In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane

In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane | 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 In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane Atsuki Nakano , Takahiro Masuya , Shinsuke Akisada , Moe Ishikawa-Fukuda , Kaoru Mitsuoka , Hideto Miyoshi , View ORCID Profile Masatoshi Murai , Ken Yokoyama doi: https://doi.org/10.1101/2025.09.19.677273 Atsuki Nakano 1 Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama , Kita-ku, Kyoto 603-8555, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takahiro Masuya 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Sakyo-ku, Kyoto 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shinsuke Akisada 1 Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama , Kita-ku, Kyoto 603-8555, Japan 3 Research and Development , Sumitomo Pharma. Co., Ltd., Osaka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Moe Ishikawa-Fukuda 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Sakyo-ku, Kyoto 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kaoru Mitsuoka 3 Research and Development , Sumitomo Pharma. Co., Ltd., Osaka, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hideto Miyoshi 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Sakyo-ku, Kyoto 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Masatoshi Murai 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Sakyo-ku, Kyoto 606-8502, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Masatoshi Murai For correspondence: yokoken{at}cc.kyoto-su.ac.jp murai.masatoshi.5s{at}kyoto-u.ac.jp Ken Yokoyama 1 Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo-Motoyama , Kita-ku, Kyoto 603-8555, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yokoken{at}cc.kyoto-su.ac.jp murai.masatoshi.5s{at}kyoto-u.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract To understand how membrane protein complexes function within biological membranes, it is essential to determine their structure in their natural membrane environment. Here, we employed cryoEM structure analysis to elucidate the structures of ATP synthase F o F 1 and respiratory Supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. On SMPs, the majority of F o F 1 was identified as dimers bound by the regulatory factor dimeric IF 1 . In addition, a tetrameric structure formed by association of F o F 1 IF 1 dimers and with a linear arrangement of the F 1 head were also identified. These structures induced a steep membrane curvature, indicating the presence of a structure on SMPs similar to that found on the tips of mitochondrial cristae. High-resolution structures of the respiratory complexes were also determined, and sub-class structures of both CI and CIII 2 were resolved. Most SCs were of the CI 1 CIII 2 CIV 3 structure, although the presence of the CI 2 CIII 2 CIV 6 mega complex was also identified. Our study enabled rapid in situ structural determination of SCs and F o F 1 ATP synthase from small amount of membrane fractions, paving the way for elucidation of the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture. Introduction Membrane proteins play essential roles in cellular functions, including signal transduction, energy conversion, and molecular transport across membranes 1 , 2 . Structural analysis of membrane proteins typically involves detergent-based extraction from biological membranes, followed by multiple purification processes. These structures of membrane protein are then determined using X-ray crystallography or cryo-electron microscopy (cryo-EM). However, during solubilization and purification, loosely bound subunits may dissociate, and essential lipids might be replaced by detergents. Additionally, higher-order super molecular structures formed by the association of multiple membrane protein complexes are likely to be lost during purification processes. Consequently, the structure of solubilized membrane proteins is likely to differ from in situ structure of membrane proteins in their native biological context. Mitochondria, intracellular energy factories, are complex organelles composed of an outer membrane and an inner membrane. The inner membrane hosts the respiratory complexes and ATP synthase, which sustain life by synthesizing ATP through the multistep process of oxidative phosphorylation 3 . The mammalian mitochondrial respiratory system comprises four enzyme complexes, designated complexes I, II, III, and IV. Complex I (NADH-ubiquinone oxidoreductase, CI) functions as a primary entry point for electrons from matrix-derived NADH, catalyzing the reduction of ubiquinone (UQ) to ubiquinol (UQH 2 ) 4 - 7 . Complex II (succinate-ubiquinone oxidoreductase, CII) provides an additional entry point for electrons through the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle, using UQ as the electron acceptor 8 , 9 . UQH 2 then donates electrons to complex III (ubiquinol-cytochrome c oxidoreductase, CIII), which reduces cytochrome c (cyt. c ) on the intermembrane space side of the inner membrane 8 , 9 . Electrons are subsequently passed to complex IV (cytochrome c oxidase, CIV), where molecular oxygen—the terminal electron acceptor—is reduced to water 10 , 11 . Electron transfer through complexes I, III, and IV is coupled to proton translocation from the matrix to the intermembrane space, generating an electrochemical proton gradient—known as the proton motive force ( pmf )—across the inner membrane, which drives ATP synthesis via the F o F 1 -ATP synthase. These respiratory chain complexes (I-IV) were first solubilized and purified separately from the mitochondrial inner membrane, and their crystal or cryoEM structures have been determined 12 - 15 . Recently, gentle solubilization of the inner mitochondrial membrane revealed that complexes I, III, and IV associate to form larger Supercomplexes (SCs), whose cryoEM structure has also been determined 16 . ATP synthase F o F 1 utilizes the pmf