Cryo-EM structures of filaments from the brains of individuals with variants G51D and H50Q in α-synuclein

Cryo-EM structures of filaments from the brains of individuals with variants G51D and H50Q in α-synuclein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cryo-EM structures of filaments from the brains of individuals with variants G51D and H50Q in α-synuclein Yang Yang, Alexey Murzin, Tiara Hinton, Sew Peak-Chew, Catarina Franco, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6490169/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Gene dosage and point mutations in SNCA , the a-synuclein gene, give rise to familial forms of Parkinson’s disease and dementia with Lewy bodies; an insertion mutation in SNCA causes juvenile-onset synucleinopathy. We previously reported the electron cryo-microscopy (cryo-EM) structures of a-synuclein filaments from the brains of individuals with Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, as well as from the brain of an individual with juvenile-onset synucleinopathy. Here we report the cryo-EM structures of a-synuclein filaments from the frontal cortex of two cases with Parkinsonism and mutation G51D in a-synuclein and those from the amygdala of a case with Parkinson’s disease and variant H50Q in a-synuclein. The G51D filaments of assembled a-synuclein consist of two identical protofilaments with the Lewy fold and island B, but without the previously identified disconnected density island A. The protofilament interface is made of residues E46, V48 and H50. Filaments with the H50Q variant comprise a single protofilament with the Lewy fold and both islands A and B. Unlike G51D, the pathogenicity of H50Q has been questioned. It remains to be seen if dimerisation of the Lewy fold may also underlie the pathogenicity of other missense mutations in a-synuclein. Moreover, filaments with a single Lewy fold have a right-handed helical twist, while the G51D, multiple system atrophy and juvenile-onset synucleinopathy filaments are left-handed, which may also be significant. Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/Protein folding/Protein aggregation Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Parkinson's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are the major synucleinopathies [ 1 ]. They share the presence of abundant filamentous inclusions that are made of a-synuclein. PD and DLB have in common the deposition of filamentous a-synuclein in Lewy bodies and Lewy neurites. By electron cryo-microscopy (cryo-EM), they share the Lewy fold, suggesting that they are mechanistically related [ 2 ]. The Lewy fold is made of a single protofilament that extends from residues 31–100 of a-synuclein, which are arranged as nine b-strands in a three-layered structure. Two regions of cryo-EM densities of unknown composition that are not connected to a-synuclein (islands A and B) are also present. MSA is associated with abundant filamentous a-synuclein inclusions in both nerve cells and glial cells, mainly oligodendrocytes (glial cytoplasmic inclusions [GCIs] or Papp-Lantos bodies [ 3 ]). By cryo-EM, two types of filaments are present that are each made of two different protofilaments [ 4 ]. Their proportions vary between brain regions and individuals. The cryo-EM structure of a-synuclein filaments from the brain of an individual with juvenile-onset synucleinopathy (JOS) uncovered a third fold of assembled a-synuclein in the human brain [ 5 ]. JOS is caused by a heterozygous mutation in SNCA , the a-synuclein gene, which gives rise to the insertion of seven amino acids after residue 22 in the amino-terminal region of a-synuclein. The JOS fold differs from the Lewy fold, but it resembles a substructure that is common to the MSA protofilament folds. We observed the presence of filaments made of either a single or two identical protofilaments with the JOS fold. Additional densities for unidentified non-proteinaceous cofactors are also present in a-synuclein filaments from human brains [ 2 , 4 , 5 ]. For MSA filaments, the extra densities are located at the protofilament interfaces. Dimeric a-synuclein protofilaments of JOS have an extra density at the protofilament interface. All a-synuclein filaments from human brains have a fuzzy coat that encompasses the disordered protein’s amino- and carboxy-terminal regions. Several dominantly inherited point mutations in SNCA have been identified in families with typical and atypical forms of PD [ 6 – 17 ]. They give rise to missense mutations in a-synuclein. In addition, gene dosage mutations (duplications and triplications) of one allele of SNCA have been shown to give rise to PD [ 18 – 20 ]. Some of these mutations also cause DLB. No SNCA mutations have been linked with MSA. The existence of point mutations and multiplications of SNCA that cause diseases with abundant a-synuclein filaments supports the notion that a-synuclein is a key player in all cases of PD and DLB. Moreover, genome-wide association studies have shown that single nucleotide polymorphisms in SNCA are associated with elevated levels of a-synuclein, which increases the risk of PD [ 21 – 23 ]. Mutation G51D in a-synuclein has been described in four different cohorts with Parkinsonism [ 11 , 12 , 14 , 15 ]. It is of particular interest, because of the rapid progression of disease and because some clinical and neuropathological characteristics overlap with those of MSA. Affected family members develop a variety of parkinsonian features with a variable levodopa response, dementia, visual hallucinations and autonomic dysfunction. Neuropathological features include CA2-CA3 hippocampal and cortical nerve cell loss, with abundant neuronal a-synuclein inclusions, together with a smaller number of GCI-like inclusions. Combined PD and MSA profiles were also reported in a family with mutation A53E in SNCA [ 13 ]. Studies using recombinant G51D a-synuclein have shown an equal or lowered aggregation propensity relative to the wild-type protein [ 11 , 24 – 26 ], impaired membrane binding ability [ 27 , 28 ] and increased secretion [ 13 ]. Variant H50Q in a-synuclein has been reported in some cases of PD [ 9 , 10 ]. Recombinant H50Q a-synuclein increases the rate of a-synuclein aggregation, secretion and toxicity [ 24 , 29 – 31 ]. However, unlike cases with the G51D mutation, the family history of cases with variant H50Q is inconsistent. Moreover, H50Q a-synuclein has also been found in population databases. It was not enriched in PD cases, arguing against the possibility of reduced penetrance and questioning its pathogenicity [ 32 ]. The structures of a-synuclein filaments from the brains of individuals with SNCA mutations that encode missense variants have not been determined. Here we report the cryo-EM structures of a-synuclein filaments extracted from the frontal cortex of two previously reported individuals with PD and mutation G51D [ 15 ] and from the amygdala of a previously unreported case of PD with variant H50Q. Results Structure of a-synuclein filaments from two cases with SNCA mutation G51D The heterozygous mutation in SNCA that encodes G51D a-synuclein [ 15 ] was confirmed for cases 1 and 2 using whole-genome sequencing. There was widespread nerve cell loss, with abundant neuronal and neuritic a-synuclein pathology. This included the neocortex, in addition to the striatum, the limbic system and the brainstem. The neuronal inclusions were annular, crescentic, globular, diffuse and neurofibrillary tangle-like. In both cases, sparse GCI-like oligodendroglial inclusions were also present in the white matter. Filaments of a-synuclein from the frontal cortex of cases 1 and 2 yielded identical structures, with resolutions of 2.3 Å for case 1 and 3.4 Å for case 2. G51D filaments comprise two identical protofilaments with the Lewy fold that are related by a 2-start (or pseudo-2 1 ) helical symmetry (Fig. 1 ; Extended Data Fig. 1 ). The protofilament interface is made of the same three residues from each protofilament: E46, V48 and H50. Like the wild-type protofilament, the G51D protofilaments contain a non-proteinaceous cofactor density in an equivalent location, but they lack island A, one of the two disconnected peptide-like densities, thus exposing the mutation site to solvent. As the densities of negatively charged carboxyl groups are generally missing from cryo-EM reconstructions, the presence of the D51 side chain is not apparent from its local density alone. There is a short stump of density, presumably corresponding to a Cb atom, that is smaller than the side chain density of the nearby A53, or of D98, the only other aspartic acid residue. This is consistent with both G51 and D51 being present in the filaments, as confirmed by mass spectrometry of the sarkosyl-insoluble fractions (Extended Data Fig. 2 ). However, the presence of D51 manifests itself through subtle differences between wild-type and G51D variants of the Lewy fold. Whereas the backbone conformations of both Lewy fold variants are nearly identical when viewed along the filament axes, they are different when viewed perpendicularly. Compared to the wild-type variant, the C-terminal part of the G51D protofilament fold is rotated relative to its N-terminal part by about 8°, with G73 in the middle serving as a hinge (Fig. 2 ). Concomitant with this rotation, the side chain conformations of residues that form salt bridges (E35-K80, E61-K96, K60-D98) between the N- and C-terminal parts are readjusted. This difference in relative orientations of the N- and C-terminal parts can also explain the different handedness of the wild-type and the G51D filaments. G51D filaments are left-handed (Extended Data Fig. 1 c), in contrast to the right-handed helical twist of wild-type filaments [ 2 ]. Other differences between the two Lewy fold variants include side chain rotamers of residues L38, Y39 and Q62 (Fig. 2 a). The rotamer of L38 compensates for the rotation of the C-terminal part in the G51D variant, maintaining the contact with residues A76 and A78 from two adjacent molecules. In wild-type filaments, the hydroxyl group of Y39 hydrogen bonds to the e-amino group of K34 that contributes to coordination of the cofactor density, whereas in G51D filaments the hydroxyl group of Y39 makes a hydrogen