Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient

Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient | 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 Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient View ORCID Profile Jun Yu , View ORCID Profile Xuefeng Zhang , View ORCID Profile Sandra Pinton , View ORCID Profile Elena Vacchi , View ORCID Profile Andrea Cavalli , Matteo Pecoraro , View ORCID Profile Giorgia Melli , View ORCID Profile Andreas Boland doi: https://doi.org/10.1101/2025.07.29.667442 Jun Yu 1 Department of Molecular and Cellular Biology, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jun Yu Xuefeng Zhang 1 Department of Molecular and Cellular Biology, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xuefeng Zhang Sandra Pinton 2 Neurodegenerative Diseases Group, Institute for Translational Research , Bellinzona, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandra Pinton Elena Vacchi 2 Neurodegenerative Diseases Group, Institute for Translational Research , Bellinzona, Switzerland 3 Faculty of Biomedical Sciences, Università della Svizzera Italiana , Lugano, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elena Vacchi Andrea Cavalli 3 Faculty of Biomedical Sciences, Università della Svizzera Italiana , Lugano, Switzerland 4 Institute for Research in Biomedicine , Bellinzona, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrea Cavalli Matteo Pecoraro 4 Institute for Research in Biomedicine , Bellinzona, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Giorgia Melli 2 Neurodegenerative Diseases Group, Institute for Translational Research , Bellinzona, Switzerland 3 Faculty of Biomedical Sciences, Università della Svizzera Italiana , Lugano, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giorgia Melli For correspondence: Andreas.Boland{at}unige.ch Giorgia.Melli{at}eoc.ch Andreas Boland 1 Department of Molecular and Cellular Biology, University of Geneva , Geneva, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andreas Boland For correspondence: Andreas.Boland{at}unige.ch Giorgia.Melli{at}eoc.ch Abstract Full Text Info/History Metrics Preview PDF Abstract Amyloid transthyretin-derived (ATTR) amyloidosis is a degenerative, systemic disease characterized by transthyretin fibril deposition in organs like the heart, kidneys, liver, and skin. We report the first cryo-EM structure of transthyretin fibrils isolated from skin tissue of a living patient carrying a rare genetic mutation (ATTRv F64S). The structure adopts a highly conserved fold previously observed in other ATTR fibrils from different tissues or genetic variants. Mass spectrometry was used to evaluate fibril content and identify post-translational modifications. The structural consistency between ATTR filaments validates non-invasive skin biopsy as a diagnostic tool. Introduction Amyloid transthyretin-derived (ATTR) amyloidosis is one of the most prevalent forms of systemic amyloidosis and encompasses two types: a genetic (ATTRv) and a sporadic wild-type (ATTRwt) form. ATTRv amyloidosis arises from pathological mutations in the TTR gene, including amino acid substitutions, duplications, and deletions 1 . Transthyretin (TTR) mutations often destabilize the native TTR fold, leading to amyloid formation in multiple organs such as the heart, kidneys, liver or skin 1 , 2 . To date, a total of 216 mutations have been identified, including 200 amyloidogenic and 16 non-amyloidogenic mutations 1 , 3 . The clinical manifestations of hereditary ATTRv amyloidosis are highly variable, however, the predominant forms are characterized by peripheral polyneuropathy and an early-onset disease 4 . In contrast, ATTRwt amyloidosis is associated with aging-related factors or unknown processes that lead to the extracellular deposition of ATTR fibrils in tissues 5 , and late-onset cardiomyopathy is typical 6 . Based on their composition and morphology, ATTR fibrils are classified into two main types. Type A fibrils are composed of full-length (127 amino acids) or fragmented TTR molecules and can be found in ATTRwt and most ATTRv variants. In contrast, type B fibrils only contain full-length TTR 7 , 8 . Importantly, different fibril types are associated with specific diseases in neurodegenerative pathologies 9 . In addition to genetic mutations, post-translational modifications (PTMs) can influence the formation and characteristics of amyloid fibrils. PTMs have been implicated in altered behaviour of many amyloid proteins, including amyloid β, tau, α-synuclein, huntingtin, and TDP43 10 . Several types of modification, including phosphorylation 11 , acetylation 12 and ubiquitination 13 have been described as modulators of aggregation rate or extent, aggregate stability, and cytotoxicity. A growing number of cryo-EM structures of ATTR fibrils, extracted from post-mortem tissues such as the heart, eyes and nerves provided critical insights into the structural organisation of ATTR amyloid fibrils 14 – 22 . All structures share a common, relatively compact and β-sheet-rich fold that has been described as spearhead-shaped 14 . Despite their overall structural homogeneity, ATTR fibrils from different tissues exhibit local variations in a region that spans amino acids G57 to G67 19 , referred to as ‘gate’ region. For example, in cardiac fibrils of ATTRv-I84S patients four distinct gate states have been observed, named open, closed, broken and absent. In contrast, in ATTRv-V30M