was likely caused by heterogeneity at either end of the Mis12c Mtw1c stalk domain (Figure S2Di) that we were unable to resolve with extensive 2D and 3D classification and 3D variability analyses. To obtain a higher-resolution structure of subdomains, we subtracted the signal arising from the poorly resolved Mis12c Mtw1c head 2 domain (Figure S2Dii) and subjected the subtracted particles to multibody refinement using masks encompassing the two domains forming the apex and base of the Mis12c Mtw1c stalk domain (Figure S2Diii). This yielded reconstructions showing the Knl1c and Spc24:Spc25 RWD domains at the apex of the Mis12c Mtw1c stalk, and head 1 at the base of the Mis12c Mtw1c stalk, at global resolutions of 4.8 Å and 4.9 Å, respectively (Figure S2Diii). The local resolutions of the reconstructions reached 4.2 Å and 4.5 Å in the central regions of the apex and base density maps, respectively (Figure S2E, F), which were sufficient to interpret with an AlphaFold2 (AF2)-predicted model ( Jumper et al., 2021 ). To obtain a composite map of the KMN junction complex, we docked the cryo-EM maps of the apex and base multi-bodies into the consensus cryo-EM map ( Figure 1B ). This composite map was used to build a model of the S. cerevisiae KMN junction complex using AF2 structure predictions and Isolde model refinement, as detailed in Methods ( Croll, 2018 ; Jumper et al., 2021 ). Cryo-EM data processing Alignment of micrograph raw movie frames and correction to a single micrograph image was performed using UCSF MotionCor2 as implemented in RELION ( Kimanius et al., 2021 ; Zheng et al., 2017 ). The contrast transfer function (CTF) of each motion corrected micrograph was estimated using CTFFIND-4.1 as implemented in RELION ( Kimanius et al., 2021 ; Rohou and Grigorieff, 2015 ). Manual particle picking of 10 micrographs was used to train a Topaz model that was used to pick the entire dataset of 26,390 micrographs ( Bepler et al., 2019 ) (Figure S2 and Table S1). The particles picked by the first Topaz model were curated by two successive rounds of 2D classification in CryoSPARC ( Punjani et al., 2017 ). 2D classification in CryoSPARC was always performed using batch sizes of 400 and 40 online-em iterations. Particles classified into class averages that resembled KMN were selected and used to train a second Topaz model in RELION that was once again used to pick all 26,390 micrographs. Particles were separated into six distinct sets, based on the appearance of 2D class averages, using two successive rounds of 2D classification in CryoSPARC. Each set of particles was used to generate an ab initio model in CryoSPARC, yielding one KMN model, one additional protein model, and four decoy noise models. All six ab initio models were used as references for a noise decoy classification step using the heterogeneous refinement job type in CryoSPARC. Particles corresponding to the model that resembled the KMN complex were subjected two further successive rounds of 2D classification, yielding 321,078 particles that corresponded to 2D class averages that resembled KMN. This set of particles was further processed in RELION for all subsequent steps. All 3D refinement and 3D classification jobs in RELION used Blush regularization, as implemented in RELION 5.0 ( Kimanius et al., 2021 ; Kimanius et al., 2024 ). The particles were used to generate an ab initio model, against which they were refined (successive unmasked and masked refinements) to reconstruct a cryo-EM density map with a resolution of 7.5 Å and discontinuous density for the Spc24 and Spc25 RWD domains and the Mis12c Mtw1c head 2 domain. To better resolve the Spc24 and Spc25 RWD domains, particles were subjected to focussed 3D classification without alignment using a mask encompassing the CC3 region of the Mis12c Mtw1c stalk domain, the Knl1c RWD domains, and the Spc24 and Spc25 RWD domains, and a T value of 25. A set of 113,688 particles corresponding to two 3D classes with improved continuity of the Spc24 and Spc25 RWD domain density were refined to reconstruct a cryo-EM density map with a resolution of 6.5 Å and discontinuous density for the Mis12c Mtw1c head 2 domain. Signal corresponding to the Mis12c Mtw1c head 2 domain was subtracted from the particle images, and the subtracted particles were refined to reconstruct a cryo-EM density map with a resolution of 6.0 Å. Two non-overlapping masks were generated for multibody refinement: one encompassed part of the CC2 region of the Mis12c Mtw1c stalk domain, the CC3 region of the Mis12c Mtw1c stalk domain, the Knl1c RWD domains, and the Spc24 and Spc25 RWD domains (‘apex mask 1’), whilst the other encompassed the Mis12c Mtw1c head 1 domains, the CC1 region of the Mis12c Mtw1c stalk domain, and the remaining segment of the CC2 region of the Mis12c Mtw1c stalk domain (‘base mask 1’). Multibody refinement using these