bond to the main chain of S42 on the opposite side of the density. In wild-type filaments, the side chain of Q62 has a folded conformation, being hydrogen bonded to the side chain of T64 in the same b-sheet. In G51D filaments, the side chain of Q62 adopts an extended conformation and makes a hydrogen bond to the main chain of V55 in the opposite b-sheet. In both wild-type and G51D filaments, the side chains of Q62 also form hydrogen-bonded ladders along the filament axes. The above differences, including the loss of island A and the formation of dimeric filaments, can be explained by the effects of the G51D mutation on the Lewy fold. The bulk of the side chain of D51 may introduce steric clashes with the residues of island A, whereas its charge probably facilitates dimerisation of the Lewy fold through electrostatic interactions. The side chain of D51 from one protofilament is linked to the cofactor-binding site of the other protofilament through a network of salt bridges, including H50 from its own protofilament and both K45 and E46 from the other protofilament; this may affect the position of the cofactor and that of the coordinating residues (Fig. 1 c,d). The loss of island A and a slight shift of the cofactor position promote minor conformational rearrangements in the filament core. Structure of a-synuclein filaments from a case with SNCA variant H50Q A heterozygous variant in SNCA that encodes H50Q a-synuclein was identified in case 3 using the Neurobooster array and confirmed by whole-genome sequencing. This case has not been described before. The clinical phenotype was consistent with idiopathic PD, with slow disease progression, a sustained levodopa response and no significant dysautonomia, pyramidal signs or dementia. Neuropathology also closely resembled that of idiopathic PD, with abundant Lewy bodies and Lewy neurites that were Gallyas-Braak silver-negative (Extended Data Fig. 3 a-f). By contrast, the neuronal inclusions from case 2 of G51D were Gallyas-Braak silver-positive (Extended Data Fig. 3 g). GCI-like inclusions were absent from the H50Q case. Small numbers of amyloid-b plaques and tau tangles were also present. By mass spectrometry, Q50 a-synuclein was detected in the sarkosyl-insoluble fraction, where it coexisted with H50 a-synuclein, indicating the presence of mixed filaments (Extended Data Fig. 4 ). Filaments of a-synuclein extracted from the amygdala of an individual with the H50Q variant in SNCA comprised a single protofilament (Fig. 3 ; Extended Data Fig. 1 ). The structure, which was determined at a resolution of 2.9 Å, was almost identical to the wild-type Lewy fold, including the same additional densities of the cofactor and peptide islands A and B (Fig. 3 b,c). Like the wild-type a-synuclein filaments [ 2 ], the H50Q filaments exhibited a right-handed helical twist (Extended Data Fig. 1 d). The only notable difference between both variants is the side chain density at the mutation site (Fig. 3 d). In the H50Q variant, this density can accommodate either the side chains of histidine or glutamine, but in a different conformation than that previously assigned to H50 in the wild-type structure and now also found in the G51D variant (Fig. 4 a-c). In the wild-type map, there is a previously unassigned density in an equivalent location between the side chains of V48 and H50, which may be the result of an alternative conformation of H50. Thus, the only apparent effect of the H50Q variant on the Lewy fold is that it selects between the two alternative side chain conformations at position 50 (H50 or Q50). In the H50Q variant, the side chain is oriented parallel to the direction of island A, which is probably not compatible with the formation of a G51D-like dimeric interface due to steric interference from E46 (Fig. 3 e). Analysis of a-synuclein filaments from the frontal cortex of the H50Q case suggests a filament structure consistent with that of filaments from the amygdala, as judged by 2D class averaging (Extended Data Fig. 5 ). Twisted and untwisted segments in H50Q and G51D a-synuclein filaments We previously reported that a-synuclein filaments extracted from the brains of cases of idiopathic PD and DLB are made of twisted and untwisted segments, with the twisted segments suitable for helical reconstruction being in a minority, but the majority untwisted segments have 2D class averages that resemble the projections of untwisted filament models with the Lewy fold [ 2 ]. H50Q filaments had the same proportion of twisted (25%) and untwisted (75%) segments as wild-type a-synuclein assemblies, with the former having a right-handed helical twist and cross-over distances of 950 Å (from class 2D segments), like what we observed in the wild-type Lewy fold. By contrast, in the dimeric G51D filaments, the proportion of untwisted segments was smaller (34%) and the twisted segments had cross-over distances of 5,500 Å (from class 2D segments) and a left-handed helical twist (Extended Data Fig. 5 ). For both H50Q and G51D filaments, the untwisted 2D class averages resembled 2D class averages of the twisted segments that were used for 3D reconstruction. It could therefore be that the helical twist is relevant for disease progression. Peptide islands in the Lewy fold and other a-synuclein filament structures The structures of G51D and H50Q filaments provided no novel clues as to what protein sequences islands A and B are made of. However, some hypotheses can be drawn from other structures. Isolated peptide-like densities are present in the JOS fold of a-synuclein filaments from the human brain [ 5 ] and in a set of in vitro assemblies, known as polymorph 2 [ 33 ]. We previously noted that the Lewy fold segment 83–92 and island B form a similar substructure than that in polymorph 2, which is made of the equivalent segment and peptide island B (Extended Data Fig. 6e in [ 2 ]). Lewy and JOS folds also share a substructure that is made of the core segment 48–59 and peptide island A (Fig. 4 d,e). In both polymorph 2 and JOS folds, island A was assigned to residues G14-G25 of a-synuclein, with V15, A17 and A19 facing the filament cores. Even though the sequence G14-G25 is compatible with islands A and B in the Lewy fold (Fig. 4 d-h), it cannot be assigned to either, because the intervening sequence is of insufficient length to connect the islands to the N-terminus of the ordered core at G31. However, a more N-terminal segment, containing residues G7, S9 and A11, which can also be modelled into the densities of islands A and B, can be connected to G31 through flexible linkers (Extended Data Fig. 6). There are no compatible segments in the C-terminal sequence outside the Lewy fold core (residues 101–140). This raises the possibility of N-terminal sequences of a-synuclein contributing to both islands A and B in the Lewy fold. Since a single protein chain cannot contribute to both islands, these islands would need to incorporate other peptides, like proteolytic fragments of a-synuclein and/or other proteins. Alternatively, they could consist of intact proteins with most of their chains being disordered. It has been reported that Synapsin III is an integral component of a-synuclein filaments from the brains of individuals with PD [ 34 ]. Discussion Here we report the structures of a-synuclein filaments extracted from the frontal cortex of two cases with missense mutation G51D in a-synuclein and the amygdala of one case with variant H50Q. Both G51D and H50Q filaments adopted essentially the same protofilament fold as the previously described Lewy fold of idiopathic PD and DLB [ 2 ]. However, they differed in the number of protofilaments: two protofilaments for G51D filaments and one protofilament for H50Q filaments. They also differed in protofilament handedness (left for G51D and right for H50Q). The expectation from previous in vitro studies has been that human brain a-synuclein filaments with these sequence variants and other pathogenic mutations would adopt folds different from those of assembled wild-type a-synuclein [ 28 , 35 – 39 ]. However, this is not the case. The Lewy fold from the human brain differs from all known folds of a-synuclein filaments assembled or seeded in vitro , including those made of mutant and post-translationally modified proteins. It follows that the reported effects of recombinant a-synuclein with missense mutations on in vitro filament assembly may not be relevant for understanding the effects of those mutations on filament assembly in the human brain. The pathological inclusions found in rodent models that express G51D a-synuclein remain to be characterised at the ultrastructural level [ 40 – 42 ]. The Lewy fold also differs from the MSA [ 4 , 43 , 44 ] and JOS [ 5 ] folds of a-synuclein filaments from the human brain (Fig. 5 ). Other PD-associated missense mutations map to either the exterior of the Lewy fold near the cofactor-binding site (A30G, A30P, E46K) or the interface with island A (A53E, A53T, A53V). In the light of our findings, it appears likely that the filaments of a-synuclein from PD cases with these mutations will also be made of variants of the Lewy fold, with the mutations at position 53 possibly leading to the displacement of island A, alteration of filament twist and protofilament dimerisation, as is the case for G51D filaments. Island A will prevent protofilament dimerisation of similar variants through the interface used by G51D filaments, because of steric clashes with the opposite protofilament. If some a-synuclein protein chains contributed to both the filament core and island A, with their linker going over the cofactor binding site and the G51D dimerisation interface, it would enforce the incompatibility of the presence of island A and the dimerisation of Lewy fold protofilaments. It would also provide a rationale for the effects of mutations A30G, A30P and E46K that may affect linker interactions. The a-synuclein filaments with variants K58N [ 45 ], T72M [ 46 ] and E83Q [ 47 ] are also compatible with the Lewy fold; K58N and E83Q map to the exterior of the Lewy fold and T72M maps to a large cavity inside that fold. Similarly, the a-synuclein variants G14R [ 48 ] and V15A [ 49 ] are compatible with the Lewy fold; their location is in the