generated by the respiratory chain complexes to rotate the hydrophobic rotor c 8 -ring of the F o domain relative to the stator complex 3 , 17 , 18 . This rotation drives the phosphorylation of ADP to synthesize ATP in the F 1 domain. The bovine mitochondria F o F 1 , initially purified as a monomer 19 , has been purified as a dimer through careful solubilization and purification, and its cryo-EM structure also determined 20 . Single-particle analysis of porcine mitochondrial inner membrane fractions solubilized with detergents has reported a tetrameric structure of F o F 1 , suggesting that multiple F o F 1 molecules associate and function as a super molecular complex in the mitochondrial inner membrane 21 . Mutations within genes encoding the respiratory complex and F o F 1 ATP synthase subunits are frequently associated with mitochondrial disorders and metabolic diseases such as diabetes, highlighting their significance as potential therapeutic targets 22 , 23 . Yet, the link between disease pathology and the higher-order architecture of these complexes remains poorly understood. Cryo-electron tomography (cryo-ET) offers a powerful approach for visualizing macromolecular structures within the context of organelle and cellular membranes. Nevertheless, the time-intensive acquisition of tilt series and the complexity of three-dimensional reconstruction have thus far limited its ability to resolve fine substructures of membrane-embedded proteins at high resolution. In this study, we determined structures on the inner mitochondria membranes through direct observation of sub-mitochondrial particles (SMPs) using cryo-EM single particle analysis. For the respiratory SCs we obtained structures with resolution comparable to that of the individual solubilized and purified complexes. We also elucidated the structure of the dimeric and tetrameric F o F 1 and obtained a high-resolution structure of the monomeric form, which can be used to construct an atomic model. Results Preparation of bovine heart SMPs and data acquisitions respiratory SCs and F o F 1 oligomer Submitochondrial particles (SMPs) were prepared from bovine heart mitochondria 24 ( Figure 1A ). The SMPs, suspended at a concentration of 7 mg protein/ml in a buffer containing 50 mM sucrose and 10 mM Tris/HCl (pH 7.4), were blotted onto a quantifoil UltraAufoil grid and flash-frozen in liquid ethane. Cryo-EM micrographs were acquired with a Titan Krios equipped with a K3 detector. A typical cryo-EM image is shown in Figure 1B . The F 1 domain of F o F 1 can be clearly observed in the micrograph where the ice thickness is relatively thin ( Figure 1B , lower panel). First, we reconstructed the 3D structures of both the F o F 1 dimer and SCs from 5k micrographs, which serves as machine learning training data for subsequent particle picking. From approximately 30,680 movies, ∼4 x 10 6 single-particle images of F o F 1 dimer and SCs were extracted and subjected to further single-particle analysis (Figure S1A and B). Download figure Open in new tab Figure 1. Cryo-EM imaging of SMPs and representative 2D class averages of F o F 1 oligomers and Supercomplexes (SCs). (A) Schematic illustration of SMP preparation from disrupted bovine heart mitochondria. Details of SMP preparation are described in the Materials and Methods section. (B) Cryo-EM micrograph of isolated SMPs (bottom, magnified view). (C) Representative 2D class averages of F o F 1 oligomers (top) and SCs on SMPs viewed from the side (upper) and top (lower). 3D reconstruction of the F o F 1 oligomer in the SMPs Using two adjacent F o F 1 dimers as templates, Topaz, a machine learning-based tool, selected 1,306 k particles from ∼4 x 10 7 particles including both F o F 1 dimer and SCs (Figure S1A). After homogeneous refinement, a structure of F o F 1 dimers connected laterally by a rod-shaped molecule, likely IF 1 , was obtained from 382 k particles (Figure S1B and 2A). Hereafter, the F o F 1 dimer connected by IF 1 is referred to as the F o F 1 –IF 1 dimer, with its left and right protomers designated F o F 1 –1 and F o F 1 –2, respectively. The resolution of the F o F 1 -IF 1 dimer structure is limited by the fluctuations of the two monomers relative to one another, with an FSC resolution of approximately 7Å. The F o domain of the F o F 1 -IF 1 dimer is embedded within the lipid bilayer ( Figure 2A ). On the matrix side, density corresponding to a domain of the c 8 -ring and the e -subunit (indicated by red and blue arrows, respectively), which is connected to the c 8 -ring, was clearly observed. Download figure Open in new tab Figure 2. Cryo-EM structures of the F o F 1 -IF1 dimer in the SMP membrane. ( A ) Cryo-EM map of the F o F 1 –IF 1 dimer, shown in side view (left panel) and from the matrix side of the membrane (right panel). In the matrix side view, the c 8 -ring and the e subunit are clearly visible. ( B ) Focused refinement maps of the two F o F 1 monomers that constitute the F o F 1 –IF 1 dimer: F o F 1 –1 and F o F 1 –2 correspond to rotational state 1 and rotational state 3, respectively. ( C ) Atomic models of the F 1 domain from F o F 1 –1 and F o F 1 –2. Side views (top panel) and top views (lower panel) are shown. In F o F 1 –1, nucleotides were modeled: no density was observed at the α E β E catalytic site, whereas ATP–Mg 2+ and ADP were bound at α T β T and α D β D , respectively. In F o F 1 –2, nucleotide modeling was not performed due to resolution limitations. ( D ) The focused refinement map of the F o domain from F o F 1 –1 (left panel), and the corresponding atomic model (right panel), with each subunit shown in a distinct color. To obtain a high resolution structure the monomeric F o F 1 , masked structural refinements were carried out on each monomer, (Figure S1B). The F o F 1 -1 and F o F 1 -2 structures were determined at resolutions of 3.3 Å and 3.7 Å, respectively ( Figure 2B , S1C), allowing the construction of atomic models ( Figure 2C ). The structure of the F o F 1 -IF 1 dimer on the SMPs is very similar to the previously published structure of the F o F 1 -IF 1 dimer unit which forms part of the solubilized tetrameric F o F 1 from porcine heart mitochondria 21 . In each monomer, the F 1 domain exhibited a distinct rotational state relative to the central axis. F o F 1 -1 was in rotational state 1, while F o F 1 -2 assumed rotational state 3 ( Figure 2C ). Using focused refinement on the membrane domain of F o F 1 , we obtained a map of the F o domain of F o F 1 -1 at a resolution of 4.1 Å. In this structure, we identified not only the transmembrane helices of both the c 8 -ring and stator a -subunit but also the long helix of the e -subunit ( Figure 2D , indicated by blue arrow), which connects the stator domain of F o with the lipid in the central pore of the c 8 -ring ( Figure 2D , indicted by red arrow). Single-particle cryo-EM analysis of the solubilized fraction of porcine heart mitochondrial SMP demonstrated the presence of a tetrameric F o F 1 structure 21 . To investigate whether a similar tetrameric structure exists in bovine heart mitochondrial SMPs, we extracted F o F 1 -IF 1 dimer images with extended pixel size from micrographs (981 x 981 Å 2 ) and reconstructed 3D images with an extended box size (Figure S1B). The resulting structures contained two sets of F o F 1 -IF 1 dimers ( Figure 3A ). When viewed laterally, the oligomeric F o F 1 exhibited a V-shaped membrane curvature, with the F o F 1 -IF 1 dimers positioned at both sides of the apex of this roughly triangular curvature ( Figure 3A , lower ). This arrangement of the F o F 1 -IF 1 dimer closely resembles the tetrameric F o F 1 structure derived from porcine heart mitochondria. In the present analysis, densities corresponding to putative F 1 structures were observed adjacent to the tetramer unit ( Figure 3A , right ). This finding suggests that, within SMPs, F o F 1 -IF 1 dimers may assemble into a linear arrangement, potentially contributing to a crista-like structure. Download figure Open in new tab Figure 3. Cryo-EM structures of F o F 1 tetramers on the SMP membrane. (A) Cryo-EM density map of the F o F 1 tetramer. The upper panel shows the overall structure, while the lower panel presents a cross-sectional side view, and the right panel displays an intact side view. Putative F 1 -head domains are indicated by blue arrows. (B) Cryo-EM map of a refined subclass of the F o F 1 tetramer obtained through 3D classification. Densities attributed to IF1 are highlighted with red ellipses. Additionally, we identified another oligomeric F o F 1 structure distinct from the tetrameric F o F 1 . In this oligomer, the arrangement of the F o F 1 -IF 1 dimer across the apex of the curved membrane was shifted by one F 1 unit ( Figure 3B ). Consequently, the state1 F 1 domains of the IF1 dimers faced each other across the apex of the membrane. This result suggests that the binding between F o F 1 -IF 1 dimers across the membrane apex is weak. 3D reconstruction of the Respiratory Supercomplexes in the SMPs As described above, we performed four rounds of heterogeneous refinement on approximately 3 million particles containing a mixture of F o F 1 oligomers and SCs, which resulted in the isolation of a subclass containing predominantly SCs, comprising 764,000 particles (Figure S1A). Homogeneous refinement of this subclass yielded a 3.5 Å-resolution structure of the SCs. Subsequent classification allowed us to distinguish two distinct populations: one consisting of SCs composed of CI and the CIII dimer (CIII 2 ), and another exhibiting an additional density adjacent to the distal end of CI (Figure S1A). As described later, this contact site corresponds to the ND5 subunit. This location of extra density coincides with that of CIV observed in previously reported SC structures from solubilized mitochondria 25 , suggesting that it represents CIV unit. We then selected the class showing clear CIV density through heterogeneous refinement, followed by focused refinement on the CIV region using the resulting 227,000 particles. This processing yielded a 2.61 Å-resolution structure of CIV within the SC. In the SC density map, two additional CIV-like densities were observed adjacent to the CIII 2 , although they were less well-defined compared to the canonical CIV density ( Figure 4A , inset). One was located in the interspace between CI and CIII 2 (location B in the inset of Figure 4A ), and the other was associated with solely CIII 2 (location C in the inset of Figure 4A ). Download figure Open in new tab Figure 4. Structure and organization of the respiratory supercomplex (SC). (A) Overall architecture of the mitochondrial respiratory SC. Cryo-EM densities of five respiratory complexes—CI-1 (blue), CIII 2 -1 (yellow), CIV A (deep pink), CIV B (pink), and CIV C (light pink)—are shown. The final composite map was generated by merging the individually refined densities, as outlined in Figure S1B. Insets display the EM density maps of SC after homogeneous refinement of 764,763 SC particles (3.5 Å, Figure S1B). (B)Comparison of EM maps corresponding to complexes CIV A , CIV B , and CIV C . (C) Superposition of the atomic model of complex IV, built from the CIV A density, onto the CIV C density map. This fitting confirms that the CIV C density corresponds to complex IV. The model is shown in a tube helix representation to highlight its correspondence with the CIV C map. Local refinement of each CIV unit, combined with subtraction of surrounding densities and particle selection via 3D classification, resulted in improved maps featuring clearly resolved transmembrane helices ( Figure 4B ). The CIV associated with distal end of CI was designated as CIV A , the one located between CI and CIII 2 as CIV B , and the one associated only with CIII 2 as CIV C . A composite map of SC and the locally refined maps of three CIV unis are shown in the Figures 4A and 4B , respectively. Membrane Curvature Induced by SCs Structural comparison of revealed a modest curvature of the membrane for the CI 1 CIII 2 in the presence of CIV A obtained from the previous porcine mitochondria study compared to the CI 1 CIII 2 SC (Figure S2B). In contrast, comparison of our CI 1 CIII 2 CIV 3 SCs map derived from SMPs with the previously reported map of the CI 1 CIII 2 CIV 1 SCs from intact porcine mitochondria showed nearly identical membrane curvature across all viewing angles (Figure S2C). These observations suggest that the membrane curvature observed in CI 1 CIII 2 CIV SCs is primarily attributable to the incorporation of CIV A . Megacomplexes in SMPs membranes Previous studies have identified a larger SCs unit, termed the respiratory megacomplex, where CI with CIV associates with CI 1 CIII 2 CIV 1 26 to form a circular complex, CI 2 CIII 2 CIV 2 27 (Figure S3D). Similar respiratory megacomplexes have also been reported on the mitochondrial inner membrane 21 , 28 . To investigate whether such mega-complexes are present in SMP membranes, we re-extracted 764,000 particles—predominantly containing SCs—using an expanded box size of 981 Å. Subsequent 2D classification yielded class averages revealed chromosome-shaped SCs (Figure S3A). This 2D projection closely resembled the previously reported CI 2 CIII 2 CIV 2 SCs identified in porcine heart mitochondria; however, four additional densities—putatively corresponding to CIV—were clearly discernible in our data, suggesting that the observed structure is more likely to represent CI 2 CIII 2 CIV 6 (Figure S3A). In this study, the predominant SCs identified on the SMPs were of the CI 1 CIII 2 CIV 3 type. In contrast, CI 2 CIII 2 CIV 2 type SCs, in which two CI units associate with a single CIII 2 (Figure S3D), were not observed on the SMPs. This absence may be due to the presence of CIV B and CIV C in the CI 1 CIII 2 CIV 3 SCs, which likely sterically hinder the association of an additional CI with CIII 2 . These findings suggest that SCs with diverse subunit compositions, including higher-order assemblies, are embedded within the SMP membranes. Additionally, 2D class averages revealed a mushroom-shaped density adjacent to the SC (Figure S3B, indicated by red arrow). A 3D reconstruction of this particle class unambiguously resolved a structure resembling the monomeric F 1 head (Figure S3B, right), implying that F o F 1 ATP synthase is distributed not only in regions of membrane curvature but also in more planar regions of the membrane. Structures of complex I in SCs Complex I (CI) is the largest protein complex in the mitochondrial respiratory system, comprising 45 distinct subunits with a total molecular mass of approximately 1 MDa 5 . It adopts a characteristic L-shaped architecture, consisting of a hydrophilic domain responsible for electron transfer and a membrane-embedded domain responsible for proton translocation ( Figure 5A and 5B ). The ubiquinone (UQ) substrate binds within a tunnel-like structure located at the interface between these two domains, known as the “UQ-accessing tunnel”, which is formed by the 49-kDa, PSST, ND1, ND6, and ND3 subunits. Download figure Open in new tab Figure 5. Structure of complex I (CI-1) in the supercomplex (SC) and conformational differences between CI-1 and CI-2. (A) Cryo-EM density map of CI-1. The density corresponding to the 14 core subunits is color-coded. (B) Atomic model of CI-1 built from the corresponding density map. The 14 core subunits are shown in color, while the 30 supernumerary (accessory) subunits are rendered in gray. (C) Comparison of subunits exhibiting density differences between CI-1 and CI-2. Overlaid densities and atomic models for each class are presented. In CI-2, certain regions of the 49-kDa, ND1, ND6, and ND3 subunits show poorly resolved densities, precluding accurate model building. Adjacent amino acid residues are annotated near regions where density is absent in CI-2. Classification focused on complex I (CI) within the SC (CI 1 CIII 2 CIV 3 ) revealed two distinct subclasses, termed CI-1 and CI-2 at a resolution of 2.55 and 2.57-Å, respectively. CI-1 displayed a well-ordered architecture at the interface between the hydrophilic and membrane-embedded domains, whereas CI-2 adopted a more disordered conformation. CI-2 showed disorder in four loop regions: the loop connecting the β1–β2 strands of the 49-kDa subunit, that between transmembrane helices (TMHs) 5–6 of ND1, that between TMHs 4–5 of ND6, and that between TMHs 1–2 of ND3 ( Figure 5C ). These structural hallmarks align with the closed and open conformations of CI, which have been extensively described and characterized by multiple research groups, where the putative UQ-accessing tunnel adopts either a relaxed or constrained state, respectively. In the CI-1 (closed) conformation, a density was detected within the UQ-accessing tunnel (Figure S4A). Although a UQ 10 molecule could be fitted into this density without significant steric clashes (Figure S4B), the local resolution is insufficient to confidently model UQ 10 . Therefore, we do not assign this density to UQ 10 , and its molecular identity remains uncertain. Overall, the CI-1 and CI-2 structures closely resemble the active- and deactive-conformations of CI observed in situ in porcine heart mitochondria (PDB IDs: 8UEO and 8UES), where the UQ-access cavities adopt closed- and open-conformations, respectively 5 . The RMSDs relative to these structures are approximately 1.11 Å for CI-1 and 1.13 Å for CI-2. Furthermore, CI-1 is nearly identical to the closed-state structure of isolated bovine heart CI (PDB ID: 7QSK) 29 , with an RMSD of ∼0.89 Å. The close structural similarity between our cryo-EM structures and those previously reported by other groups—derived from both in situ and isolated preparations—suggests that CI retains a high degree of structural integrity even during biochemical procedures, including membrane solubilization and