masks yielded two cryo-EM density maps with global resolutions (GS-FSC) of 4.8 Å (‘apex body 1’) and 4.9 Å (‘base body 1’), respectively. The local resolutions of the reconstructions reached 4.2 Å and 4.5 Å in the central regions of the apex and base density maps, respectively (Figure S2E, F), which were sufficient to interpret with an AlphaFold2 (AF2)-predicted model ( Jumper et al., 2021 ). To obtain a composite map of the KMN junction complex, the cryo-EM maps of the apex and base multi-bodies were docked into the consensus cryo-EM map ( Figure 1B ). This composite map was used to build a model of the S. cerevisiae KMN junction complex using AF2 structure predictions and Isolde model refinement ( Croll, 2018 ; Jumper et al., 2021 ) as detailed below in ‘Cryo-EM molecular model building’. To better resolve the Mis12c Mtw1c head 2 domain, particles were subjected to focussed 3D classification without alignment using a mask encompassing the Mis12c Mtw1c head 1 and head 2 domains and the CC1 region of the Mis12c Mtw1c stalk domain (Figure S4 and Table S1). A set of 18,160 particles corresponding to a 3D class with continuous density for the Mis12c Mtw1c head 2 domain were refined to reconstruct a cryo-EM density map with a resolution of 7.5 Å that retained discontinuous density for the Spc24 and Spc25 RWD domains. Signal corresponding to the Spc24 and Spc25 RWD domains was subtracted from the particle images, and the subtracted particle images were used to reconstruct a cryo-EM density map with a resolution of 7.2 Å. Two non-overlapping masks were generated for multibody refinement: one encompassed part of the CC2 region of the Mis12c Mtw1c stalk domain, the CC3 region of the Mis12c Mtw1c stalk domain, and the Knl1c RWD domains (‘apex mask 2’), whilst the other encompassed the Mis12c Mtw1c head 1 and head 2 domains, the CC1 region of the Mis12c Mtw1c stalk domain, and the remaining segment of the CC2 region of the Mis12c Mtw1c stalk domain (‘base mask 2’). Multibody refinement using these masks reconstructed a cryo-EM density map for the Mis12c Mtw1c head 1 and head 2 domains and relevant sections of the stalk domain with a global resolution (GS-FSC) of 6.5 Å (‘base body 2’). Cryo-EM molecular model building AF2 multimer structure predictions of Mis12c Mtw1c :Spc24:Spc25 and Knl1c:Mis12c Mtw1c , performed using full-length, wild type proteins apart from Knl1 Spc105 (from which residues 1-444 were excluded) and Dsn1 (from which residues 1-278 were excluded), were used to build a model for the KMN junction complex. A composite map of the KMN junction complex was obtained by docking ‘apex body 1’ and ‘base body 1’ into the cryo-EM density map reconstructed by the preceding refinement and used for model building. The AF2 structure predictions were divided into five regions for model building. ‘Region one’, corresponding to Spc24 and Spc25 RWD domains interacting with Mis12 Mtw1_Spc24-helix and Dsn1 Cterm-helix helical motifs, comprised Spc24 143-213 , Spc25 124-221 , Mis12 Mtw1_228-252 , and Dsn1 556-576 . ‘Region two’, corresponding to the RWD domains of ZWINT Kre28 and Knl1 Spc105 interacting with the Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix helical motifs, comprised Knl1 Spc105_719-917 , ZWINT Kre28_280-368 , Mis12 Mtw1_274-289 , and Nnf1 184-201 . ‘Region three’, corresponding to all of the CC3 and most of the CC2 regions of the Mis12c Mtw1c stalk domain, comprised Dsn1 483-547 , Nnf1 139-180 , Nsl1 143-216 , and Mis12 Mtw1_132-222 . ‘Region four’, corresponding to the remainder of the CC2 and all of the CC1 regions of the Mis12c Mtw1c stalk domains, comprised Dsn1 431-482 , Nnf1 115-138 , Nsl1 97-142 , and Mis12 Mtw1_116-131 . ‘Region 5’, comprising the Mis12c Mtw1c head 1 domain, comprised Nnf1 1-114 and Mis12 Mtw1_1-115 . Isolde, as implemented in Chimera X, was used to remodel and refine these five regions of the AF2 structure predictions into the composite map of the KMN junction complex ( Croll, 2018 ; Goddard et al., 2018 ). C-terminal residues of Nnf1, Mis12 Mtw1 , Dsn1, and Nsl1 in ‘region five’ and ‘region four’ were manually reconnected with the N-terminal residue of the corresponding chain in ‘region four’ and ‘region three’, respectively, and refined into the density in Coot ( Emsley et al., 2010 ) to yield ‘region tff’. The model comprising ‘region one’, ‘region two’, and ‘region tff’ was subsequently refined and remodelled into the composite map using Isolde to yield the final model of the KMN junction complex. To build the experimental model of the S. cerevisiae KMN junction complex with head 2 we docked the KMN junction complex (without head 2) and the AF2 prediction of the S. cerevisiae Mtw1 Mis12 head2 domain (Dsn1 residues 259-353 and Nsl1 residues 1-84), and the AF2 prediction of Kl Mtw1 Mis12c , into the 6.5 Å KMN junction base body 2 cryo-EM map (Figure S4C