potential linker between island A and the core of the Lewy fold. Nevertheless, all known missense variants in a-synuclein are found in the region comprising the seven imperfect repeats (residues 7–87) that are lipid-binding domains [ 50 , 51 ], suggesting that those mutations may also influence the lipid-binding properties of a-synuclein. Mutation G51D in a-synuclein has been reported to result in clinical and neuropathological characteristics reminiscent of both PD and MSA [ 12 , 15 ]. Like the glial cytoplasmic and neuronal inclusions of MSA, and unlike the neuronal inclusions of PD and DLB [ 52 ], the neuronal inclusions from case 2 of G51D were Gallyas-Braak silver-positive; those from the H50Q case were silver-negative. Even though it is not known what underlies Gallyas-Braak silver positivity, these findings show that a-synuclein inclusions with distinct filament structures give rise to different staining patterns. This is also supported by propagation studies in M83 transgenic mice that used G51D a-synuclein seeds prepared from the temporal cortex of cases 1 and 2 and concluded that the a-synuclein conformer specified by mutation G51D resembles more closely a PD-associated than an MSA-associated strain [ 53 ]. Both mutant and wild-type a-synucleins are found in dimeric G51D filaments. A seed may form from the mutant protein, with both wild-type and mutant proteins being incorporated subsequently. In conclusion, we show that mutation G51D in SNCA leads to the formation of left-handed filaments that are made of two identical protofilaments with the Lewy fold, in the absence of island A. By contrast, variant H50Q gives rise to right-handed filaments with a single protofilament with the Lewy fold. Dimerisation of a-synuclein protofilaments with the Lewy fold, or the formation of left-handed filaments, may be a general characteristic of filaments from cases with disease-causing missense mutations in SNCA. Materials And Methods Ethics Brain tissues from cases 1-3 were donated to the Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology, with informed consent and the study was approved by the National Health Services Health Research Authority Ethics Committee, London-Central (reference no. 23/LO/0044). The cryo-EM study was approved by the Cambridgeshire Research Ethics Committee (09/HO308/163). Clinical history We determined the cryo-EM structures of a-synuclein filaments extracted from the frontal cortex of two previously described individuals with mutation G51D in a-synuclein and a family history of atypical PD [15]. Case 1 (family two, patient III), presented with resting tremor of the right hand, anxiety and depression at age 69. A diagnosis of PD was made. This female individual died aged 75. Case 2 (family 2, patient IV) was the son of case 1. At age 46, he presented with resting tremor of the right hand and a depressed mood; a diagnosis of PD was made. Death occurred at age 52. We also determined the cryo-EM structures of a-synuclein filaments extracted from the amygdala of a novel case of PD with variant H50Q in a-synuclein without a family history. This female patient (referred to as case 3) developed resting tremor in her right hand, dragging of her right foot and micrography at age 44. Neurological examination three years later revealed hypomimia, bradykinesia, as well as upper and lower limb cogwheel rigidity. The patient was diagnosed with PD. The symptoms responded well to levodopa and the patient subsequently developed motor fluctuations. Non-motor symptoms included constipation, urinary urgency and hesitancy, anxiety, depression and insomnia. The patient experienced freezing of gait by age 55 and recurrent falls by age 56. By age 67, she required a wheelchair for mobility, followed by dysphagia at age 73 and death at age 78. Genetics DNA was extracted from the frontal cortex of cases 1 and 2 and the amygdala of case 3; it was sequenced using Illumina short-read whole genome sequencing, with a mean coverage of 30x. Paired-end reads of 150 bp were aligned to the human reference genome (GRCh38 build) using the functional equivalence pipeline [54]. Sample processing and variant calling were performed using DeepVariant v.1.6.1 [55] and joint genotyping was performed using GLnexus v.1.4.3 with the preset DeepVariant WGS configuration [56]. Quality control was performed according to the quality metrics defined by the Accelerating Medicines Partnership Parkinson’s disease program (AMP-PD; https://amp-pd.org), and variant annotation was performed with Ensembl Variant Effect Predictor [57,58]. DNA from each sample was also genotyped with the Illumina Infinium Global Diversity Single Nucleotide Polymorphism Array with custom content for neurodegenerative diseases (Neurobooster) [59]. Neuropathology Pathological examination of the human brains was performed following standard Queen Square Brain Bank protocols. For histological analysis, 8 mm thick sections were cut from formalin-fixed paraffin-embedded tissue blocks sampled from representative brain regions. Immunohistochemical staining for a-synuclein (MA1-90342, Thermo Scientific, 1:500), amyloid-b (M0872, Dako, Abnova 1:500) and phosphorylated tau (MN1020, Thermo Scientific, 1:600) was performed on a Menarini automated staining platform following the manufacturers’ guidelines. Biotinylated secondary antibodies were used with horseradish peroxidase-conjugated streptavidin complex and diaminobenzidine as the chromogen. To visualise inclusions, sections from insular cortex were also silver-impregnated according to Gallyas-Braak [60,61]. All sections were counterstained with haematoxylin. Filament extraction from the human brain For cryo-EM analysis, sarkosyl-insoluble material was extracted from the frontal cortex (cases of G51D and H50Q) and the amygdala (case of H50Q), essentially as described [62]. Briefly, tissues were homogenised in 20 vol (w/v) extraction buffer consisting of 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 10-20% sucrose and 1 mM EGTA. Homogenates were brought to 2% sarkosyl and incubated for 60 min at 37° C. Following a 10 min centrifugation at 10,000 g, the supernatants were spun at 100,000 g for 60 min. The final pellets were resuspended in 100 ml/g of 20 mM Tris-HCl, pH 7.4, 50 mM NaCl. Mass spectrometry Mass spectrometry was performed as described [63]. Sarkosyl-insoluble pellets were resuspended in 200 ml hexafluoroisopropanol. Following a 3 min sonication at 50% amplitude (QSonica), they were incubated at 37° C for 2h and centrifuged at 3,000 g for 5 min; the supernatants were transferred to new tubes before being dried by vacuum centrifugation. They were resuspended in 4M urea and 50 mM ammonium bicarbonate (ambic), before being reduced with 5 mM dithiothreitol at 37° C for 30 min and alkylated in the dark at room temperature for 30 min with 10 mM iodoacetamide. LysC (Promega) was then added to the samples for 2h at 25°C. They were diluted to 1.5 M urea with 50 mM ambic and incubated with trypsin (Promega) at 30°C overnight. Trypsin digestion was stopped by the addition of formic acid to 0.5%, followed by centrifugation at 16,000 g for 5 min. The supernatants were desalted using C18 stage tips (3M Empore) packed with Poros oligo R3 (Thermo Scientific) resin that were produced in-house. Bound peptides were eluted stepwise with 30, 50 and 80% acetonitrile in 0.5% formic acid and partially dried in a SpeedVac (Savant). The peptide mixtures were analysed by LC-MS/MS using a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer, as described [64]. Cryo-EM Holey carbon grids (Quantifoil AuR1.2/1.3, 300 mesh) were glow-discharged with an Edwards (S150B) sputter coater at 30 mA for 30 s. Three ml aliquots were applied to the grids and blotted for 3-5 s with filter paper at 100% humidity and 4° C using a Thermo Fisher Vitrobot Mark IV. Datasets of the G51D case 1 and the H50Q case were acquired on a Thermo Fisher G4 microscope, equipped with a Falcon-4i detector and a Selectris-X energy filter (Thermo Fisher Scientific) with a slit width of 10 eV to remove inelastically scattered electrons. The G51D case 2 dataset was collected on a Titan Krios G3 microscope with a Gatan K3 detector in superresolution counting mode, using a Bioquantum energy filter with a slid width of 20 eV. Images were recorded with a total dose of 40 electrons per Å 2 . Data processing All super-resolution frames were gain-corrected, binned by a factor of 2, aligned, dose-weighted and then summed into a single micrograph using RELION’s own implementation of MotionCor2 [65]. Contrast transfer function (CTF) parameters were estimated using CTFFIND-4.1 [66]. Subsequent image-processing steps were performed using helical reconstruction methods in RELION [67,68]. Filaments were picked manually. Reference-free 2D classification was performed to identify homogeneous segments for further processing. Initial 3D reference models were reconstructed de novo from 2D class averages [69] using an estimated rise of 4.75 Å and helical twists according to the observed cross-over distances of filaments in the micrographs. To increase the resolution of the reconstructions, Bayesian polishing and CTF refinement were performed [70]. Blush regularisation was applied to improve the quality of the reconstructions [71]. Final 3D reconstructions, after auto-refinement, were sharpened using the standard post-processing procedures in RELION, and resolutions calculated from Fourier shell correlations at 0.143 between the two independently refined half-maps, using phase-randomisation to correct for convolution effects of a generous, soft-edged solvent mask. Further details of the data acquisition and processing are given in Supplementary Table 1. Model building and refinement Atomic models were built in Coot [72] using shared substructures of the Lewy fold (PDB: 8A9L) as template. Coordinate refinements were performed using Servalcat [73]. Final models were obtained using refinement of only the asymmetric unit against the half-maps in Servalcat . Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. References 54. Regier, A.A. et al. Functional equivalence of genome sequencing analysis pipelines enables harmonized variant calling across human Genetics projects. Nature Commun. 