fibrils from the eye only one gate type was observed, called blocking gate 15 , 19 . ATTRwt cardiac fibrils from five patients and ATTRv fibrils (V20I, P24S, V30M, G47E, T60A, V122I and V122ι1) from various tissues show a closed gate near a polar channel. Polymorphism has also been observed in the number of protofilaments. Most ATTR fibrils consist of a single protofilament under cryo-EM conditions, with the exceptions of ATTRv-V122ι1 cardiac fibrils that contain one or two protofilaments and ATTRv-V30M fibrils from the eye that are formed by multiple protofilaments 15 , 22 . Recently, we showed that skin biopsy is an extremely sensitive, minimally invasive test for detecting and typing ATTR amyloidosis 23 . In this study, we use immunohistochemistry, mass spectrometry, and cryogenic electron microscopy (cryo-EM) to describe the molecular composition and the structural characteristics of amyloid fibrils extracted from ankle and thigh tissues from skin biopsies of a living ATTRv-F64S patient. The ATTR fibril structure of this genetic variant has not been determined yet. Our work reveals that ATTRv-F64S fibrils contain one protofilament and, less frequently, two protofilaments. The single protofilament adopts a near-identical fold to that of ATTRwt and most ATTRv fibrils, featuring a closed gate. Our structure is the first high-resolution structure of ATTR fibrils derived from a skin biopsy of a living patient, demonstrating that sufficient quantities of amyloid fibrils can be extracted from minimal amount of skin tissue (between 5-10 milligrams). The structural conservation of ATTR fibrils across various tissues, including skin, further corroborates skin biopsy as a minimally invasive test for detecting, typing and determining the structure of ATTR amyloid fibrils. Results Characterisation of ATTR fibrils from ankle and thigh skin biopsies of a living patient In a first step we quantified the abundance of ATTR amyloid fibrils in two different skin sections, namely from ankle and thigh tissue. Filaments were stained with Congo red dye that results in red or pink deposits that can be observed by brightfield (BF) microscopy ( Extended Data Fig. 1a, BF). Using polarized light (PL) the characteristic birefringence of amyloid fibrils was detected in both tissues ( Extended Data Fig. 1a, PL) 23 . When comparing ankle and thigh tissue, a stronger Congo red staining and higher birefringence was detected in ankle tissue from this patient. PGP9.5 (Protein Gene Product 9.5) antibody staining showed reduced intraepidermal nerve fibre density in both samples, confirming small fibre neuropathy ( Extended Data Fig. 1a, PGP9.5). We next extracted ATTR fibrils from ankle and thigh tissue obtained from a living ATTRv-F64S patient by skin biopsy. Fibril extraction and purification was performed using a water extraction protocol previously shown to preserve the structure of the amyloid fibrils 24 . Quantification of isolated fibrils from the two tissue samples was assessed by silver-stained SDS-PAGE gels. The gels showed two or more bands running at approximately 12-15 kDa, indicating the presence of full-length (127 amino acids) and fragmented TTR. Consistent with our quantification in tissue sections, we observed much stronger intensities of TTR monomer bands in the sample isolated from ankle compared to thigh tissue ( Extended Data Fig. 1b, dashed box). A higher abundance of fibrils was also detected in ankle tissue versus thigh tissue using negative-stain microscopy ( Extended Data Fig. 1c ). Characterisation of ATTRv-F64S fibrils by mass spectrometry Next, minute quantities of fibrils from ankle and thigh tissue (300 ng and 30 ng, respectively) were analysed using bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS) with high-sensitivity acquisition on a trapped ion mobility (TIMS) mass spectrometer. TTR was readily identified in both samples with 20 and 22 unique peptides, respectively. This resulted in a near full coverage (92.9%) of the TTR mature form, with only the first nine amino acids missing ( Extended Data Fig. 2a , b and Supplementary File 1 ). An N-terminal free semi-specific search further increased the coverage to a maximum of 98.4%, including three additional peptides that further confirmed the presence of full-length TTR within the fibrils ( Extended Data Fig. 2c , d and Supplementary File 1 ). For peptides encompassing the F64S mutation site, both wild-type and mutant sequences were detected. When analysing their relative intensities, we consistently observed higher intensities for peptides carrying the F64S mutation compared to wild-type peptides ( Fig. 1a-c ). Because the mutation could in principle alter the ionization properties of the peptides, we also analysed a piece of ankle skin biopsy without performing fibril extraction, to inspect bulk epidermal TTR by LC-MS/MS. In this setting, we indeed observed similar intensity levels of wild-type and mutant peptides, in line with the patient’s heterozygosity for the TTR-F64S mutation ( Extended Data Fig. 3 and Supplementary File 1 ). Our results suggest a higher fraction of mutant versus wild-type TTR in ATTRv-F64S fibrils, in line with a destabilisation effect of the native TTR fold by this mutation, eventually leading to amyloid formation. We then examined the fibril data for post-translational modifications (PTMs), including acetylation, methylation, phosphorylation and ubiquitination. Using a deep learning prediction