and Table S1). The Kl Mtw1 Mis12c model accounted for nearly all density in the cryo-EM map, whereas for the S. cerevisiae model of head 2, some density regions were not assigned, mainly a helical-like density associated with Dsn1 head 1 (Dsn1 AI , Sc residues 229-255), the Dsn1 CC1-head2 linker ( Sc residues 354-365) and the Nsl1 CC1-head2 linker ( Sc residues 85-96) (Figure S5A, B, E). We used the Kl Mtw1 Mis12c model (Figure S5G, H) to guide complete building of an experimental model of Sc Mtw1 Mis12c ( Figure 6B , C). Model refinement Refinement was performed using the Phenix package ( Liebschner et al., 2019 ) (Table S1). AlphaFold2 structure predictions Local installations of AlphaFold2 ( Jumper et al., 2021 ) were used for all protein structure predictions in this study. The Colabfold implementation AF2 ( Mirdita et al., 2022 ) of AF2 was used for all structure predictions other than for the Kluyveromyces lactis Mis12c Mtw1c ( Kl Mis12c Mtw1c ), which used the Colabfold 3 version. Unless otherwise stated, wild-type, full-length protein sequences deposited in Uniprot were used for all protein structure predictions. Isothermal titration calorimetry experiments Dsn1 228-254 and Dsn1 228-254,S240E,S250E peptides were synthesised by Alta BioScience with N-terminal acetylation and C-terminal GSAW extension and amidation. The final sequence of Dsn1 228-254 was PHYIQRTRERKKSISHSQRGRRLSMLASGSAW; and Dsn1 228-254,S240E,S250E was PHYIQRTRERKKSISHSQRGRRLEMLASGSAW. All protein complexes and peptides used in a given experiment were dialysed for a minimum of 16 h and/or buffer exchanged by size exclusion chromatography into the same batch of ITC buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP). All experiments were performed using an Auto-iTC200 instrument (Malvern Instruments) at 25 ° C. In each experiment, 360 μL of protein complex was pipetted into the calorimeter cell. The protein complex loaded into the calorimeter cell contained Knl1c:Mis12c Mtw1c with Knl1 Spc105_HB-RWD and Dsn1 1′1-257 and either wildtype Mis12 Mtw1 (K HB-RWD M Dsn1Δ257 ) or Mis12 Mtw1_E69A,E73A,E77A mutant (K HB-RWD M Dsn1Δ257_Mis12- 3A ). Wild-type constructs of all other Knl1c and Mis12c Mtw1c subunits were included. Titration of either the Dsn1 228-254 or Dsn1 228-254,S240E,S250E peptide into the cell was then performed by an additional injection of 0.5 μL, followed by 19 further injections of 2.0 μL each. Heat arising from the initial injection was discarded, and the MicroCal PEAQ-ITC analysis software 1.0.0.1258 (Malvern instruments) was subsequently used to fit the change in the amount of heat released, integrated over the entire titration, to a single-site binding model. Concentrations of the protein complex and peptide used for each experiment are provided in the relevant figure panel. Titrations were performed in duplicate or triplicate and reported K D and n values were calculated as an average from all replicate results. CENP-U Ame1_1-33 (MDRDTKLAFRLRGSHSRRTDDIDDDVIVFKTPNW-amide), CENP-U Ame1_1-33-Mut (MDRDTKLAFRLRGSHSRRTDDIDDAAAVAKAPNW-amide) and CENP-C Mif2_1-38 (MDYMKLGLKSRKTGIDVKQDIPKDEYSMENIDDFFKDD-amide) peptides were synthesized by Alta BioScience. Peptides and proteins (K HB-RWD M and K HB-RWD M 2E ) were dialysed for 16 h at 4 ° C against a buffer of 25 mM HEPES (pH 7.5), 100 mM NaCl, 0.5 mM TCEP. The ITC experiments were performed in triplicate as described above except at 20 ° C. Investigation of the function of Mis12c Mtw1c motifs that form interfaces with Knl1c To test the effect of deleting the Mis12c Mtw1c motifs that form interfaces with Knl1c on Mis12c Mtw1c assembly and interaction with Ndc80c, 4 μM concentration of wildtype Mis12c Mtw1c or one of the three Mtw1c mutants (Mis12c Mtw1c_Mis12ΔC , contained Mis12 Mtw1_Δ272-C ; Mis12c Mtw1c_Nnf1ΔC , contained Nnf1 Δ180-C ; Mis12c Mtw1c_Mis12ΔC-Nnf1ΔC: contained Mis12 Mtw1_Δ272-C and Nnf1 Δ180-C ) were either individually diluted into KMN buffer, or mixed with 4 μM concentration of wildtype Ndc80c in KMN buffer. Mixtures of Ndc80c and wildtype or mutant Mis12c Mtw1c were incubated on ice for 30 min to reconstitute the complex. 100 μL of individual protein complexes or reconstitution mixtures were loaded onto a Superose 6 3.2/300 size exclusion chromatography column (Cytiva) and 100 μL eluate fractions were collected. Aliquots of each eluate fraction were mixed 1:1 (volume/volume) with 2xLDS-PAGE loading buffer (Thermo Fisher Scientific), supplemented with DTT, boiled for 3 min at 95 ° C, and analysed by SDS-PAGE. To test the effect of deleting the Mis12c Mtw1c motifs that form interfaces with Knl1c on the interaction between Knl1c and Mis12c Mtw1c , suspension cultures of High-5 insect cells were infected with one of two P3 suspension cultures, or two P3 cultures in combination, that were amplifying one of two sets of viruses. The first virus