9, 4038 (2018). 55. Poplin, R. et al. A universal SNP and small-indel variant caller using deep neural networks. Nature Biotechnol. 36, 983-987 (2018). 56. Yun, T. et al. Accurate, scalable cohort variant calls using DeepVariant and GLnexus. Bioinformatics 36, 5582-5589 (2021). 57. Iwaki, H. et al. Accelerating Medicines Partnership: Parkinson’s disease. Genetic Resource. Mov. Disord. 36, 1795-1804 (2021). 58. McLaren, W. et al. The Ensembl Variant Effect Predictor. Genome Biol. 17, 122 (2016). 59. Bandres-Ciga, S. et al. NeuroBooster array: A genome-wide genotyping platform to study neurological disorders across diverse populations. Mov. Disord. 39, 2039-2048 (2024). 60. Braak, H. et al. Silver impregnation of Alzheimer’s neurofibrillary changes counterstained for basophilic material and lipofuscin pigment. Stain Technol. 63, 197-200 (1988). 61. Gallyas, F. Silver staining of Alzheimer’s neurofibrillary changes by means of physical development. Acta Morphol. Acad. Sci. Hung. 19, 1-8 (1971). 62. Tarutani, A. et al. Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods. Acta Neuropathol. Commun. 6, 29 (2018). 63. Näslund, J. et al. Relative abundance of Alzheimer Aβ amyloid peptide variants in Alzheimer disease and normal aging. Proc. Natl. Acad. Sci. U.S.A. 91, 8378-8382 (1994). 64. Yang, Y. et al. Cryo-EM structures of amyloid-β filaments from human brains. Science 357, 167-172 (2022). 65. Zheng, S.Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nature Meth. 14, 331-332 (2017). 66. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216-221 (2015). 67. He, S. & Scheres, S.H.W. Helical reconstruction in RELION. J. Struct. Biol. 193, 163-176 (2017). 68. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, 42166 (2018). 69. Scheres, S.H.W. Amyloid structure determination in RELION 3.1. Acta Crystallogr. D 76, 94-101 (2020). 70. Zivanov, J., Nakane, T. & Scheres, S.H.W. A Bayesian approach to beam-induced motion correction in cryo-EM single particle analysis. IUCrJ 6, 5-17 (2019). 71. Kimanius, D. et al. Data-driven regularization lowers the size barrier of cryo-EM structure determination. Nature Meth. 21, 1216-1221 (2024). 72. Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1055-1064 (2020). 73. Yamashita, K. et al. Cryo-EM single particle structure refinement and map calculation using Servalcat. Acta Crystallogr. D 77, 1282-1291 (2021). Declarations Data availability Cryo-EM maps were deposited to the EM Data Bank under accession codes EMD-70252 and EMD-70253. Corresponding refined atomic models were deposited to the PDB under accession codes PDB:9O9J and PDB: 9O9K. Please address requests for materials to the corresponding authors. Source data are provided with this paper. Acknowledgements We thank the patients and their families without whose generous donations this study would not have been possible. We thank the staff of the Queen Square Brain Bank for their help with material preparation and Sebastian Brandner for facilitating the digitisation of histology slides. We are grateful to Max Jacobsen and Kelsey Cox for help with neuropathology. We also thank the Electron Microscopy Facility of the Medical Research Council (MRC) Laboratory of Molecular Biology for help with cryo-EM; and Jake Grimmett, Toby Darling and Ivan Clayson for support with high-performance computing. This work was financed by the MRC as part of U.K. Research and Innovation (UKRI) (MC_UP_A025_1013 to S.H.W.S. and MC_1051284291 to M.G.). It was also supported by the Indiana University School of Medicine (B.G.). Genetic data were obtained from the Global Parkinson’s Genetics Program, which is funded through the Aligning Science across Parkinson’s (ASAP) initiative and implemented by the Michael J. Fox Foundation for Parkinson’s Research (https://gp2.org). For a list of members, see https://gp2.org Author contributions P.W.C., R.R., H.M., H.H., B.G. and Z.J. identified participants and performed neuropathology and DNA sequencing. Y.Y., S.P.C. and C.F. performed mass spectrometry. Y.Y. and T.H. collected cryo-EM data. Y.Y., A.G.M. and T.H. analysed cryo-EM data. S.H.W.S. and M.G. supervised the project. All authors contributed to the writing of the manuscript. Competing interests The authors declare no competing interests. References Scheres, S.H.W., Ryskeldi-Falcon, B. & Goedert M. Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids. Nature 621, 701–710 (2023). Yang Y. et al. Structures of a-synuclein filaments from human brains with Lewy pathology. Nature 610, 791–795 (2022). Krismer, F. et al. Multiple system atrophy: advances in pathophysiology, diagnosis, and treatment. Lancet Neurol. 23, 1252–1266 (2024). Schweighauser, M. et al. Structures of a-synuclein filaments from multiple system atrophy. Nature 585, 464–469 (2020). Yang, Y. et al. New SNCA mutation and structures of a-synuclein filaments from juvenile-onset synucleinopathy. Acta Neuropathol. 145, 561–572 (2023). Polymeropoulos, M. et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047 (1997). Krüger, R. et al. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nature Genet. 18, 106–108 (1998). Zarranz, J.J. et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173 (2004). Proukakis, C. et al. A novel alpha-synuclein missense mutation in Parkinson’s disease. Neurology 80, 1062–1064 (2012). Appel-Cresswell, S. et al. Alpha-synuclein H50Q, a novel pathogenic mutation for Parkinson’s disease. Mov. Disord. 28, 811–813 (2013). Lesage, S. et al. G51D a-synuclein mutation causes a novel Parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459–471 (2013). Kiely, A.P. et al. a-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson’s disease and multiple system atrophy? Acta Neuropathol. 125, 753–769 (2013). Pasanen, P. et al. A novel a-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson’s disease-type pathology. Neurobiol. Aging 35, 2180.e1-e5 (2014). Tokutake, T. et al. Clinical and neuroimaging features of patient with early-onset Parkinson’s disease with dementia carrying SNCA p.G51D mutation. Parkinsonism Relat. Disord. 20, 262–264 (2014). Kiely, A.P. et al. Distinct clinical and neuropathological features of G51D SNCA mutation cases compared with SNCA duplication and H50Q mutation. Mol. Neurodegeneration 10, 41 (2015). Yoshino, H. et al. Homozygous alpha-synuclein p.A53V in familial Parkinson’s disease. Neurobiol. Aging 57, 248.e7-248.e12 (2017). Liu, H. et al. A novel SNCA A30G mutation causes familial Parkinson’s disease. Mov. Disord. 36, 1624–1633 (2021). Singleton, A.B. et al. a-Synuclein locus triplication causes Parkinson’s disease. Science 302, 841 (2003). Chartier-Harlin, M.C. et al. Alpha-synuclein locus duplication as a cause of familial Parkinson’s disease. Lancet 364, 1167–1169 (2004). Ibáñez, P. et al. Causal relation between alpha-synuclein gene duplication and familial Parkinson’s disease. Lancet 364, 1169–1171 (2004). Nalls, M.A. et al. Identification of novel risk loci, causal insights, and heritable risk for Parkinson’s disease: a meta-analysis of genome-wide association studies. Lancet Neurol. 18, 1091–1102 (2019). Pan, H. et al. Genome-wide association study using whole-genome sequencing identifies risk loci for Parkinson’s disease in Chinese population. N.P.J. Parkinson’s Dis. 9, 22 (2023). Andrews, S.V. et al. The genetic drivers of juvenile, young, and early-onset Parkinson’s disease in India. Mov. Disord. 39, 339–349 (2024). Rutherford, N.J. et al. Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of a-synuclein. J Neurochem 131, 859–867 (2014). Fares, M.B. et al. The novel Parkinson’s disease linked mutation G51D attenuates in vitro aggregation and membrane binding of a-synuclein and enhances its secretion and nuclear localization in cells. Hum. Mol. Genet. 23, 4491–4509 (2014). Flagmeier, P. et al. Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of alpha-synuclein aggregation. Proc. Natl. Acad. Sci. U.S.A. 113, 10328–10333 (2016). Stefanovic, A.N. et al. Oligomers of Parkinson’s disease-related alpha-synuclein mutants have similar structures but distinctive membrane permeabilization properties. Biochemistry 54, 3142–3150 (2015). Sun, Y. et al. The hereditary mutation G51D unlocks a distinct fibril strain transmissible to wild-type a-synuclein. Nature Commun. 12, 6252 (2021). Guan, Y. et al. Pathogenic mutations differentially regulate cell-to-cell transmission of alpha-synuclein. Front. Cell. Neurosci. 14, 159 (2020). Khalaf, O. et al. The H50Q mutation enhances a-synuclein aggregation, secretion and toxicity. J. Biol. Chem. 289, 21856–21876 (2014). Porcari, R. et al. The H50Q mutation induces a 10-fold decrease in the solubility of a-synuclein. J. Biol. Chem. 290, 2395–2404 (2015). Blauwendraat, C. et al. Insufficient evidence for pathogenicity of SNCA His50Gln (H50Q) in Parkinson’s disease. Neurobiol. Aging 64, 159.e5-e8 (2018). Guerrero-Ferreira, R. et al. Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy. eLife 8, e48907 (2019). Longhena, F. et al. Synapsin III is a key component of a-synuclein fibrils in Lewy bodies of PD brains. Brain Pathol. 28, 875–888 (2018). Boyer, D.R. et al. Structures of fibrils formed by a-synuclein hereditary disease mutation H50Q reveal new polymorphs. Nature Struct. Mol. Biol. 26, 1044–1052 (2019). Zhao, K. et al. Parkinson’s disease associated mutation E46K of alpha-synuclein triggers the formation of a distinct fibril structure. Nature Commun. 11, 2643 (2020). Boyer, D.R. et al. The alpha-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure. Proc. Natl. Acad. Sci. USA 117, 3592–3602 (2020). Sun, Y. et al. Cryo-EM structure of full-length a-synuclein amyloid fibril with Parkinson’s disease familial A53T mutation. Cell Res. 30, 360–362 (2020). Sun, C. et al. Cryo-EM structure of amyloid fibril formed by a-synuclein hereditary A53E mutation reveals a distinct protofilament interface. J. Biol. Chem. 299, 104566 (2023). Morley, V. et al. In vivo 18 F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D a-synuclein mutation. Front. Neurosci. 17, 1095761 (2023). Nuber, S. et al. Generation of G51D and 3D mice reveals decreased a-synuclein tetramer-monomer ratios promote Parkinson’s disease phenotypes. N.P.J. Parkinson’s Dis. 10, 47 (2024). Kim, Y. et al. Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice. Proc. Natl. Acad. Sci. U.S.A. 121, e2406479121 (2024). Yan, N.L. et al. Cryo-EM structure of a novel a-synuclein filament subtype from multiple system atrophy. FEBS Lett. 599, 33–40 (2025). Cullinane, P.W. et al. Identical seeding characteristics and cryo-EM filament structures in FTLD-synuclein and typical multiple system atrophy. Neuropathol. Appl. Neurobiol. 51, e70013 (2025). Al-Azzini, M. et al. A novel alpha-synuclein K58 missense variant in a patient with Parkinson’s disease. Medrxiv (2024). Fevga, C. et al. A new a-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson’s disease. Parkinsonism Relat. Disord. 89, 63–72 (2021). Kapasi, A. et al. A novel SNCA E83Q mutation in a case of dementia with Lewy bodies and atypical frontotemporal lobar degeneration. Neuropathology 40, 620–626 92020). Brücke, C. et al. A novel G14R missense variant is associated with atypical neuropathological features. Medrxiv (2024). Cali, F. et al. Interpreting genetic variants: Hints from a family cluster of Parkinson’s disease. J. Parkinson’s Dis. 9, 203–206 (2019). Perrin, R.J. et al. Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins. J. Biol. Chem. 289, 21856–21876 (2001). Snead, D. & Eliezer, D. Alpha-synuclein function and dysfunction in cellular membranes. Exp. Neurobiol. 23, 292–313 (2014). Uchihara, T. et al. Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: Comparison between Gallyas-Braak and Campbell-Switzer methods. Acta Neuropathol. 110, 255–260 (2005). Lau, H.H.C. et al. The G51D SNCA mutation generates a slowly progressive a-synuclein strain in early-onset Parkinson’s disease. Acta Neuropathol. Commun. 11, 72 (2023). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryDatas.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6490169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449540813,"identity":"c1fcd528-32f0-4f5d-b68e-38b742e17c0e","order_by":0,"name":"Yang Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYFAD9gYGhgTStPAcIFmLBLHqDY6fPfyCsc0uTz7yjeGHBww2+fIOhLScyUuzYGxLLja8nWMMtCjNcuMBQloO5JgZMLYxJ26cnZYA1HLYwLCBkJbzb0Ba6hM3zjyW/IM4LTdyjB8wth1OnC/BfAxsizwBHQySN96YMSScO564gSf5mEWCQZqBASEtfOdzjD98KKtOnN9+sPnmjwobA3lCDlM4wMAmkcgGCgewO2EMPABoJvMHhj9gBlxkFIyCUTAKRgEKAAAoPUP8AWjRBQAAAABJRU5ErkJggg==","orcid":"","institution":"Van Andel Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":449540814,"identity":"7ddb83e6-8961-4aff-9079-499abbef6260","order_by":1,"name":"Alexey Murzin","email":"","orcid":"","institution":"MRC Laboratory of Molecular Biology","correspondingAuthor":false,"prefix":"","firstName":"Alexey","middleName":"","lastName":"Murzin","suffix":""},{"id":449540815,"identity":"3eabc1e2-217d-4082-b058-3419a20dcfc8","order_by":2,"name":"Tiara Hinton","email":"","orcid":"","institution":"Van Andel Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Tiara","middleName":"","lastName":"Hinton","suffix":""},{"id":449540816,"identity":"9e081fad-848d-4623-849e-9f22986e64cb","order_by":3,"name":"Sew Peak-Chew","email":"","orcid":"","institution":"MRC LMB","correspondingAuthor":false,"prefix":"","firstName":"Sew","middleName":"","lastName":"Peak-Chew","suffix":""},{"id":449540817,"identity":"fa898af6-d43f-4b4a-9e46-4fe0b535abd2","order_by":4,"name":"Catarina Franco","email":"","orcid":"","institution":"MRC LMB","correspondingAuthor":false,"prefix":"","firstName":"Catarina","middleName":"","lastName":"Franco","suffix":""},{"id":449540818,"identity":"6e90972a-0297-42ec-93eb-6a9e919eba8a","order_by":5,"name":"Patrick Cullinane","email":"","orcid":"","institution":"Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Cullinane","suffix":""},{"id":449540819,"identity":"5b4c5515-aa1b-4863-bfaa-41429a71eb2b","order_by":6,"name":"Raquel Real","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Raquel","middleName":"","lastName":"Real","suffix":""},{"id":449540820,"identity":"12502cf3-d669-49f8-82f9-047fd706a629","order_by":7,"name":"Huw Morris","email":"","orcid":"https://orcid.org/0000-0002-5473-3774","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Huw","middleName":"","lastName":"Morris","suffix":""},{"id":449540821,"identity":"9e1237a4-966b-413d-849f-842ca5ede406","order_by":8,"name":"Henry Houlden","email":"","orcid":"https://orcid.org/0000-0002-2866-7777","institution":"UCL Queen Square Institute of Neurology and The National Hospital for Neurology and Neurosurgery","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"","lastName":"Houlden","suffix":""},{"id":449540822,"identity":"0f1cc19a-2e05-417b-bbc7-50cec9c6efde","order_by":9,"name":"Bernardino Ghetti","email":"","orcid":"","institution":"Department of Pathology and Laboratory Medicine, Indiana University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bernardino","middleName":"","lastName":"Ghetti","suffix":""},{"id":449540823,"identity":"5670e8b6-fbaf-4d98-b6ce-285d454c4f45","order_by":10,"name":"Zane Jaunmuktane","email":"","orcid":"https://orcid.org/0000-0001-7738-8881","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Zane","middleName":"","lastName":"Jaunmuktane","suffix":""},{"id":449540824,"identity":"ec14098d-3cdf-4573-94d4-5d9c259f7791","order_by":11,"name":"Sjors Scheres","email":"","orcid":"https://orcid.org/0000-0002-0462-6540","institution":"MRC Laboratory of Molecular Biology","correspondingAuthor":false,"prefix":"","firstName":"Sjors","middleName":"","lastName":"Scheres","suffix":""},{"id":449540825,"identity":"e8fb008c-6fe6-4159-aac1-997c3b61c3bc","order_by":12,"name":"Michel Goedert","email":"","orcid":"https://orcid.org/0000-0002-5214-7886","institution":"MRC Laboratory of Molecular Biology","correspondingAuthor":false,"prefix":"","firstName":"Michel","middleName":"","lastName":"Goedert","suffix":""}],"badges":[],"createdAt":"2025-04-20 15:40:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6490169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6490169/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82359118,"identity":"227b416d-efb9-40fc-a401-560efb3470ca","added_by":"auto","created_at":"2025-05-09 11:26:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":875228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structures of G51D a-synuclein filaments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Amino acid sequence of human a-synuclein, with the G51D mutation indicated in bold. The protofilament core-forming residues (G31-L100) are underlined in cyan. Thick arrows indicate b-strands.\u003c/p\u003e\n\u003cp\u003eb, Cross-sections through the cryo-EM reconstructions, perpendicular to the helical axis and with a projected thickness of approximately one rung, are shown for a-synuclein filaments from both cases with \u003cem\u003eSNCA\u003c/em\u003emutation G51D. Scale bar, 1 nm.\u003c/p\u003e\n\u003cp\u003ec, Cryo-EM density map and atomic model of filaments with G51D mutant a-synuclein. Island B is shown in grey.\u003c/p\u003e\n\u003cp\u003ed, Electrostatic networks around the dimerisation interface. The G51D fold is shown in cyan and water molecules are in red. Interactions are indicated by dashed lines. Boxes indicate the network regions.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/1c9daa85a709385614a68999.png"},{"id":82359116,"identity":"2c9724df-c027-4aa3-8157-823e3e91f8fb","added_by":"auto","created_at":"2025-05-09 11:26:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":452666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the Lewy folds of G51D and wild-type a-synuclein filaments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Superposition of the axial projections of the Lewy folds, shown in sticks. The G51D variant is in cyan and the wild-type variant is in orange. The interior residues with different side chain rotamers, L38, Y39 and Q62, are labelled.\u003c/p\u003e\n\u003cp\u003eb, A view of the superposed backbones, tilted by 45°. Islands A and B are omitted for clarity.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/9cee7cf7f11e888120c28786.png"},{"id":82359924,"identity":"ee39cdf3-555a-43a5-86b7-af79a75df67b","added_by":"auto","created_at":"2025-05-09 11:34:53","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":297085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structures of H50Q a-synuclein filaments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Amino acid sequence of human a-synuclein, with the H50Q variant indicated in bold. The protofilament core-forming residues (G31-L100) are shown in green. Thick arrows indicate b-strands.\u003c/p\u003e\n\u003cp\u003eb, Cross-sections through the cryo-EM reconstructions, perpendicular to the helical axis and with a projected thickness of approximately one rung, are shown for a-synuclein filaments from a case with \u003cem\u003eSNCA\u003c/em\u003evariant H50Q. Scale bar, 1 nm.\u003c/p\u003e\n\u003cp\u003ec, Cryo-EM density map and atomic model of filaments with H50Q a-synuclein. Islands A and B are shown in grey.\u003c/p\u003e\n\u003cp\u003ed, Cryo-EM density map and atomic model of the Lewy fold. Islands A and B are shown in grey.\u003c/p\u003e\n\u003cp\u003ee, Superposition of the H50Q Lewy fold and the Lewy fold of idiopathic PD. The H50Q Lewy fold is shown in magenta and the Lewy fold of idiopathic PD in orange.\u003c/p\u003e\n\u003cp\u003ef, Superposition of the H50Q and G51D Lewy folds. The H50Q fold is shown in magenta and the G51D fold in cyan.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/aa24531ebee0bd44bb7f40ac.jpeg"},{"id":82359132,"identity":"81ba147f-8a7e-47ee-b7e8-f0149f629bb5","added_by":"auto","created_at":"2025-05-09 11:26:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1465469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeptide islands in the a-synuclein filaments with Lewy, JOS and polymorph 2 folds.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM densities and models of island A and adjacent core segments of wild-type (a), G51D variant (b) and H50Q (c) variant of the Lewy fold and the JOS fold (d). (e), Superposition of the shared substructure of Lewy and JOS folds, made of segment 48-59 and island A, onto the map of the H50Q variant. Cryo-EM densities and models of island B and adjacent structures of the G51D variant of the Lewy fold (f) and the island and adjacent structures of the polymorph 2 fold (g). (h), Superposition of shared substructure of Lewy and polymorph 2 folds, made of segment 83-93 and the island B, onto the map of the G51D variant.