module of the MSFragger search tool, we confidently identified fourteen PTMs on nine sites. These sites were filtered for their localization probability and spectral similarity to predicted MS/MS spectra above 80% ( Extended Data Table 1 ). Eight of the PTM sites were present in fibrils extracted from ankle and thigh tissues (ten hits if the similarity score is reduced to 79%), and were consistently found in independent scans, with a maximum of 21 spectral matches. K15 ubiquitination was the only PTM identified from a single spectrum, however, this modification has also been described in the PhosphoSitePlus database 25 . Other previously identified modifications that we corroborate in this study include K15 acetylation, S52 phosphorylation and Y105 phosphorylation 25 – 27 . Of note, three PTMs were identified in peptides that include the F64 mutation site. Of these, T49 phosphorylation and K70 acetylation were specifically assigned to F64S-TTR, while S52 was found in the wild-type and mutated TTR form. These results show that comprehensive and reliable PTM analysis can be performed on ATTR fibrils extracted from skin biopsies in an unbiased manner without prior enrichment for modified peptides. Download figure Open in new tab Figure 1. Mass spectrometry analysis and cryo-EM reconstruction of amyloid fibrils from skin tissue of an ATTRv-F64S amyloidosis patient. a-b , Extracted LC-MS ion chromatogram of the wild-type (dark green) and F64S-mutant (pink) peptides in fibrils from (a) ankle and (b) thigh skin tissues. c, TIC-normalized peak areas indicate a higher intensity of the F64S-mutant peptide in fibrils from both biopsy sites. d, A representative cryo-EM micrograph of ATTRv-F64S fibrils. Scale bar, 500 Å. e, Central slice of the 3D map of ATTRv-F64S fibrils. Scale bar, 50 Å. f, Cryo-EM density map of the amyloid fibrils. Left, side view of the reconstructed fibril map. Right, close-up views (top and side) of the map with the helical rise indicated. g, High-resolution electron density map and stick model of a single fibril layer (top view), consisting of an N-terminal fragment (C10 to A36) and a C-terminal fragment (G57 to N124). The disordered region (residues 37-56) of the ATTRv-F64S fibril structure is shown as dashed line. F64S mutation is indicated by an arrow. A hydrogen bond is shown with a yellow dashed line. Cryo-EM structure of ATTRv-F64S fibrils ATTR fibrils obtained from roughly 5-10 mg of ankle tissue allowed the preparation of a total of four EM grids. Based on visual inspection of the micrographs, the imaged fibrils appeared largely uniform in morphology, although aberrant, low-abundance ATTR fibrils may also exist ( Fig. 1d and Extended Data Fig. 1c ). Automated filament picking was performed using a modified Topaz pipeline 28 , followed by helical reconstruction in RELION 29 . Two-dimensional (2D) class averages revealed a spacing of ∼4.8 Å, as previously described 14 and showed two types of fibrils: a single twisted protofilament and a twisted dimer ( Extended Data Figs. 4a, b ). Three-dimensional (3D) classification of single protofilament segments yielded one class with high-resolution features ( Fig. 1e-f ). A subsequent 3D reconstruction refined to a resolution of approximately 2.8 Å ( Fig. 1g , Extended Data Figs. 4c, d and Extended Data Table 2 ). Due to a limited number of twisted-dimer fibrils obtained during data collection, reconstruction of a high-resolution structure was unsuccessful for this fibril type. Therefore, our analysis focused solely on the single protofilament. The fibril exhibits a helical twist of – 1.36° and a rise of 4.78 Å ( Extended Data Table 2 ). A structure model was built into the density map showing two separate density regions corresponding to residues C10 to A36 and G57 to N124 ( Fig. 1g ). The region spanning A37 to H56 was not resolved in the structure, indicating local flexibility. Each fibril layer adopts a relatively planar conformation and consists of β-stands, which showing high similarity to ATTRwt fibrils (PDB: 8ADE 16 ) extracted from cardiac tissue ( Fig. 2a ). The ATTRv-F64S fibril contains ten β-stands with β1-β3 located in the N-terminal fragment and β4-β10 in the C-terminal fragment. Hydrophobicity analysis of a single fibril layer revealed three primary hydrophobic grooves formed by residues in (i) the N-terminal fragment (β1-β3), (ii) the C-terminal fragment spanning residues Y78 to A97 (β6-β7) and (iii) the core region involving β3, β5 and β8 ( Fig. 2b ). Extensive hydrophobic interactions, as well as hydrogen bonding and ρ-ρ stacking interactions, contribute to the fibril stability as observed in other ATTR fibrils 18 , 19 . Electrostatic surface potential analysis indicated two negatively charged patches involving four glutamate residues near β4 and two glutamate residues in a loop between β6 and β7 strands ( Fig. 2c ). The latter residues were previously shown to be forming the twist-dimer interface of ATTRv-V30M fibrils 15 . Download figure Open in new tab Figure 2. Structural comparison of ATTR fibrils. a , Schematic representation of the secondary structure elements of ATTRv-F64S fibril. Top, ATTRv-F64S fibril b-sheet organization. Bottom, wild-type ATTR (PDB: 8ADE 16 ) fibril shown for comparison. Arrows represent b-stands and dashed lines indicate unresolved regions of the fibril protein. Amino acid numbers of the TTR protein are indicated below. The structure of ATTRv-F64S fibril is shown as