encoded ZWINT Kre28_RWD fused to a C-terminal twin-strep-tag II and Knl1 Spc105_RWD fused to a N-terminal tag comprising tandem maltose binding protein (MBP) and monomeric enhanced green fluorescent protein (mEGFP) tags. The second set of viruses encoded either wildtype Mis12c Mtw1c , or one of the Mis12c Mtw1c_Mis12ΔC , Mis12c Mtw1c_Nnf1ΔC , or Mis12c Mtw1c_Mis12ΔC-Nnf1ΔC mutants. All viruses encoding Mis12c Mtw1c contained genes that expressed Dsn1 Δ1-278 . The insect cell cultures were harvested 48 h after infection, when cell viabilities had dropped to 70-75%. All subsequent steps were performed at 4 ° C or on ice. Cells were lysed by sonication in a lysis buffer containing 50 mM HEPES pH 8.5, 200 mM NaCl, 8 mM benzamidine, 7.5% glycerol, 1 mM TCEP, 0.5 mM EDTA, 0.2 mM PMSF that was supplemented with cOmplete EDTA-free protease inhibitor tablets and benzonase. The lysate was cleared by ultracentrifugation at 38,000 x g for one h in a JA-25.50 rotor. A sample of the cleared supernatant was withdrawn and mixed 1:9 (volume/volume) with lysis buffer, mixed with 10 volume equivalents of 2xLDS-PAGE loading buffer (Thermo Fisher Scientific) supplemented with DTT, and boiled for 5 min at 95 ° C for analysis as an input sample by SDS-PAGE. Cleared supernatants were subsequently filtered and incubated whilst rolling with 0.5 mL of a 50% slurry of Strep-Tactin Superflow plus resin (Qiagen) in a wash buffer containing 50 mM HEPES pH 8.0, 200 mM NaCl, 5% glycerol, 0.5 mM EDTA, 1 mM TCEP for 90 min. Resin was collected by centrifugation at 500 x g for 3 min and washed with 10 mL of wash buffer. Resin washes were repeated, as above, a further four times. Resin was collected a final time by centrifugation, as above, and bound complexes were eluted with wash buffer containing 2.5 mM d-desthiobiotin. The resin was collected by centrifugation, as above, and 10 μL of eluate was withdrawn and mixed with an equal volume of 2xLDS-PAGE loading buffer (Thermo Fisher Scientific) that was supplemented with DTT. This sample of eluate was then boiled for 5 min and analysed by SDS-PAGE. Investigation of Dsn1 N-terminal intrinsically disordered domain function in Mis12c Mtw1c auto-inhibition To test the effect of deleting or mutating regions of the Dsn1 N-terminal disordered domain on Mis12c Mtw1c auto-inhibition, 4 μM concentrations of Knl1c:Mis12c Mtw1c complexes containing Knl1 Spc105_HB-RWD and different Dsn1 constructs were mixed with 4 μM concentrations of either CENP-C Mif2_1-63 or CENP-QU in KMN buffer. Knl1c:Mis12c Mtw1c complexes used contained one of the following Dsn1 constructs: K HBRWD M, contained full-length and wildtype Dsn1; K HB-RWD M S240E,S250E , contained Dsn1 S240E,250E ; K HB-RWD M Dsn1Δ226 , contained Dsn1 Δ1-226 ; K HB-RWD M Dsn1Δ257 , contained Dsn1 Δ1-257 ; K HB-RWD M Dsn1Δ226,S240E,S250E, contained Dsn1 Δ1-226,S240E,S250E . Protein mixtures were incubated together on ice for 30 min to attempt to reconstitute a protein complex. 100 μL of individual proteins or protein complexes or reconstitution mixtures were loaded onto a Superose 6 3.2/300 size exclusion chromatography column (Cytiva) equilibrated in (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP) and 100 μL eluate fractions were collected. Aliquots of each indicated eluate fraction was mixed 1:1 (volume/volume) with 2xLDS-PAGE loading buffer supplied by Thermo Fisher Scientific, supplemented with DTT, boiled for 3 min at 95 ° C, and analysed by SDS-PAGE. Yeast strain construction We constructed S. cerevisiae strains encoding the OsTir1 ubiquitin ligase at the URA3 locus and a mAID 3 -FLAG 5 C-terminal tag at either the endogenous NNF1 (encoding Nnf1 ) or MTW1 (encoding Mis12 Mtw1 ) loci. Yeast strains constructed for this study are listed in Table S2. To construct strains encoding Mis12 Mtw1 -mAID 3 -FLAG 5 ( MTW1-mAID 3 -FLAG 5 allele) or Nnf1-mAID 3 -FLAG 5 ( NNF1-mAID 3 FLAG 5 allele), a degron cassette encoding mAID 3 -FLAG 5 and a KanMX gene that provided resistance to G418 were amplified from plasmid pST1933 ( Tanaka et al., 2015 ) through a polymerase chain reaction (PCR) that used Q5 polymerase and oligonucleotide primers containing 48 bp 5’ extensions with homology to the relevant locus in the yeast genome. BY26972 strain yeast cells (Table S2) from an exponentially growing suspension culture were transformed with the cassette according to the previously detailed LiAc protocol ( Gietz and Schiestl, 2007 ). In brief, cells were harvested by centrifugation, washed thrice with sterile Millipore water (MPW), and incubated with 360 μL transformation buffer (100 mM lithium acetate, 33% PEG 3350, 100 μg salmon sperm DNA) containing the degron cassette. Cells were transformed with the cassette through a 45 min heat shock at 42 ° C, and allowed to recover by standing in 1 mL of YEPD medium for 3 h at 22 ° C. Transformants were then