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/5c2a51ebb7df6f97c7c38ccb.png"},{"id":82359124,"identity":"83b7c106-b731-43ba-a78d-311f1e0d4ff1","added_by":"auto","created_at":"2025-05-09 11:26:53","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of different variants of the Lewy fold, the MSA fold and the JOS fold of assembled a-synuclein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic of secondary structure elements in filaments with known variants of the Lewy fold, the MSA fold and the JOS fold, depicted as a single rung (N-terminal region of a-synuclein in orange, hydrophobic region in green and C-terminal region in blue; thick connecting lines with arrowheads indicate β-strands). Islands of unknown sequence identity are shown in grey. The extra densities are depicted in dark blue. The charged residues that co-ordinate these densities or form fold-supporting salt bridges are highlighted with coloured circles. The salt bridge between E46 from one protofilament and H50 from the other in the G51D variant of the Lewy fold is also indicated. Residue numbers with apostrophes indicate those from the second protofilament.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/272a3b01360acd0e7b731818.jpeg"},{"id":82395759,"identity":"f7293991-0174-42cb-a699-8cf5461795f3","added_by":"auto","created_at":"2025-05-09 20:17:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3867329,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/a854209c-3ef2-462c-89e3-f505b5be6136.pdf"},{"id":82359119,"identity":"67189725-a709-47a0-b252-ef9ea9996a8b","added_by":"auto","created_at":"2025-05-09 11:26:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6704425,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryDatas.docx","url":"https://assets-eu.researchsquare.com/files/rs-6490169/v1/13a6c1874c099e91dda7af65.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cryo-EM structures of filaments from the brains of individuals with variants G51D and H50Q in α-synuclein","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are the major synucleinopathies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They share the presence of abundant filamentous inclusions that are made of a-synuclein. PD and DLB have in common the deposition of filamentous a-synuclein in Lewy bodies and Lewy neurites. By electron cryo-microscopy (cryo-EM), they share the Lewy fold, suggesting that they are mechanistically related [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The Lewy fold is made of a single protofilament that extends from residues 31\u0026ndash;100 of a-synuclein, which are arranged as nine b-strands in a three-layered structure. Two regions of cryo-EM densities of unknown composition that are not connected to a-synuclein (islands A and B) are also present.\u003c/p\u003e \u003cp\u003eMSA is associated with abundant filamentous a-synuclein inclusions in both nerve cells and glial cells, mainly oligodendrocytes (glial cytoplasmic inclusions [GCIs] or Papp-Lantos bodies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]). By cryo-EM, two types of filaments are present that are each made of two different protofilaments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Their proportions vary between brain regions and individuals.\u003c/p\u003e \u003cp\u003eThe cryo-EM structure of a-synuclein filaments from the brain of an individual with juvenile-onset synucleinopathy (JOS) uncovered a third fold of assembled a-synuclein in the human brain [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. JOS is caused by a heterozygous mutation in \u003cem\u003eSNCA\u003c/em\u003e, the a-synuclein gene, which gives rise to the insertion of seven amino acids after residue 22 in the amino-terminal region of a-synuclein. The JOS fold differs from the Lewy fold, but it resembles a substructure that is common to the MSA protofilament folds. We observed the presence of filaments made of either a single or two identical protofilaments with the JOS fold.\u003c/p\u003e \u003cp\u003eAdditional densities for unidentified non-proteinaceous cofactors are also present in a-synuclein filaments from human brains [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For MSA filaments, the extra densities are located at the protofilament interfaces. Dimeric a-synuclein protofilaments of JOS have an extra density at the protofilament interface. All a-synuclein filaments from human brains have a fuzzy coat that encompasses the disordered protein\u0026rsquo;s amino- and carboxy-terminal regions.\u003c/p\u003e \u003cp\u003eSeveral dominantly inherited point mutations in \u003cem\u003eSNCA\u003c/em\u003e have been identified in families with typical and atypical forms of PD [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. They give rise to missense mutations in a-synuclein. In addition, gene dosage mutations (duplications and triplications) of one allele of \u003cem\u003eSNCA\u003c/em\u003e have been shown to give rise to PD [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Some of these mutations also cause DLB. No \u003cem\u003eSNCA\u003c/em\u003e mutations have been linked with MSA. The existence of point mutations and multiplications of \u003cem\u003eSNCA\u003c/em\u003e that cause diseases with abundant a-synuclein filaments supports the notion that a-synuclein is a key player in all cases of PD and DLB. Moreover, genome-wide association studies have shown that single nucleotide polymorphisms in \u003cem\u003eSNCA\u003c/em\u003e are associated with elevated levels of a-synuclein, which increases the risk of PD [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutation G51D in a-synuclein has been described in four different cohorts with Parkinsonism [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is of particular interest, because of the rapid progression of disease and because some clinical and neuropathological characteristics overlap with those of MSA. Affected family members develop a variety of parkinsonian features with a variable levodopa response, dementia, visual hallucinations and autonomic dysfunction. Neuropathological features include CA2-CA3 hippocampal and cortical nerve cell loss, with abundant neuronal a-synuclein inclusions, together with a smaller number of GCI-like inclusions. Combined PD and MSA profiles were also reported in a family with mutation A53E in \u003cem\u003eSNCA\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies using recombinant G51D a-synuclein have shown an equal or lowered aggregation propensity relative to the wild-type protein [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], impaired membrane binding ability [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and increased secretion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVariant H50Q in a-synuclein has been reported in some cases of PD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recombinant H50Q a-synuclein increases the rate of a-synuclein aggregation, secretion and toxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, unlike cases with the G51D mutation, the family history of cases with variant H50Q is inconsistent. Moreover, H50Q a-synuclein has also been found in population databases. It was not enriched in PD cases, arguing against the possibility of reduced penetrance and questioning its pathogenicity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe structures of a-synuclein filaments from the brains of individuals with \u003cem\u003eSNCA\u003c/em\u003e mutations that encode missense variants have not been determined. Here we report the cryo-EM structures of a-synuclein filaments extracted from the frontal cortex of two previously reported individuals with PD and mutation G51D [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and from the amygdala of a previously unreported case of PD with variant H50Q.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eStructure of a-synuclein filaments from two cases with\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003emutation G51D\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe heterozygous mutation in \u003cem\u003eSNCA\u003c/em\u003e that encodes G51D a-synuclein [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] was confirmed for cases 1 and 2 using whole-genome sequencing. There was widespread nerve cell loss, with abundant neuronal and neuritic a-synuclein pathology. This included the neocortex, in addition to the striatum, the limbic system and the brainstem. The neuronal inclusions were annular, crescentic, globular, diffuse and neurofibrillary tangle-like. In both cases, sparse GCI-like oligodendroglial inclusions were also present in the white matter.