a cartoon. b , Hydrophobicity representation of a single fibril layer (top view). c , Electrostatic surface representation of a single fibril layer (top view). Electrostatic potentials are contoured from –10 (red) to +10 kTe - 1 (blue). Figures are prepared in Chimera X 34 . d , Structural comparison of ATTR fibrils, including wild-type and variant forms. Left, Views of structural alignment between ATTRv-F64S fibril (tomato red) and ATTRwt fibrils (light blue) extracted from heart (PDB ID: 8ADE 16 , 8E7D 18 , 8G9R 18 , 8GBR 18 and 8E7H 18 ). Middle, Views of structural alignment between ATTRv-F64S fibril and cardiac ATTRv fibrils (wheat, PDB ID: 8PKE 17 , 8E7I, 6SDZ 14 , 8PKF 17 , 8E7E 19 , 8E7J 19 , 8TDN 19 , 8TDO 19 and 8PKG 17 , including V20I, P24S, V30M, G47E, I84S and V122I mutants.). Right, structural alignment of ATTRv-F64S fibril with ATTRv-V30M fibril (sky blue, PDB ID: 7OB4 15 ) from the eye. Structural consistency of ATTR fibrils across various tissue types Structural alignments were performed on all available ATTR fibril structures in the Protein Data Bank (PDB), including five ATTRwt fibrils, nine ATTRv fibrils derived from cardiac tissue, one ATTRv fibril from the eye and the structure from ankle skin tissue part of this study ( Fig. 2d ). To assess structural variability, the backbone displacement was estimated by calculating the root mean square deviation (RMSD) of the Cα atoms ranging from 0.594 Å to 1.398 Å across the aligned structures ( Extended Data Table 3 ). The structural alignment indicated that the overall fold is conserved throughout all ATTR fibrils extracted from different tissues, despite local variations around the gate region ( Fig. 2d ). As in all ATTRwt fibrils from cardiac tissue and most ATTRv fibrils, the gate conformation of the ATTRv-F64S fibril is in a closed state. Exceptions are ATTRv-I84S fibrils exhibiting four different conformations (absent, broken, closed and open), and ATTRv-V30M fibrils from the human eye in a blocking state 15 , 19 . Interestingly, the F64S mutation, which is located within the gate region of the fibril structure, appears to have no effect on the gate conformation. In sum, our findings suggest that ATTRv-F64S fibrils derived from skin tissue are nearly identical to other ATTR fibrils across different tissues and different genetic variants. To the best of our knowledge, this is the first amyloid fibril structure that has been determined from a living patient, and we anticipate that this work will guide the development of strain-specific therapeutic strategies for ATTR amyloidosis. Discussion Recent evidence supports a high diagnostic accuracy of skin biopsies for ATTRv even in early and presymptomatic stages of the disease and intraepidermal nerve fibre density (IENFD) correlates with clinical findings of neuropathy 30 . In this study, we aimed to characterize ATTR fibrils extracted from the tissue of a patient with ATTRv-F64S polyneuropathy. To this end, we extracted ATTRv amyloid fibrils from ankle and thigh tissue obtained by skin biopsy. We found that fibrils were more abundant in the ankle tissue than in the thigh tissue in the examined tissue material, suggesting variations in ATTR fibril deposition throughout the body. Using bottom-up proteomics, we identified wild-type and F64S-mutated TTR contributing to the fibril’s composition, with the mutant form being detected at a higher proportion. Therefore, our analysis indicates that the mutant form of TTR may promote fibril formation. We also identify fourteen PTMs on nine sites, ten of which have not been previously described. Some modifications, such as T49 phosphorylation and K70 acetylation, have only been detected in mutant TTR and may be involved in fibril formation ( Extended Data Table 1 ). We mapped these modifications onto the tetrameric and filament structures of transthyretin ( Extended Data Fig. 5 ). Our cryo-EM analysis of a skin-derived sample revealed two types of ATTRv-F64S fibrils: a predominant single protofilament and a low-abundance twisted dimer under current conditions ( Extended Data Figs. 4a and b ). Similar variations in fibril morphology have been reported in ATTRv-V40I and ATTRv-V122ι1 cardiac fibrils, as well as in ATTRv-V30M fibrils from the eye 15 , 22 , 24 . These findings suggest that polymorphism is a common feature of ATTR fibrils deposited within different tissues or organs, like other amyloid disorders, such as light-chain (AL) amyloidosis 24 . The ATTRv-F64S fibril structure adopts a fold similar to other ATTR fibrils from heart, eye, and nerves, despite varying mutations and patient phenotypes. The largest variations occur in the gate region (residues G57–G67), with five observed gate states, the closed state being most common ( Fig. 2d ). Previous studies have suggested that mutations that destabilize the monomeric TTR fold are a potential driver of ATTR fibril formation. The crystal structure of dimeric TTR (PDB: 4TLT 31 ) reveals that F64 engages in extensive hydrophobic interactions ( Extended Data Fig. 4e ). Therefore, mutations of F64 to S, V, I and L – all variants identified in the TTR gene 1 –likely destabilize the monomeric TTR fold. Using FoldX to calculate the change in Gibbs free energy (ΔG) for specific mutants show that all described mutants have a positive ΔG, indicating that these mutations indeed destabilize the wild-type fold ( Extended Data Fig. 4e ) 32 . Notably, F64S has the highest change in