selected by plating yeast cells on YEPD agar containing 500μg/mL G418 (Sigma, A1720-5G) and grown at 30 ° C for 3–4 days. Individual colonies were picked and restreaked on fresh YEPD agar containing 500 μg/mL G418 twice, successively. Integration of the cassette was then screened by PCR amplification using Q5 polymerase and two primers directed against the 3’ end of the relevant gene and an internal region of the degron cassette. PCR products obtained by this method were sequenced to validate in-frame integration of the DNA sequences encoding the mAID 3 -FLAG 5 tags at the 3’ end of the relevant gene. Strains expressing Mis12 Mtw1 -mAID 3 -FLAG 5 or Nnf1-mAID 3 -FLAG 5 and an allele to be tested for its ability to rescue cell death were constructed as follows. Yeast strains expressing Mis12 Mtw1 -mAID 3 -FLAG 5 or Nnf1 1 -mAID 3 -FLAG 5 were transformed, as above, with pRS304 (encoding MTW1 alleles) or pRS305 (encoding NNF1 alleles) plasmids that were cleaved with a restriction enzyme in a single site within the LEU2 ( pRS304 plasmids) or TRP1 ( pRS305 plasmids) selection marker. The plasmids contained the following alleles encoding the listed gene products: MTW1 , full-length and wildtype Mis12 Mtw1 ; mtw1 ΔC , Mis12 Mtw1_Δ272-C , NNF1 , full-length and wildtype Nnf1; nnf1 ΔC , Nnf1 Δ180-C . Transformants were selected for by plating yeast cells on leucine dropout ( MTW1 or mtw1 ΔC transformants) or tryptophan dropout ( NNF1 or nnf1 ΔC transformants) agar media containing 500 μg/mL G418. Integration of the cassette at the LEU2 or TRP1 locus was screened using two PCR reactions that amplified across the 5’ and 3’ recombination junctions. The first PCR used a pair of oligonucleotide primers targeted against a region of the genome 5’ of LEU2 or TRP1 and against an internal region of the integrated cassette, and the second PCR used a pair of primers targeted against a region of the genome 3’ of LEU2 or TRP1 and against another internal region of the cassette. Positive transformants were stored in 50% glycerol at-80 °C for later use. All strains grew well on YEPD plates in the absence of IAA, showing that the incorporation of the mAID 3 -FLAG 5 tag at the endogenous MTW1 and NNF1 loci, and rescue alleles at the exogenous loci, did not adversely affect cell growth (Figure S3E). Auxin depletion assays 10 mL suspension cultures of each yeast strain were grown overnight at 30 ° C. The optical density at 600 nm of each suspension culture was measured in triplicate, yeast cells were subsequently diluted to OD 600nm = 0.1, and then serially diluted 1:9 (volume/volume) over 4 further dilutions. 4 μL of each dilution was then applied to agar plates containing either YEPD medium, or YEPD medium and 0.5 mM indole-3-acetic acid (IAA). Cells were grown at 30 ° C for 3 days and imaged. Western blotting To validate expression levels of the gene products of the MTW1 (Mis12 Mtw1 ) and NNF1 (Nnf1) alleles integrated at ectopic sites, 40 mL of each cell culture was grown in YEPD until the culture reached an OD 600nm between 0.7 and 2.0. At this point, 1×10 7 ( MTW1-mAID 3 -FLAG 5 strains) or 1.4×10 7 ( NNF1-mAID 3 -FLAG 5 strains) cells were withdrawn from the culture and processed for western blotting as described below. Depletion of MTW1-mAID 3 -FLAG 5 and NNF1-mAID 3 -FLAG 5 gene products by addition of IAA was monitored and validated by growing 40 mL suspension cultures of each strain in YEPD medium until the culture attained an OD 600nm of between 0.7 and 2.0. This was followed by addition of either IAA to a final concentration of 0.5 mM, or of an equal volume of phosphate buffered saline (PBS) as a negative control. A volume of suspension culture containing 1×10 7 MTW1-mAID 3 -FLAG 5 strains) or 1.4×10 7 ( NNF1-mAID 3 -FLAG 5 ) cells was then withdrawn from each culture at the indicated time points and processed for western blotting as described below. All subsequent steps were performed at 22 ° C, unless otherwise indicated. Yeast cells were collected by centrifugation at 3,000 x g for 3 min, resuspended in 1 mL of sterile Millipore water (MPW), and pelleted by centrifugation at 12,000 x g for 1 min. Whole cell extracts were then prepared by incubation of the cell pellets in 50 μL of 0.2 M NaOH for 5 min, centrifugation for 1 min at 12,000 x g , a wash of the cell pellet with 1 mL of MPW, and a final collection of the pellet by centrifugation as above. Pellets were then boiled for 3 min at 95 ° C in 50 μL of 1xLDS-PAGE loading buffer supplemented with DTT. The pellet was collected by centrifugation, and 10 μL of each supernatant was applied to an SDS-PAGE gel to separate proteins through application of a constant 210 V for 45 min. Proteins were transferred from the SDS-PAGE gel onto a TransBlot Turbo minigel membrane using a Trans-blot Turbo transfer system. Membranes were blocked with 3% milk suspension ( NNF1 gene