\u003c/p\u003e \u003cp\u003eFilaments of a-synuclein from the frontal cortex of cases 1 and 2 yielded identical structures, with resolutions of 2.3 \u0026Aring; for case 1 and 3.4 \u0026Aring; for case 2. G51D filaments comprise two identical protofilaments with the Lewy fold that are related by a 2-start (or pseudo-2\u003csub\u003e1\u003c/sub\u003e) helical symmetry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The protofilament interface is made of the same three residues from each protofilament: E46, V48 and H50. Like the wild-type protofilament, the G51D protofilaments contain a non-proteinaceous cofactor density in an equivalent location, but they lack island A, one of the two disconnected peptide-like densities, thus exposing the mutation site to solvent. As the densities of negatively charged carboxyl groups are generally missing from cryo-EM reconstructions, the presence of the D51 side chain is not apparent from its local density alone. There is a short stump of density, presumably corresponding to a Cb atom, that is smaller than the side chain density of the nearby A53, or of D98, the only other aspartic acid residue. This is consistent with both G51 and D51 being present in the filaments, as confirmed by mass spectrometry of the sarkosyl-insoluble fractions (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the presence of D51 manifests itself through subtle differences between wild-type and G51D variants of the Lewy fold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhereas the backbone conformations of both Lewy fold variants are nearly identical when viewed along the filament axes, they are different when viewed perpendicularly. Compared to the wild-type variant, the C-terminal part of the G51D protofilament fold is rotated relative to its N-terminal part by about 8\u0026deg;, with G73 in the middle serving as a hinge (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Concomitant with this rotation, the side chain conformations of residues that form salt bridges (E35-K80, E61-K96, K60-D98) between the N- and C-terminal parts are readjusted. This difference in relative orientations of the N- and C-terminal parts can also explain the different handedness of the wild-type and the G51D filaments. G51D filaments are left-handed (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), in contrast to the right-handed helical twist of wild-type filaments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther differences between the two Lewy fold variants include side chain rotamers of residues L38, Y39 and Q62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The rotamer of L38 compensates for the rotation of the C-terminal part in the G51D variant, maintaining the contact with residues A76 and A78 from two adjacent molecules. In wild-type filaments, the hydroxyl group of Y39 hydrogen bonds to the e-amino group of K34 that contributes to coordination of the cofactor density, whereas in G51D filaments the hydroxyl group of Y39 makes a hydrogen bond to the main chain of S42 on the opposite side of the density. In wild-type filaments, the side chain of Q62 has a folded conformation, being hydrogen bonded to the side chain of T64 in the same b-sheet. In G51D filaments, the side chain of Q62 adopts an extended conformation and makes a hydrogen bond to the main chain of V55 in the opposite b-sheet. In both wild-type and G51D filaments, the side chains of Q62 also form hydrogen-bonded ladders along the filament axes.\u003c/p\u003e \u003cp\u003eThe above differences, including the loss of island A and the formation of dimeric filaments, can be explained by the effects of the G51D mutation on the Lewy fold. The bulk of the side chain of D51 may introduce steric clashes with the residues of island A, whereas its charge probably facilitates dimerisation of the Lewy fold through electrostatic interactions. The side chain of D51 from one protofilament is linked to the cofactor-binding site of the other protofilament through a network of salt bridges, including H50 from its own protofilament and both K45 and E46 from the other protofilament; this may affect the position of the cofactor and that of the coordinating residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d). The loss of island A and a slight shift of the cofactor position promote minor conformational rearrangements in the filament core.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructure of a-synuclein filaments from a case with\u003c/b\u003e \u003cb\u003eSNCA\u003c/b\u003e \u003cb\u003evariant H50Q\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA heterozygous variant in \u003cem\u003eSNCA\u003c/em\u003e that encodes H50Q a-synuclein was identified in case 3 using the Neurobooster array and confirmed by whole-genome sequencing. This case has not been described before. The clinical phenotype was consistent with idiopathic PD, with slow disease progression, a sustained levodopa response and no significant dysautonomia, pyramidal signs or dementia. Neuropathology also closely resembled that of idiopathic PD, with abundant Lewy bodies and Lewy neurites that were Gallyas-Braak silver-negative (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f). By contrast, the neuronal inclusions from case 2 of G51D were Gallyas-Braak silver-positive (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). GCI-like inclusions were absent from the H50Q case. Small numbers of amyloid-b plaques and tau tangles were also present. By mass spectrometry, Q50 a-synuclein was detected in the sarkosyl-insoluble fraction, where it coexisted with H50 a-synuclein, indicating the presence of mixed filaments (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFilaments of a-synuclein extracted from the amygdala of an individual with the H50Q variant in \u003cem\u003eSNCA\u003c/em\u003e comprised a single protofilament (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The structure, which was determined at a resolution of 2.9 \u0026Aring;, was almost identical to the wild-type Lewy fold, including the same additional densities of the cofactor and peptide islands A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c). Like the wild-type a-synuclein filaments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], the H50Q filaments exhibited a right-handed helical twist (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The only notable difference between both variants is the side chain density at the mutation site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In the H50Q variant, this density can accommodate either the side chains of histidine or glutamine, but in a different conformation than that previously assigned to H50 in the wild-type structure and now also found in the G51D variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003eIn the wild-type map, there is a previously unassigned density in an equivalent location between the side chains of V48 and H50, which may be the result of an alternative conformation of H50. Thus, the only apparent effect of the H50Q variant on the Lewy fold is that it selects between the two alternative side chain conformations at position 50 (H50 or Q50). In the H50Q variant, the side chain is oriented parallel to the direction of island A, which is probably not compatible with the formation of a G51D-like dimeric interface due to steric interference from E46 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Analysis of a-synuclein filaments from the frontal cortex of the H50Q case suggests a filament structure consistent with that of filaments from the amygdala, as judged by 2D class averaging (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTwisted and untwisted segments in H50Q and G51D a-synuclein filaments\u003c/h2\u003e \u003cp\u003eWe previously reported that a-synuclein filaments extracted from the brains of cases of idiopathic PD and DLB are made of twisted and untwisted segments, with the twisted segments suitable for helical reconstruction being in a minority, but the majority untwisted segments have 2D class averages that resemble the projections of untwisted filament models with the Lewy fold [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. H50Q filaments had the same proportion of twisted (25%) and untwisted (75%) segments as wild-type a-synuclein assemblies, with the former having a right-handed helical twist and cross-over distances of 950 \u0026Aring; (from class 2D segments), like what we observed in the wild-type Lewy fold. By contrast, in the dimeric G51D filaments, the proportion of untwisted segments was smaller (34%) and the twisted segments had cross-over distances of 5,500 \u0026Aring; (from class 2D segments) and a left-handed helical twist (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For both H50Q and G51D filaments, the untwisted 2D class averages resembled 2D class averages of the twisted segments that were used for 3D reconstruction. It could therefore be that the helical twist is relevant for disease progression.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePeptide islands in the Lewy fold and other a-synuclein filament structures\u003c/h3\u003e\n\u003cp\u003eThe structures of G51D and H50Q filaments provided no novel clues as to what protein sequences islands A and B are made of. However, some hypotheses can be drawn from other structures. Isolated peptide-like densities are present in the JOS fold of a-synuclein filaments from the human brain [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and in a set of \u003cem\u003ein vitro\u003c/em\u003e assemblies, known as polymorph 2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We previously noted that the Lewy fold segment 83\u0026ndash;92 and island B form a similar substructure than that in polymorph 2, which is made of the equivalent segment and peptide island B (Extended Data Fig.\u0026nbsp;6e in [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]). Lewy and JOS folds also share a substructure that is made of the core segment 48\u0026ndash;59 and peptide island A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,e). In both polymorph 2 and JOS folds, island A was assigned to residues G14-G25 of a-synuclein, with V15, A17 and A19 facing the filament cores.\u003c/p\u003e \u003cp\u003eEven though the sequence G14-G25 is compatible with islands A and B in the Lewy fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-h), it cannot be assigned to either, because the intervening sequence is of insufficient length to connect the islands to the N-terminus of the ordered core at G31. However, a more N-terminal segment, containing residues G7, S9 and A11, which can also be modelled into the densities of islands A and B, can be connected to G31 through flexible linkers (Extended Data Fig.\u0026nbsp;6). There are no compatible segments in the C-terminal sequence outside the Lewy fold core (residues 101\u0026ndash;140).\u003c/p\u003e \u003cp\u003eThis raises the possibility of N-terminal sequences of a-synuclein contributing to both islands A and B in the Lewy fold. Since a single protein chain cannot contribute to both islands, these islands would need to incorporate other peptides, like proteolytic fragments of a-synuclein and/or other proteins. Alternatively, they could consist of intact proteins with most of their chains being disordered. It has been reported that Synapsin III is an integral component of a-synuclein filaments from the brains of individuals with PD [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we report the structures of a-synuclein filaments extracted from the frontal cortex of two cases with missense mutation G51D in a-synuclein and the amygdala of one case with variant H50Q. Both G51D and H50Q filaments adopted essentially the same protofilament fold as the previously described Lewy fold of idiopathic PD and DLB [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, they differed in the number of protofilaments: two protofilaments for G51D filaments and one protofilament for H50Q filaments. They also differed in protofilament handedness (left for G51D and right for H50Q).