ΔG. The lack of detailed structural data on distinct ATTRwt and ATTRv amyloid fibrils impedes our understanding of the disease mechanisms. This and other studies demonstrate an overall conserved ATTR fibril fold with local variations around the gate region across various tissues and organs. We show that minimally invasive skin biopsies can be conducted to characterize the fibril composition, post-translational modifications, and to determine the three-dimensional structure by cryo-EM. Therefore, skin biopsies of living patients are a powerful tool for tracking amyloidosis progression at the molecular level. Wild-type ATTR deposition in systemic organs is a common ageing-related phenomenon 33 , and ultrastructural characterization of pathogenic fibrils in skin could guide the development of strain-specific therapeutic strategies for ATTR amyloidosis in the future, paving the way towards personalized care. Figure legends Download figure Open in new tab Extended Data Figure 1. Characterization and extraction of ATTR fibrils from skin tissue. a , Amyloid deposits in skin biopsies stained with Congo red. Top, 50 μm thin sections were examined under bright-field (BF) microscopy. Magnification 4x, scale bar 200 μm. Bottom, Congo red deposits with characteristic birefringence under polarized light (PL) are shown from the inserts (dashed rectangles). Magnification 10x, scale bar 200 μm. b , Representative immunofluorescence images showing a reduced intraepidermal innervation in ankle and thigh tissue. Thin sections were stained with anti-PGP9.5 antibody (green) and the nuclear stain DAPI (blue). Sections were analysed with an inverted fluorescence microscope. PGP9.5-positive fibres crossing the dermal-epidermal junction (dotted line) were counted according to published protocols 23 . Magnification 40x, scale bar 50 μm. c, Silver stained SDS-PAGE gel of samples from washing and extraction steps during amyloid fibril extraction from an ATTRv-F64S patient. Fibrils were extracted from 5–10 mg of ankle and thigh skin tissues. The TTR protein is indicated by a dashed box. d , Negative staining micrographs of ATTRv-F64S fibrils from ankle and thigh tissues. Scale bar, 500 Å. Download figure Open in new tab Extended Data Figure 2. LC-MS/MS analysis of ATTR fibrils extracted from skin tissue. a-b , Alignment of the LC-MS/MS identified peptides in fibrils extracted from ankle ( a ) or thigh ( b ) biopsies with the sequence of mature transthyretin, yielding a 92.9% coverage in both sites. c and d , Analysis of the N-terminus of transthyretin using an N-free semi-specific search of the LC-MS/MS data. The alignment shows an increased total coverage of the mature form of the TTR protein to 98.4% in fibrils extracted from the ankle biopsy ( c ), and to 93.7% in fibrils extracted from thigh biopsy ( d ). Download figure Open in new tab Extended Data Figure 3. LC-MS/MS analysis of a bulk skin biopsy sample from ankle. a , Annotated MS/MS spectra of the wild-type (top) and F64S-mutant (bottom) peptides in bulk skin fibrils. b , Extracted LC-MS ion chromatogram of the wild-type (dark green) and F64S-mutant (pink) peptides. c , TIC-normalized peak areas show similar intensity levels between wild-type and mutant peptides. Download figure Open in new tab Extended Data Figure 4. Cryo-EM analysis of ATTRv-F64S fibrils. a , Representative two-dimensional (2D) class averages of single twisted protofilaments extracted from ankle skin. Scale bar, 100 Å. b , Representative two-dimensional (2D) class averages of twist-dimer ATTRv-F64S fibrils from ankle skin. Scale bar, 100 Å. c , Gold standard Fourier Shell Correlation (FSC) curve of the cryo-EM map. The FSC curve between the cryo-EM map and the atomic coordinates was calculated using Mtriage 35 . d , Local resolution map of ATTRv-F64S fibril color coded according to the local resolution ranging from 2.6 to 3.8 Å. e , The F64S mutation likely destabilizes the TTR protein. Left, Structure of the TTR tetramer shown as a cartoon (PDB ID: 4TLT 31 ). Middle, close-up view of hydrophobic interactions between F64 and surrounding residues (distance < 4 Å) in a single monomer. Right, Energy differences between wild-type TTR protein and four F64-related mutants that have been identified in patients, which were calculated using FoldX 32 . Download figure Open in new tab Extended Data Figure 5. PTM sites identified by MS mapped on the tetrameric and fibril structures. a , PTM sites are mapped onto the ATTR-F64S fibril structure. The disordered region (residues 37-56) is shown as a dashed line. T49 and S52 are shown as black dots and all other residues identified are shown as sticks. b , PTM sites are mapped onto the structure of the TTR tetramer (PDB ID: 4TLT 31 ). Materials and Methods Skin biopsy A three mm-diameter punch skin biopsy was performed on the distal leg, 10 cm above the lateral malleolus, and on the thigh, 10 cm above the knee, as previously described 23 . The biopsy was conducted from a patient with ATTRv-F64S polyneuropathy, and a disease duration of three years. The skin samples were flash-frozen and stored at –80°C until further analysis. To quantify amyloid deposits in skin tissue, 50-µm thin sections were stained with Congo red solution 23 and examined under bright-field (BF) microscopy (Zeiss Axio Lab.A1 Microscope, AxioCam ERc 5 s, Oberkochen, Germany). To evaluate small fibre neuropathy in the skin tissue, at least three non-consecutive 50 μm thin tissue