product blots) or 5% milk suspension ( MTW1 gene product blots) in PBS-T (Phosphate buffered saline containing 0.2% Tween-20) for 30 min at 22 ° C. Membranes were then probed with the primary antibody detailed below overnight at 4 ° C in PBS-T to detect the protein of interest. To detect MTW1-mAID 3 -FLAG 5 and NNF1-mAID 3 -FLAG 5 gene products, a 1:1000 (volume/volume) dilution of anti-FLAG antibody (Cell Signalling, D6W5B) was used. To detect NNF1-V5 3 and nnf1 1′C -V5 3 or MTW1-HA 3 and mtw1 1′C expressed from the ectopically integrated loci, 1:3333 dilutions of anti-V5 (Proteintech, 14440-1-AP) or anti-HA (Abcam, ab1424) antibodies, respectively, were used. To detect alpha tubulin as a loading control, anti-α-tubulin (Bio-Rad, MCA78G) antibody was used. Membranes were washed four times with PBS-T before incubation with a 1:3333 (volume/volume) dilution of HRP-conjugated secondary antibody in PBS-T by rolling at 21 ° C for 1.5–2.5 h. Membranes were washed a further four times with PBS-T before visualisation of the secondary antibody using chemiluminescence using the ECL Prime Western blotting detection kit (Amersham) and imaging using a ChemiDoc imaging system (Bio-Rad). Multiple sequence alignment Multiple sequence alignments were performed with Clustal Omega using default settings and sequences full-length, wildtype protein sequences deposited in Uniprot unless otherwise indicated ( Sievers et al., 2011 ). Sequence alignments were visualised using the Jalview workbench using a conservation score of 10 ( Waterhouse et al., 2009 ). Crosslinking mass-spectrometry Protein cross linking reactions on the S. cerevisiae K HB-RWD M complex (100 μL at 0.5 mg/mL) were carried out with sulfo-SDA (2 mM concentration). Sulfo-SDA and the protein complex were mixed and incubated on ice for 5 min before being cross linked for 10 sec with 365 nm UV radiation from a home build UV LED setup. Crosslinking reactions were quenched with the addition of ammonium bicarbonate to a final concentration of 50 mM. The quenched solution was reduced with 5 mM DTT and alkylated with 20 mM iodoacetamide. The SP3 protocol, as described ( Batth et al., 2019 ; Hughes et al., 2019 ), was used to clean-up and buffer exchange the reduced and alkylated protein. In brief, for the SP3 protocol; proteins are washed with ethanol using magnetic beads for protein capture and binding. The proteins were resuspended in 100 mM NH 4 HCO 3 and were digested with trypsin (Promega, UK) at an enzyme-to-substrate ratio of 1:20, and protease max 0.1% (Promega, UK). Digestion was carried out overnight at 37 ° C. Clean-up of peptide digests was carried out with HyperSep SpinTip P-20 (Thermo Fisher Scientific) C18 columns, using 60% Acetonitrile as the elution solvent. Peptides were then evaporated to dryness via Speed Vac Plus (Savant, USA). Dried peptides were resuspended in 30% acetonitrile and were fractionated via SEC using a Superdex 30 Increase 3.2/300 column (Cytiva) at a flow rate of 20 μL/min using 30% (v/v) ACN 0.1 % (v/v) TFA as a mobile phase. Fractions are taken every 5 min, and the 2 nd to 7 th fractions containing cross linked peptides are collected. Dried peptides were suspended in 3% (v/v) acetonitrile and 0.1 % (v/v) formic acid and analysed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC to deliver a flow of 300 nL/min. Peptides were trapped on a C18 Acclaim PepMap100 5 μm, 0.3 μm x 5 mm cartridge (Thermo Fisher Scientific) before separation on Aurora Ultimate C18, 1.7 μm, 75 μm x 25 cm (Ionopticks, Melbourne). Peptides were eluted on optimised gradients of 90 min and interfaced via an EasySpray ionisation source to a tribrid quadrupole Orbitrap mass spectrometer (Orbitrap Eclipse, Thermo Fisher Scientific) equipped with FAIMS. MS data were acquired in data dependent mode with a Top-25 method, high resolution scans full mass scans were carried out (R = 120,000, m/z 400 – 1550) followed by higher energy collision dissociation (HCD) with stepped collision energy range 21, 30, 34 % normalised collision energy. The tandem mass spectra were recorded (R=60,000, isolation window m/z 1, dynamic exclusion 50 s). Mass spectrometry measurements were cycled for 3 s durations between FAIMS CV-45, and-60 V. Crosslinking mass-spectrometry data analysis Xcalibur raw files were converted to MGF files using ProteoWizard and cross links were analysed by XiSearch ( Chambers et al., 2012 ; Mendes et al., 2019 ). Search conditions used 3 maximum missed cleavages with a minimum peptide length of 5. Variable modifications used were carbmidomethylation of cysteine (57.02146 Da) and methionine oxidation (15.99491 Da). False discovery rate was set to 5%. Crosslinking mass-spectrometry data was visualised and analysed using XiVIEW ( Combe et al., 2024 ). Computational methods The UCSF Chimera-X package was used to visualise cryo-EM density maps, AF2 structure predictions, previously deposited structures retrieved from the protein data bank, and crosslinking mass-spectrometry data superimposed onto the structural models ( Goddard et al., 2018 ; Pettersen et al., 2021 ). Data Deposition Protein coordinates and cryo-EM maps were deposited at RCSB and EMDB, respectively (Supplementary Table S1). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE ( Perez-Riverol et al., 2025 ) partner repository with the dataset identifier PXD072662. Funding UKRI/Medical Research Council MC_UP_1201/6 (D.B.) Cancer Research UK C576/A25675 (D.B.) Cancer Research UK C576/A14109 (D.B.): CR-UK PhD studentship (N.N.T). Supplementary Video Captions Video S1. Overview of the S. cerevisiae KMN junction complex. Video shows the molecular details of the S. cerevisiae KMN junction complex as shown in Figures 1 - 3 , detailing the overall architecture of the complex ( Figure 1 ); details of the Mis12c Mtw1c interface with Knl1c Spc105c ( Figure 2 ); and details of the Mis12c Mtw1c interface with Ndc80c ( Figure 3 ). Video S2. Predicted conformational change of Dsn1 AI and head 2 accompanying Sc Mis12c Mtw1c activation by Dsn1 AI phosphorylation. Video shows proposed conformational change of Dsn1 AI and head 2 caused by Aurora B Ipl1 -phosphorylation of Ser240 and Ser250 ( Figure 7 ). Phospho-Ser240 would cause electrostatic repulsion with the negatively-charged surface of Mis12c Mtw1c head 1 as well as steric clashes with Nsl1 of head 2. Displacement of Dsn1 AI and head 2 releases the CENP-C Mif2 -binding site on Mis12c Mtw1c mainly mediated through head 1. The Mis12c Mtw1c -CENP-C Mif2 complex is based on an AF2 model. Download figure Open in new tab Figure S1: Preparation of S. cerevisiae KMN complex sample for Cryo-EM and Validation of in vivo protein depletion and in vivo rescue allele expression A) Subcomplex and subunit domain organisation of the S. cerevisiae KMN complex proteins. Coiled-coil (CC), calponin homology (CH), helical bundle (HB), N-terminal intrinsically disordered region (N-IDR) and RING-WD40-DEAD box helicase (RWD) domains are highlighted. B) Coomassie brilliant blue stained SDS-PAGE gels of the purified KMN subcomplexes. C-E) Coomassie brilliant blue stained SDS-PAGE gels of isolated C) N and D) K HB-RWD M subcomplexes, or E) reconstituted K HB-RWD MN complexes after separation using 10-30% glycerol gradients. F) Coomassie brilliant blue stained SDS-PAGE gels of the reconstituted K HB-RWD MN complex crosslinked with a 0.0–0.2% glutaraldehyde gradient over the course of a 10–30% glycerol gradient using the GraFix methodology ( Stark, 2010 ). Crosslinking was repeated twice with identical results. Fractions corresponding to the glycerol gradient volume 2.00 – 2.75 mL were pooled for cryo-EM sample preparation. Fractions corresponding to the glycerol gradient volume 2.00 – 2.75 mL were pooled for cryo-EM sample preparation. G) Immunoblots of whole cell extracts (WCEs) of the indicated yeast strains treated for the indicated duration with PBS or 0.5 mM Indole-3-acetic acid (IAA). Membranes were blotted with anti-FLAG primary antibody to detect NNF1-mAID 3 -FLAG 5 gene products (top panel) or anti-α-tubulin primary antibody as a loading control (bottom panel). H) Immunoblots of WCEs of the indicated yeast strains treated for the indicated duration with PBS or 0.5 mM Indole-3-acetic acid (IAA). Membranes were blotted with anti-FLAG primary antibody to detect MTW1-mAID 3 -FLAG 5 gene products (top panel) or anti-α-tubulin primary antibody as a loading control (bottom panel). I) Immunoblots of WCEs of the indicated yeast strains. Membranes were blotted with anti-V5 primary antibodies to detect the gene products expressed from the NNF1 or nnf1 ΔC variant alleles (top panel), or with anti-α-tubulin primary antibody as a loading control (bottom panel). J) Immunoblots of WCEs of the indicated yeast strains. Membranes were blotted with anti-HA primary antibodies to detect the gene products expressed from the indicated MTW1 or mtw1 ΔC variant alleles (top panel), or with anti-α-tubulin primary antibody as a loading control (bottom panel). Download figure Open in new tab Figure S2: Cryo-EM data processing to reconstruct the KMN junction complex A) Representative motion corrected cryo-EM movie obtained after data acquisition. A total of 26,390 cryo-EM movies were obtained after data acquisition. B) Representative 2D class averages generated using CryoSparc during the 2D classification step following noise decoy classification. C) Angular distribution of views plot of the particles that contributed to the ‘apex body 1’ and ‘base body 1’ reconstructions. D) Cryo-EM data processing scheme used to reconstruct higher resolution cryo-EM density maps for the KMN junction complex, as described in the Materials and methods section. E-F) Cryo-EM density maps coloured according