sonication, highlighting its intrinsic robustness. Structures of complex III dimer in SCs Within the SCs (CI 1 CIII 2 CIV 3 ) of SMPs, complex III (CIII) exists as a two-fold symmetric dimer (CIII 2 ), with each monomer comprising 11 distinct subunits ( Figures 6A and 6B ). CIII 2 associates with CI through multiple contacts: CI-B15 (NDUFB4) and CI-B22 (NDUFB9) with CIII–core protein 1 on the matrix side, and CI-B14.7 (NDUFA11) with CIII-subunit 8 within the membrane bilayer (Figure S5A). Download figure Open in new tab Figure 6. Structure of CIII 2 in the supercomplex (SC) and conformational differences in the ISP domain between CIII 2 -1 and CIII 2 -2. (A) Cryo-EM density map of CIII 2 -1. The density corresponding to each subunit is shown in a different color. (B) Atomic model of CIII 2 -1, with subunits color-coded as in panel (A). (C) Structural comparison of CIII 2 -1 and CIII 2 -2. The overall views show the relative positioning of CIII 2 and CI within the SC. Insets present enlarged views of the ISP head domains, where CIII 2 -1 (light pink) is overlaid with CIII 2 -2 (gray, left panel), and vice versa (right panel). These overlays reveal a marked conformational difference in ISP1: in CIII 2 -2, the 2Fe– 2S cluster of ISP1 is repositioned 17.7 Å closer to the heme c 1 of cytochrome c 1 compared to its position in CIII 2 -1. Focused classification of the cryo-EM data resolved CIII 2 into two structural classes, CIII 2 -1 and CIII 2 -2, at resolutions of 2.23 Å and 2.42 Å, respectively (Figure S1C). For both classes, complete atomic models were built, including all amino acid residues and cofactors. The overall architectures of CIII 2 -1 and CIII 2 -2 were nearly identical, except for the conformation of the iron–sulfur protein (ISP), a core catalytic subunit containing a 2Fe–2S cluster responsible for electron transfer from heme b H of cytochrome b to heme c 1 of cytochrome c 1 . In CIII 2 -1, the ISPs adopt symmetric conformations, whereas in CIII 2 -2, they exhibit asymmetry ( Figure 6C ). Specifically, in CIII 2 -2, one solvent-exposed Rieske domain—the mobile part of the ISP—remains in the same position as in CIII 2 -1, while the other shifts toward heme c 1 ( Figure 6C ). Previous cryo-EM studies by Wieferig and Kühlbrandt on CIII 2 isolated from Yarrowia lipolytica under varying redox conditions demonstrated that the Rieske domain adopts multiple conformations to facilitate electron transfer between hemes b L and c 1 via the 2Fe–2S cluster 30 . They also reported both symmetric and asymmetric CIII 2 dimers, depending on the relative positions of the two Rieske domains. Although it remains debated whether the two ISPs operate independently or in a coordinated manner, our structures of bovine CIII 2 replicate the asymmetric conformation observed in the Yarrowia enzyme, suggesting that each Rieske domain moves independently, potentially reflecting the redox state of each monomer. Structures of complex IV in SCs Among the three complex IV (CIV) units present within the SC (CI 1 CIII 2 CIV 3 ), local refinement of CIV A —positioned adjacent to the distal end of complex I via interactions between the CI-ND5 and CIV A -Cox7A subunits (Figure S5B)—yielded a density map at 2.61 Å resolution, enabling atomic model construction of CIV A ( Figure 7A ). Consistent with recent findings, including the in situ cryo-EM structure of porcine mitochondrial CIV 16 , an additional density corresponding to the 14th subunit, NDUFA4, was identified in all three CIV units ( Figures 7B ). Download figure Open in new tab Figure 7. Structure of CIV A in the supercomplex (SC) and interaction between CIV C and CIII 2 . (A) Cryo-EM density map of CIV A . Each subunit is color-coded. (B) Close-up view of the newly identified NDUFA4 subunit. The atomic model is superimposed on the corresponding EM density (shown in gray). (C) Atomic model of CIV A , with subunits color-coded as in panel (A). (D) Interface between CIV C and CIII 2 within the SC. The left panel shows a top-down view of the interface between CIII 2 and CIV C , while the right panel presents a side view. The model of CIV C (shown in a tube helix representation) is superimposed on the corresponding EM density map. To clarify the orientation of CIV C within the SC, the subunit NDUFA5 is highlighted in red. The second CIV unit, CIV B (2.86 Å resolution), was located in the interspace between CI and CIII 2 , forming contacts with both complexes—specifically between CI-39-kDa and CIV B -Cox5A on the matrix side, and between CIII-subunit 9 and CIV B -Coq6A2 within the membrane bilayer (Figure S5C). The third CIV unit, CIV C (3.98 Å resolution), was positioned adjacent to CIII 2 . Although its resolution was lower than that of CIV A and CIV B , the TMHs remained clearly discernible, allowing confident identification (Figure S1C). At the interface between CIV C and CIII 2 , the TMH of CIV C -Cox6A2 was located in close proximity to CIII 2 -subunit 9 ( Figures 7C ). However, due to the limited resolution of CIV C , it was not possible to observe how it associates with CIII 2 at the amino acid level—whether through direct protein–protein interactions or via lipid-mediated association. Discussion F o F 1 oligomers in SMPs membrane In this study, we successfully determined the structures of oligomeric F o F 1 ATP synthase and respiratory SCs located in the mitochondrial inner membrane without the need for extraction and isolation using detergent. We determined the oligomeric structure of F o F 1 on SMPs, revealing the existence of dimers, in which two F o F 1 monomers are linked by dimeric IF 1 ( Figure 2 ). Using focused refinement on each monomer, we obtained the structure of monomeric F o F 1 at a resolution sufficient to construct an atomic model. Furthermore, we identified a tetrameric structure with an additional F o F 1 -IF 1 dimer opposite side to the originally identified F o F 1 -IF 1 dimer ( Figure 3A ). In the tetrameric F o F 1 