sections were stained each location using a primary antibody against the pan axonal marker protein gene product 9.5 (PGP9.5, Abcam, Cambridge UK, 1:1000) and the nuclear stain DAPI (Sigma-Aldrich, Saint Louis USA, 1:5000). Fluorescence imaging was performed using an inverted fluorescence microscope (Nikon Eclipse Ti-E, Tokyo, Japan). PGP9.5-positive fibres crossing the dermal-epidermal junction were counted according to published protocols 36 . Fibril extraction from the skin tissue Amyloid fibrils were extracted from human skin tissue using a modified water extraction protocol 24 . In brief, approximately 5-10 mg frozen skin tissues from ankle or thigh biopsy of a living patient carrying a point mutation F64S were thawed at room temperature and subsequently diced. Both skin samples were processed equally in all subsequent steps. Diced tissues from thigh or ankle were separately transferred to 1.5 ml Eppendorf tubes and washed with 0.5 mL Tris-calcium buffer (20 mM Tris, 140 mM NaCl, 2 mM CaCl 2 , 0.1 % NaN 3 , pH 8.0). The resulting suspensions were homogenized using a Kimble pellet pestle (Sigma-Aldrich) and centrifuged for 5 min at 3100 × g. All centrifugation steps were performed at 4 °C. The washing procedure was repeated five more times. Pellets were resuspended in 1 mL freshly prepared collagenase digestion buffer supplemented with protease inhibitor cocktail tablet (PIC) (cOmplete EDTA-free, Roche Diagnostics) and 5 mg/mL crude collagenase from Clostridium histolyticum (Sigma-Aldrich). The suspension was incubated overnight at 37 °C with shaking at 150 rpm. Next, the samples were centrifuged at 3100 × g for 30 min. Pellets were resuspended in 0.2 mL Tris-EDTA buffer (20 mM Tris, 140 mM NaCl, 10 mM EDTA, 0.1% NaN 3 , pH 8.0), homogenized, and centrifuged for 10 min at 3100 × g . This washing step was repeated four more times. For the extraction of amyloid fibrils, all pellets were resuspended in 0.2 mL of ice-cold water and centrifuged for 10 min at 3100 × g . The fibril-containing supernatant was collected, and the extraction step was repeated four more times. Amyloid fibrils extracted ankle and thigh tissues were concentrated by ultracentrifugation at 100,000 x g for one hour. Final pellets were resuspended in 30 µL ice-cold water and used for cryo-EM and mass spectrometry analyses. Negative staining analyses In an initial step, amyloid fibrils extracted from ankle and thigh tissues were analysed by negative-stain transmission electron microscopy. Briefly, 3 μL of each sample were applied onto 400-mesh copper grids with carbon film (Electron Microscopy Sciences) that had been glow discharged for 20 s. The samples were incubated for one minute, after which excess solution was removed by blotting using Whatman® Filter Paper. Grids were then washed twice with water and subsequently stained with 2% uranyl acetate for one minute, followed by blotting to remove excess stain. Grids were examined using an in-house Thermo Fisher Scientific (TFS) Talos L120C G2 transmission electron microscope. Cryo-EM sample preparation and data collection Skin tissue from ankle biopsy contained significantly more amyloid fibrils than skin tissue from thigh biopsy. Consequently, cryo-EM analyses were limited to fibrils extracted from ankle skin tissue. 4 µL of concentrated fibril extraction were applied onto glow-discharged holey carbon grids (Quantifoil R1.2/1.3, 300 mesh). Grids were front blotted for three to four seconds with an additional movement of 1 mm (95% humidity at 15 °C) before being plunged into liquid ethane using an EM GP2 automatic plunge freezer (Leica). The grids were stored in liquid nitrogen until data collection. The cryo-EM dataset was acquired using a Thermo Fisher Scientific (TFS) Titan Krios transmission electron microscope at an accelerating voltage of 300 kV. A total of 9,763 movies from two grids were collected on a Falcon 4i direct electron detector (equipped with a Selectris X filter) at a nominal magnification of 165,000 x, resulting in a pixel size of 0.726 Å. Data were collected using EPU (TFS) software, with five images recorded per hole, with a set defocus range of −0.6 and −2.0 µm and a total electron dose of 40 e − /Å 2 . Data acquisition was monitored using on-the-fly preprocessing in CryoSPARC v.4.2.1 37 . Helical reconstruction Beam-induced motion correction for all movies was performed using RELION’s own implementation of the UCSF motioncor2 program 29 . Contrast Transfer Function (CTF) parameters were estimated by Gctf 38 . All further processing steps were carried out using RELION 4.0 29 . Fibrils were auto-picked with a binarization threshold of –6 using a modified version of the Topaz module 28 . Segments were extracted using a box size of 384 or 512 pixels with an inter-box distance of 14.2 Å. The segments were then downscaled to 96 or 128 pixels, resulting in a downscaled pixel size of 2.904 Å. Segments were subjected to iterative rounds of reference-free two-dimensional (2D) classification using regularization parameter T = 3, and 100 classes to remove junk molecules. Final high-quality classes contained a total of 135,574 segments. After one final round of 2D classification, 15 images were selected to generate an initial model using the relion_helix_inimodel2d program 28 by optimizing crossover distances. The best initial model was used as a reference for three-dimensional (3D) classification of re-extracted segments with a box size of 280 