to local resolution of the E) ‘apex body 1’ and F) ‘base body 1’ multibody derived reconstructions that were used to build molecular models (top). Fourier shell correlation (FSC) plot of the E) ‘apex body 1’ and F) ‘base body 1’ multibody derived reconstructions that were used to build the model of the KMN junction complex (bottom). Download figure Open in new tab Figure S3: Biochemical analysis of Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix function A) SEC elution chromatograms of Mis12c Mtw1c containing either full-length and wild-type proteins, or with full-length and wild-type proteins apart from deletion of Mis12 Mtw1 residues 272-289 (Mis12 Mis12_Δ272-C : Mis12c Mtw1c_Mis12ΔC ), deletion of Nnf1 residues 180-201 (Nnf1 Δ180-C : Mis12c Mtw1c_ Nnf1 ΔC ), or deletion of both Mis12 Mtw1 residues 272-289 and Nnf1 residues 180-201 (Mis12c Mtw1c_Mis12ΔC-Nnf1ΔC ). B) Coomassie brilliant blue stained SDS-PAGE gels of all of the experiments presented in panel A . C) SEC elution chromatograms of the Mtw1c:Ndc80c interaction reconstitutions. Attempts at reconstituting Mis12c Mtw1c , Mis12c Mtw1c_Mis12ΔC , Mis12c Mtw1c_Nnf1ΔC , and Mis12c Mtw1c_Mis12ΔC- Nnf1 ΔC with full-length, unmodified Ndc80c are presented alongside the elution profile of Ndc80c alone. D) Coomassie brilliant blue stained SDS-PAGE gels of the experiments in panel C . E) Analysis of mtw1 ΔC and nnf1 ΔC mutants in yeast strain backgrounds with endogenous MTW1 and NNF1 gene products translationally fused to mAID 3 -FLAG 5 tag. Cell growth was investigated by plating 1:9 serial dilutions onto either YEPD plates or YEPD plates supplemented with 0.5 mM Indole-3-acetic acid (IAA). - = no rescue allele was expressed. mAID = monomeric auxin inducible degron. Download figure Open in new tab Figure S4: Cryo-EM data processing to reconstruct Mis12c Mtw1c head 2 domain A) Cryo-EM data processing scheme used to reconstruct cryo-EM density maps containing continuous density for the Mis12c Mtw1c head 2 domain, as described in the Materials and methods section. B) Fourier shell correlation (FSC) plot for ‘base body 2’. C) Cryo-EM density map for ‘base body 2’ that was used to model the Mis12c Mtw1c head 2 domain coloured according to local resolution. D) Angular distribution of views plot for the particles that contributed to the reconstruction of ‘base body 2’. Download figure Open in new tab Figure S5: Modelling of contacts between Mis12c Mtw1c head 1, head 2 and Dsn1 N-IDR domains A) Cryo-EM density map with improved occupancy for the Mis12c Mtw1c head 2 domain (Figure S4) interpreted using the model of Mis12c Mtw1c stalk and head 1 domains built in this study and the AF2 model of S. cerevisiae head 2 presented in panels C and D . (6A) and (6D) highlight regions of the map that are shown in close up in Figures 6A and 6D , respectively. B) Experimental model of S. cerevisiae Mis12c Mtw1c head 1 and head 2 domains interacting at the base of the S. cerevisiae Mis12c Mtw1c stalk domain. C) AF2 structure prediction of the S. cerevisiae Mis12c Mtw1c head 2 domain from the Mis12c Mtw1c :Spc24:Spc25 prediction coloured by chain. Mis12c Mtw1c head 1 and stalk domains, and Spc24:Spc25 are not shown. D) AF2 model presented in panel C coloured by residue pLDDT score. The predicted alignment error (PAE) score plot is shown on the right of the panel. E) AF2 structure prediction of S. cerevisiae Mis12c Mtw1c ( Sc Mis12c Mtw1c ) coloured by chain docked into the cryo-EM map. The prediction of the head 2 position and orientation does not fit the cryo-EM density. F) AF2 model presented in panel E coloured by residue pLDDT score. The predicted alignment error (PAE) score plot is shown on the right of the panel. G) AF2 structure prediction of the K. lactis Mis12c Mtw1c ( Kl Mis12c Mtw1c ) coloured by chain. The prediction was performed using all full-length and wild-type protein sequences apart from K. lactis Dsn1 ( Kl Dsn1), for which residues 1-149 were excluded to obtain predictions for the interaction between Kl Dsn1 201-230 and Kl Mis12c Mtw1c head 1 domain. H) AF2 model presented in panel G coloured by residue pLDDT score. The predicted alignment error (PAE) score plot is shown on the right of the panel. View this table: View inline View popup Download powerpoint Table S1: Cryo-EM data collection, refinement and validation statistics View this table: View inline View popup Download powerpoint Table S2: Genotypes of S. cerevisiae strains used within this study. Acknowledgments. We are grateful to the LMB EM Facility for help with the EM data collection; J. Grimmett, T. Darling and I. Clayson for scientific computing; J. Shi and K. Turton for help with insect cell expression; and to T. Dendooven, D. Kimanius and S. Yatskevich for discussions. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising. 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