structure obtained in this study, the F o F 1 -IF 1 dimer pairs are associated through their membrane domains, inducing a steep membrane curvature ( Figure 3A , lower). Additionally, density corresponding to the F 1 heads was observed on both sides of the reconstructed F o F 1 tetramer ( Figure 3A , right). These findings suggest that the F o F 1 tetramer structures align laterally, indicating the presence of a curved structure on the SMPs membrane similar to the cristae tips in mammalian mitochondria. The involvement of the F o F 1 tetramer structure in membrane curvature has been previously suggested by detergent solubilized F o F 1 tetramer structures, and our results provide direct evidence supporting this hypothesis, in a mammalian biological membrane. Our analysis also revealed a multimeric structure in which the F o F 1 -IF 1 dimer was offset by one F o F 1 unit ( Figure 3B ), suggesting association of F o F 1 -IF 1 dimers through the F o domain was weak at cristae tips. In ciliates, a stable F o F 1 dimer structure formed via interactions between the peripheral stalks and F o domains has been reported 30 , and this dimer configuration is known to contribute to cristae tip formation in the mitochondria of these organisms. In contrast, here the interaction between the F o F 1 monomers at the tip of curved structure in the F o F 1 tetramer appears to rely solely on the F o domain. Our observation of the offset F o F 1 -IF 1 dimer structure implys that the architecture of cristae tips is inherently dynamic. Indeed, cristae morphology has been shown to undergo dramatic changes depending on the metabolic state of mammalian mitochondria, and the fluidity of F o F 1 multimeric assemblies may play a role in facilitating such structural remodeling 31 . In addition, we have also obtained cryoEM images showing the presence of a monomeric F 1 heads near the SCs (Figure S3). While most F o F 1 complexes are located at the tips of cristae-like membrane structures, the presence of such orphan F o F 1 is suggestive of the fluidity of mitochondrial cristae structures. Structural organization of respiratory supercomplexes We also successfully obtained a high-resolution 3D structure of the respiratory SCs directly from SMPs. The predominant SC exhibited a stoichiometry of CI 1 CIII 2 CIV 3 , in which three CIV units are bound to the CI 1 CIII 2 core. This organization differs from SCs derived from solubilized mitochondrial membrane, which typically contain only a single CIV unit, as well as from the in situ structure of CI 1 CIII 2 CIV 2 observed in porcine heart mitochondria, where the second CIV is positioned between the ND6 subunit of CI and the cytochrome b subunit of CIII 2 16 . Moreover, 2D class averages revealed a novel megacomplex with the stoichiometry CI 2 CIII 4 CV 6 (Figure S3A). In contrast, the structure corresponding to CI 2 CIII 2 CV 2 —previously reported in in situ structural analyses of porcine heart mitochondria 16 —were not identified. In the CI 2 CIII 2 CV 2 complex (Figure S3D), the two CI units are bridged by a shared CIII 2 ; therefore, binding of CIV to CIII 2 may sterically hinder the formation of this arrangement. As noted in previous studies of porcine mitochondrial SCs 16 , solubilization of membrane proteins using detergents may disrupt SC architecture by simultaneously stripping off tightly bound phospholipids that are essential for stabilizing interactions among respiratory complexes. In this study, structural determination was achieved without solubilizing the mitochondrial inner membrane, enabling the identification of novel SCs, CI 1 CIII 2 CIV 3 and CI 2 CIII 4 CV 6 . This detergent-free approach may facilitate the discovery of previously unidentified SCs, not only within the mitochondrial inner membrane but also across other organellar or plasma membranes. Structures of ubiquinone-accessing tunnel in Complex I Compared to the F o F 1 oligomer, the respiratory SCs were resolved at higher resolution, enabling the construction of atomic models for each respiratory complex. Within the SCs, complex I (CI) was successfully classified into closed and open conformations ( Figure 5C and Figure S4), consistent with those previously reported for the isolated enzyme. Notably, these conformations likely correspond to the biochemically defined active and deactive states of CI, respectively; however, this correspondence remains a subject of ongoing debate 5 , 6 . In the closed conformation structure, the narrow ubiquinone (UQ)-accessing tunnel is occupied by a density likely corresponding to UQ (Figure S4), which appears to hinder the binding of additional ligands. Nevertheless, our chemical biology studies using bovine SMPs revealed that bulky synthetic ligands—larger than the tunnel diameter in the closed conformation—can still access the UQ-binding sites 32 , 33 . This observation suggests that the open conformation, whose complete structure remains unresolved, permits access for such ligands. These findings support the possibility that the open conformation of CI is competent for ligand binding. Further investigations integrating structural analyses of SCs in bovine SMPs with our chemical biology approaches provide insights into the relationship between closed/open conformations and the active/inactive states of CI, thereby advancing our understanding of the mechanisms underlying their structural transitions. Perspective Recent studies have reported that metabolic processes are regulated through alterations in the composition of mitochondrial respiratory SCs 28 . The isolation of mitochondria from small biopsy specimens, particularly from patients with mitochondrial disorders or metabolic diseases such as diabetes, holds promise for uncovering structural correlations between SCs and disease pathophysiology. Notably, given that cyt c 1 is released into the cytosol during the early stages of apoptosis, in situ structural analysis of SCs and F o F 1 oligomer