pixels. The first round of 3D classification was performed using four classes and a regularization parameter of T = 40, resulting in 12,633 segments that contributed to a high-resolution reconstruction. Two more rounds of 3D classification were performed on these segments, using a single class and increasing T values (40 and 80), with local optimization of the helical twist and rise parameters. In the final round of 3D classification, β-strands were well separated, and large side chains could be resolved. The model and data were then used for high-resolution gold-standard 3D refinement. Iterative Bayesian polishing and CTF refinement were performed on these segments to improve the resolution further. The final ATTR fibril map converged with a helical rise of 4.78 Å and a helical twist of −1.36°. The final map refined to a resolution of 2.82 Å, using standard post-processing by applying a soft mask consisting of 30% of the box size. Model building and refinement The cryo-EM structure of ATTRv-V30M (PDB: 6SDZ 14 ) was used as an initial reference for model building. The initial model was fitted into the cryo-EM map using Chimera X 34 , followed by manual and iterative building in Coot 39 , before real-space refined using PHENIX 40 . Model validation was performed with MolProbity 41 . The FSC curve between the cryo-EM map and the atomic coordinates was calculated using Mtriage 35 . Structural figures were generated in Chimera X. All relevant statistics are summarized in Extended Data Table 2 . Liquid chromatography – tandem mass spectrometry (LC-MS/MS) Protein extraction and enzymatic digestion: 300 ng or 30 ng of extracted fibrils, either from the ankle or the thigh, were dissolved in 50 µL of 8 M urea and 50 mM ammonium bicarbonate (ABC) by sonication at 4 °C in a water bath (Bioruptor, Diagenode; 15 cycles of 30 seconds on and 30 seconds off in high mode). Proteins were reduced with 10 mM dithiothreitol for 20 minutes at room temperature, followed by alkylation with 50 mM iodoacetamide for 30 minutes at room temperature. Protein digestion was performed by adding LysC (Wako Fujifilm, 1:100 w/w) for two hours at room temperature, after which the digestion buffer was diluted to 2 M urea and 50 mM ABC for overnight digestion with trypsin at room temperature (Promega, 1:100 w/w). Digestion was stopped using a solution of 2% acetonitrile (ACN) and 0.3% trifluoroacetic acid (TFA) and samples were cleared after by centrifugation at maximum speed for 5 minutes. Digested peptides were purified using C18 StageTips 42 and eluted with 80% ACN, 0.5% acetic acid. The elution buffer was removed by vacuum centrifugation and purified peptides were resuspended in 2% ACN, 0.5% acetic acid, 0.1% TFA for single-shot LC-MS/MS measurements. For bulk skin analysis, the skin material from the ankle was first thawed on ice before washed once with PBS buffer to remove any remaining blood. The tissue was then manually homogenized in 250 µL of 4% SDS (100 mM TRIS, pH 7.6), prior to sonication in a water bath at 4 °C (Bioruptor, Diagenode; 15 cycles of 30 seconds on and 30 seconds off in high mode). The tissue was incubated for 10 min at 95 °C (Thermomixer, Eppendorf, 800 rpm). The lysate was alkylated with 50 mM iodoacetamide for 30 minutes at room temperature. After clearing the lysate by centrifugation (5 min, 4 °C, 13,000 rpm), proteins were precipitated overnight at –20°C in 80% acetone. Proteins were pelleted by centrifugation (20 min, 4 °C, 13,000 rpm), washed with 80% acetone, and dried at 40 °C. The protein pellet was then resuspended in 8 M urea and 50 mM ABC by sonication in a water bath. Protein digestion and peptide purification were performed as described for the extracted fibrils. LC-MS/MS analysis Peptides were separated on a nanoElute2 HPLC system (Bruker) coupled to a timsTOF HT mass spectrometer (Bruker) via a nanoelectrospray source (Captive spray source, Bruker). 500 ng of bulk skin peptides or the entire purified fibril digests were resuspended in a mixture of water and 0.1% formic acid and loaded onto an in-house column (inner diameter: 75 µm; length: 25 cm) packed with ReproSil-Pur C18-AQ 1.9 µm resin (Dr. Maisch HPLC GmbH). The column was kept at 50 °C in a column oven, and the samples were eluted over ninety (bulk skin) or sixty minutes (fibrils) using a linear gradient between 2 and 35% ACN/0.1% formic acid at a flow rate of 300 nL/min. The mass spectrometer was operated in a data-dependent (DDA)-PASEF mode with 10 PASEF ramps. For the bulk biopsy analysis, the accumulation and ramp times were set to 100 ms, covering a 100-1700 m/z range and a 0.70-1.50 Vs/cm 2 mobility range, for an estimated cycle time of 1.17 seconds. The signal intensity threshold was set to 1,200 and precursors were actively excluded for 0.4 minutes after reaching a target intensity of 14,500. For fibril analysis, the accumulation and ramp times were set at 166 ms, covering a 100-1700 m/z range and a 0.60-1.60 Vs/cm 2 mobility range, for an estimated cycle time of 1.89 seconds. The signal intensity threshold was set to 1,000 and precursors were actively excluded for 0.4 minutes after reaching a target intensity of 20,000. High-sensitivity detection for low sample amounts was activated. For both analyses, the collision energy was ramped up linearly from 20 eV at 0.60 Vs/cm 2 to 59 eV at 1.60 Vs/cm 2 . Bruker’s default active precursor region filter was used to select precursors with charge up to 5 for