from apoptotic cells may provide critical insights into the molecular mechanisms governing initiation of apoptosis. Collectively, these advances underscore the potential of native-state structural biology to deepen our understanding of mitochondrial function and its implications in human health and disease. Author contributions A.N., and K.Y. designed, performed and analyzed the experiments. A.N., T.M., S.A., M.I., analyzed the data and contributed to preparation of the samples. K.M. provided technical support and conceptual advice. A.N., H.M., M.M., K.Y. designed and supervised the experiments and wrote the manuscript. All authors discussed the results and commented on the manuscript. The authors declare no conflicts of interest associated with this manuscript. All data is available in the manuscript or in the supplementary materials. Competing interests We declare no completing interests. Data and materials availability The Cryo-EM density maps and models generated in this study have been deposited in the EM and protein database under accession codes: EMDB 65580, EMDB 65581, EMDB 65583, EMDB 65584, EMDB 65585, EMDB 65586, EMDB 65587, EMDB 65577, EMDB 65578, EMDB 65579, PDB 9W2U, PDB 9W2V, PDB 9W2X, PDB 9W2Y, PDB 9W2Z, PDB 9W2R, PDB 9W2S, PDB 9W2T. The deposited PDB files, cryo-EM density maps, and validation reports have been uploaded to Figshare. Material and Methods Preparation of SMPs Bovine heart mitochondria were isolated according to the method described by Smith 34 . Heart muscle from one bovine heart, obtained from slaughterhouse within several hours post-slaughter, was minced using a meat grinder at 4 °C to yield approximately 1 kg of tissue. The mince was disrupted using a mechanical blender in a buffer containing 250 mM sucrose, 1.0 mM sodium succinate, 0.2 mM EDTA, and 10 mM Tris-HCl (pH 7.8) at a ratio of 300 g mince/L of buffer. The pH was subsequently adjusted to 7.4 with 6.0 M KOH. The homogenate was centrifuged at 680 × g for 20 min at 4 °C to remove unbroken cells and nuclei. The resulting supernatant was neutralized to pH 7.4 with 1.0 M KOH and centrifuged at 10,000 × g for 20 min at 4°C. The pellet was carefully homogenized using a tight-fitting Teflon-glass homogenizer (100 ml capacity) and centrifuged at 30,700 × g for 15 min at 4°C. This homogenization and centrifugation step was repeated, and the final pellet containing both light- and heavy-mitochondria was resuspended in the same buffer (∼50 ml) and stored at −80 °C as intact mitochondria until use. The protein concentration was determined using the Biuret method. Typically, 1,500-2,000 mg of mitochondria were obtained from one bovine heart. Submitochondrial particles (SMPs) were prepared as described by Matsuno-Yagi and Hatefi 24 . Frozen mitochondria were thawed and adjusted to a final protein concentration of 40 mg of protein/ml with a buffer containing 250 mM sucrose and 10 mM Tris/HCl (pH 7.5). The suspension was supplemented with 1.0 mM potassium succinate, 1.5 mM ATP, 10 mM MgCl 2 , 10 mM MnCl 2 , followed by a sonication using a TOMY UR-200P (power setting 8, 30 sec, 5 cycles, with 3 min intervals) on ice. After sonication, the pH was adjusted to 7.5 with 1.0 M KOH and the sample centrifuged for 15 min at 34,400 x g at 4°C. The clear supernatant was carefully collected and subjected to ultracentrifugation at 200,000 x g for 45 min at 4°C. The resulting brown pellet was rinsed with a buffer containing 250 mM sucrose and 10 mM Tris/HCl (pH 7.5) and homogenized in the same buffer at a protein concentration of 30-40 mg/ml. The SMP suspension was snap-frozen in liquid nitrogen in small aliquots (100 µL) and stored at −80°C. Cryo-grid preparation and image acquisition for SMPs SMP suspensions (8 mg/ml) were used for preparation of cryogrids using Quantifoil UltraAufoil R1.2/1.3 grids. Before blotting, the grids were treated with an ion bombardier for 1 minute of glow discharge. Using a FEI Vitrobot (Thermo Fisher Scientific), 3 µl of SMP solution were applied to a grid using a blot time of 2.5 s, blot force 10, and a drain time of 0.5 s at 25°C and 100% humidity. Cryo-grids were imaged using a Titan Krios G2 (Thermo Fisher Scientific) (UHV EM at Osaka University) equipped with a K3 electron detector (Gatan). For data collection using the Titan Krios G2 (Thermo Fisher Scientific), cryo-EM movies were collected automatically at a nominal magnification of 29,000x corresponding to a calibrated pixel size of 0.84 Å/pix using image shift based data collection with the SerialEM software employing a defocus range of −0.8 to −1.8 μm. Image data was collected in 50 frames in CDS mode at a total electron dose of 50 electrons/Å 2 . Structural analysis The detailed single particle analysis workflow is summarized in Figure S1. Single particle analysis was performed by RELION ver 4.0 35 and CryoSPARC ver 4.7 36 . Particle metadata in cryoSPARC (.cs) format were converted to RELION .star files using the PyEM script csparc2star.py. Beam-induced drift motion was corrected in 30,680 movies using MotionCor2 37 , CTF parameters were estimated with CTFFIND4 38 . Approximately 300 particles of F o F 1 dimer or SC were manually picked and used to train Topaz. Using the trained model, 3,636,680 particles were picked from 5,000 micrographs, extracted with a box size of 150 pixels (4.36 Å/pix), and 2D classification was performed. Particles of F o F 1 dimer or SCs were selected and initial maps of F o F 1 dimer and SCs were created by Ab-initio reconstruction, respectively. Heterogeneous Refinements were then performed with all the particles picked by topaz to obtain F o F 1 dimer or SCs structures. The topaz models were further trained using 3000 particles of the both F o F 1 dimer and SCs particles curated by Heterogeneous Refinement. Using both models, a total of 40,298,925 particles were picked and extracted from 30,680 micrographs. These particles were divided into eight subsets, with obvious junk particles excluded by 2D classification, yielding 30,421,492 particles. Heterogeneous refinement was performed four times using either the F o F 1 dimer or SCs, and multiple junk classes resulting from Ab-initio reconstruction as reference volumes. To obtain high-precision CTF parameters, the 764,363 particles of the SCs were processed with C2 symmetry imposed on the Complex III region, followed by Local refinement and combined Global/Local CTF refinement. Iteration of this process yielded a structure of 2.3 Å resolution for the CIII 2 domain. The particles with CTF parameters refined in the CIII 2 region were then used to proceed with structural analysis on the other respiratory chain proteins. After local refinement in the CI, 3D classification was performed to classify the CI as open or closed, and the respective structures were obtained. For the CIII 2 , symmetry expansion was performed on C2, and after local refinement, 3D classification was performed using a mask including the Rieske domain to classify the CIII 2 -1 and CIII 2 -2. For CIV A , CI 1 CIII 2 and CI 1 CIII 2 CIV A each was classified by Heterogeneous Refinement, followed by Local Refinement and then 3D classification to remove low-resolution classes to obtain the structure. For CIV B and CIV C , Heterogeneous Refinement classified structures with stronger densities of CIV B and CIV C . Then, CI 1 CIII 2 CIV A domain were signal subtracted to improve alignment in Local Refinement. The structures of CIV B and CIV C were obtained by removing the junk class by 3D classification. For structural analysis of the megacomplex and SCs-F o F 1 , we re-extracted at a 981pixel box size (3.27 Å/pix) and limited the alignment resolution to 20-30 Å in the 2D classification and clear 2D class-averaged images of both the megacomplex and SC-F o F 1 were obtained. By performing Heterogeneous Refinement of these particles with the structure of SCs as a reference, followed by Homogeneous refinement, the 3D structures of the megacomplex and SC-F o F 1 were obtained. For analysis of the F o F 1 oligomer, Heterogeneous Refinement yielded 328,822 particles, and the structure of the F o F 1 -IF 1 dimer was obtained by Homogeneous Refinement. First, CTF refinement and Local refinement were performed on the F 1 -1 domain, followed by local refinement and 3D classification in F o -1, F 1 -2, and F o -2 to obtain the structure of each. To obtain structural information of a broader region of the F o F 1 oligomer, particles were re-extracted using a box size of 981 pixels (3.27 Å/pixel), followed by local refinement and 3D classification focused on the region encompassing the F o F 1 –IF 1 dimer and adjacent F o F 1 units. Through 3D classification, a distinct class was identified corresponding to a tetrameric assembly in which two F o F 1 –IF 1 dimers are paired. Using this structure as a reference, heterogeneous refinement further resolved two conformational states of the F o F 1 tetramer. Model building The atomic model of F o F 1 monomer and focused F o domain were built from the Cryo-EM structure of bovine ATP synthase PDB 6ZPO, 6ZQN, and 6ZBB. For the model construction of SCs, we used bovine CI structure of PDB7QSK, bovine CIII structure of 1SQB, and CIV structure of 6JY3. Rigid body fitting was performed by ChimeraX 39 , and after manual modification of the entire model by ISOLDE 40 , a refinement by phenix.real_space_refinement 41 was performed. The refinement model was evaluated with MolProbity 42 , and Phenix.real_space_refinement. Manual correction with COOT and ISOLDE and phenix.real_space_refinement were repeated until model parameters improved. Acknowledgements We are grateful to all the members of the Yokoyama Lab for their continuous support and technical assistance. We are thankful to Dr. Jun-ichi Kishikawa (Kyoto Institute of Technology) for the structural analysis. Our research was supported by Grant-in-Aid for Scientific Research (JSPS KAKENHI) Grant Numbers 23H02453 to K.Y., 24K08729 to T.M. and 25K01958 to M.M., Takeda Science foundation funding to K.Y., The Uehara Memorial foundation to M.M, and Tokyo Kasei Chemical Promotion foundation to M.M. Our research was also supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP17am0101001 (support number 1312), and Grants-in-Aid from the “ARIMS” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to KY. Funder Information Declared JSPS KAKENHI) , 23H02453 , 24K08729 , 25K01958 Takeda Science foundation funding References 1. ↵ Levental , I. & Lyman , E. Regulation of membrane protein structure and function by their lipid nano-environment . Nat Rev Mol Cell Biol 24 , 107 – 122 ( 2023 ). 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Acta Crystallogr D Biol Crystallogr 66 , 12 – 21 ( 2010 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted September 19, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane Atsuki Nakano , Takahiro Masuya , Shinsuke Akisada , Moe Ishikawa-Fukuda , Kaoru Mitsuoka , Hideto Miyoshi , Masatoshi Murai , Ken Yokoyama bioRxiv 2025.09.19.677273; doi: https://doi.org/10.1101/2025.09.19.677273 Share This Article: Copy Citation Tools In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane Atsuki Nakano , Takahiro Masuya , Shinsuke Akisada , Moe Ishikawa-Fukuda , Kaoru Mitsuoka , Hideto Miyoshi , Masatoshi Murai , Ken Yokoyama bioRxiv 2025.09.19.677273; doi: https://doi.org/10.1101/2025.09.19.677273 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Biochemistry Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17681) Bioengineering (13890) Bioinformatics (41930) Biophysics (21446) Cancer Biology (18586) Cell Biology (25493) Clinical Trials (138) Developmental Biology (13374) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15607) Genomics (22498) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88584) Paleontology (666) Pathology (2831) Pharmacology and Toxicology (4823) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9823) Zoology (2271)