fragmentation. LC-MS/MS Data analysis Raw data were analysed using FragPipe 21.1 43,44 . MSFragger 4 was used to search MS/MS spectra against the Human UNIPROT database (June 2019), with an additional entry for the F64S-mutated TTR sequence. Cleavage type was set to “enzymatic” with the “stricttrypsin” cleavage rule. The minimum peptide length was set to seven amino acids, the maximum to 50, and the mass range between 500 and 5,000 Da. For the regular search, N-terminal protein acetylation and methionine oxidation were set as variable modifications and cysteine carbamidomethylation as a fixed modification. Precursor and fragment mass tolerance were set to 20 ppm, and a false discovery rate (FDR) of 1% was required at the PSM, ion, peptide, and protein levels. PSMs were rescored with MSBooster using deep learning prediction and validated with Percolator using a minimum probability threshold of 0.5. Label-free quantification was carried out with IonQuant 1.10.12. The same parameters as described before were used for the semispecific N-terminal search, except the cleavage type was changed to “semi_N_term”. Four separate searches were performed for the analysis of post-translational modifications by adding the following variable modifications: 1) serine/threonine/tyrosine phosphorylation; 2) lysine/arginine monomethylation and dimethylation, and lysine trimethylation; 3) lysine acetylation; 4) lysine ubiquitination. PTM site localization was enabled by running PTM-Prophet with a minimum probability threshold of 0.5. Results were further filtered based on the localization probability of each PTM and the spectral similarity to predicted spectra, which were set at a minimum of 0.8. Ion chromatogram extraction and peak area quantification were performed using Skyline 23.1.0.455. Author contributions J.Y. and X.Z. extracted fibrils from skin tissue, prepared cryo-EM samples, and determined the cryo-EM structure. P.M. carried out mass spectrometry analysis. E.V. and S.P. were responsible for processing the skin biopsies and performing the immunohistochemical analysis. G.M. was responsible for the conceptualization of the project. G.M. and A.B. directed the project. J.Y., M.P., G.M. and A.B. wrote the paper with contributions from all other authors. Data availability Structural data have been deposited to the worldwide Protein Data Bank (wwPDB) and the Electron Microscopy Data Bank (EMDB) with the accession codes: 9HYW and EMD-52519, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 45 partner repository with the dataset identifier PXD065685. Declaration of Interests The authors declare no competing interests. View this table: View inline View popup Download powerpoint Extended Data Table 1 Post-translational modifications analysis of ATTRv-F64S fibrils. Sites are filtered for both localization probability of the modification site and spectral similarity to predicted MS/MS spectra above 80%. The PTM sites are highlighted in red, while the F64S mutation is shown in green. Reported with * are two sites just below the spectral similarity threshold that was applied. View this table: View inline View popup Download powerpoint Extended Data Table 2 Cryo-EM data collection, refinement, and validation statistics. View this table: View inline View popup Download powerpoint Extended Data Table 3 Cryo-EM structures of ATTR amyloid fibrils (wild type and variants). Acknowledgments We would like to express our deep gratitude to the patient providing the biological material needed to conduct this research study. We thank A. Reynaud and Y. Pfister for technical assistance; All group members from the Melli and Boland groups for their input and discussion; The computing department at the University of Geneva for providing the infrastructure to perform cryo-EM analysis; N. Roggli for maintaining computing in the Molecular and Cellular Biology department; C. Bauer, A. Howe, and S. Barrass at the DCI-Geneva (aka as cryoGEnic) and E. Uchikawa, B. Beckert, S. Nazarov, and A. Myasnikov from DCI-Lausanne – all for their excellent support in EM data collection and analysis. K. Muir and R. Loewith for critical reading of the manuscript. We are grateful for the for the generous funding support of our research projects from the Swiss National Science Foundation (SNSF) (TMSGI3_211581), the Fondation Roger de Spoelberch, the Fondation pour la recherche en biologie et médecine, and an AFRI-EOC Research Support Grant 2023–2024. 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Share Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient Jun Yu , Xuefeng Zhang , Sandra Pinton , Elena Vacchi , Andrea Cavalli , Matteo Pecoraro , Giorgia Melli , Andreas Boland bioRxiv 2025.07.29.667442; doi: https://doi.org/10.1101/2025.07.29.667442 Share This Article: Copy Citation Tools Structure of ATTRv-F64S fibrils isolated from skin tissue of a living patient Jun Yu , Xuefeng Zhang , Sandra Pinton , Elena Vacchi , Andrea Cavalli , Matteo Pecoraro , Giorgia Melli , Andreas Boland bioRxiv 2025.07.29.667442; doi: https://doi.org/10.1101/2025.07.29.667442 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 (41929) Biophysics (21446) Cancer Biology (18586) Cell Biology (25492) Clinical Trials (138) Developmental Biology (13374) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22497) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88584) Paleontology (666) Pathology (2831) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)