\u003c/p\u003e \u003cp\u003eThe expectation from previous \u003cem\u003ein vitro\u003c/em\u003e studies has been that human brain a-synuclein filaments with these sequence variants and other pathogenic mutations would adopt folds different from those of assembled wild-type a-synuclein [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, this is not the case. The Lewy fold from the human brain differs from all known folds of a-synuclein filaments assembled or seeded \u003cem\u003ein vitro\u003c/em\u003e, including those made of mutant and post-translationally modified proteins. It follows that the reported effects of recombinant a-synuclein with missense mutations on \u003cem\u003ein vitro\u003c/em\u003e filament assembly may not be relevant for understanding the effects of those mutations on filament assembly in the human brain. The pathological inclusions found in rodent models that express G51D a-synuclein remain to be characterised at the ultrastructural level [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The Lewy fold also differs from the MSA [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and JOS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] folds of a-synuclein filaments from the human brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther PD-associated missense mutations map to either the exterior of the Lewy fold near the cofactor-binding site (A30G, A30P, E46K) or the interface with island A (A53E, A53T, A53V). In the light of our findings, it appears likely that the filaments of a-synuclein from PD cases with these mutations will also be made of variants of the Lewy fold, with the mutations at position 53 possibly leading to the displacement of island A, alteration of filament twist and protofilament dimerisation, as is the case for G51D filaments. Island A will prevent protofilament dimerisation of similar variants through the interface used by G51D filaments, because of steric clashes with the opposite protofilament.\u003c/p\u003e \u003cp\u003eIf some a-synuclein protein chains contributed to both the filament core and island A, with their linker going over the cofactor binding site and the G51D dimerisation interface, it would enforce the incompatibility of the presence of island A and the dimerisation of Lewy fold protofilaments. It would also provide a rationale for the effects of mutations A30G, A30P and E46K that may affect linker interactions. The a-synuclein filaments with variants K58N [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], T72M [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and E83Q [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] are also compatible with the Lewy fold; K58N and E83Q map to the exterior of the Lewy fold and T72M maps to a large cavity inside that fold. Similarly, the a-synuclein variants G14R [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and V15A [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] are compatible with the Lewy fold; their location is in the potential linker between island A and the core of the Lewy fold.\u003c/p\u003e \u003cp\u003eNevertheless, all known missense variants in a-synuclein are found in the region comprising the seven imperfect repeats (residues 7\u0026ndash;87) that are lipid-binding domains [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], suggesting that those mutations may also influence the lipid-binding properties of a-synuclein.\u003c/p\u003e \u003cp\u003eMutation G51D in a-synuclein has been reported to result in clinical and neuropathological characteristics reminiscent of both PD and MSA [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Like the glial cytoplasmic and neuronal inclusions of MSA, and unlike the neuronal inclusions of PD and DLB [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], the neuronal inclusions from case 2 of G51D were Gallyas-Braak silver-positive; those from the H50Q case were silver-negative. Even though it is not known what underlies Gallyas-Braak silver positivity, these findings show that a-synuclein inclusions with distinct filament structures give rise to different staining patterns. This is also supported by propagation studies in M83 transgenic mice that used G51D a-synuclein seeds prepared from the temporal cortex of cases 1 and 2 and concluded that the a-synuclein conformer specified by mutation G51D resembles more closely a PD-associated than an MSA-associated strain [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Both mutant and wild-type a-synucleins are found in dimeric G51D filaments. A seed may form from the mutant protein, with both wild-type and mutant proteins being incorporated subsequently.\u003c/p\u003e \u003cp\u003eIn conclusion, we show that mutation G51D in \u003cem\u003eSNCA\u003c/em\u003e leads to the formation of left-handed filaments that are made of two identical protofilaments with the Lewy fold, in the absence of island A. By contrast, variant H50Q gives rise to right-handed filaments with a single protofilament with the Lewy fold. Dimerisation of a-synuclein protofilaments with the Lewy fold, or the formation of left-handed filaments, may be a general characteristic of filaments from cases with disease-causing missense mutations in \u003cem\u003eSNCA.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissues from cases 1-3 were donated to the Queen Square Brain Bank for Neurological Disorders, UCL Institute of Neurology, with informed consent and the study was approved by the National Health Services Health Research Authority Ethics Committee, London-Central (reference no. 23/LO/0044). The cryo-EM study was approved by the Cambridgeshire Research Ethics Committee (09/HO308/163).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical history\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe determined the cryo-EM structures of a-synuclein filaments extracted from the frontal cortex of two previously described individuals with mutation G51D in a-synuclein and a family history of atypical PD [15]. Case 1 (family two, patient III), presented with resting tremor of the right hand, anxiety and depression at age 69. A diagnosis of PD was made. This female individual died aged 75. Case 2 (family 2, patient IV) was the son of case 1. At age 46, he presented with resting tremor of the right hand and a depressed mood; a diagnosis of PD was made. Death occurred at age 52. We also determined the cryo-EM structures of a-synuclein filaments extracted from the amygdala of a novel case of PD with variant H50Q in a-synuclein without a family history. This female patient (referred to as case 3) developed resting tremor in her right hand, dragging of her right foot and micrography at age 44. Neurological examination three years later revealed hypomimia, bradykinesia, as well as upper and lower limb cogwheel rigidity. The patient was diagnosed with PD. The symptoms responded well to levodopa and the patient subsequently developed motor fluctuations. Non-motor symptoms included constipation, urinary urgency and hesitancy, anxiety, depression and insomnia. The patient experienced freezing of gait by age 55 and recurrent falls by age 56. By age 67, she required a wheelchair for mobility, followed by dysphagia at age 73 and death at age 78.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from the frontal cortex of cases 1 and 2 and the amygdala of case 3; it was sequenced using Illumina short-read whole genome sequencing, with a mean coverage of 30x. Paired-end reads of 150 bp were aligned to the human reference genome (GRCh38 build) using the functional equivalence pipeline [54]. Sample processing and variant calling were performed using DeepVariant v.1.6.1 [55] and joint genotyping was performed using GLnexus v.1.4.3 with the preset DeepVariant WGS configuration [56]. Quality control was performed according to the quality metrics defined by the Accelerating Medicines Partnership Parkinson\u0026rsquo;s disease program (AMP-PD; https://amp-pd.org), and variant annotation was performed with Ensembl Variant Effect Predictor [57,58]. DNA from each sample was also genotyped with the Illumina Infinium Global Diversity Single Nucleotide Polymorphism Array with custom content for neurodegenerative diseases (Neurobooster) [59].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuropathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePathological examination of the human brains was performed following standard Queen Square Brain Bank protocols. For histological analysis, 8 mm thick sections were cut from formalin-fixed paraffin-embedded tissue blocks sampled from representative brain regions. Immunohistochemical staining for a-synuclein (MA1-90342, Thermo Scientific, 1:500), amyloid-b (M0872, Dako, Abnova 1:500) and phosphorylated tau (MN1020, Thermo Scientific, 1:600) was performed on a Menarini automated staining platform following the manufacturers\u0026rsquo; guidelines. Biotinylated secondary antibodies were used with horseradish peroxidase-conjugated streptavidin complex and diaminobenzidine as the chromogen. To visualise inclusions, sections from insular cortex were also silver-impregnated according to Gallyas-Braak [60,61]. All sections were counterstained with haematoxylin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFilament extraction from the human brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cryo-EM analysis, sarkosyl-insoluble material was extracted from the frontal cortex (cases of G51D and H50Q) and the amygdala (case of H50Q), essentially as described [62]. Briefly, tissues were homogenised in 20 vol (w/v) extraction buffer consisting of 10 mM Tris-HCl, pH 7.4, 0.8 M NaCl, 10-20% sucrose and 1 mM EGTA. Homogenates were brought to 2% sarkosyl and incubated for 60 min at 37\u0026deg; C. Following a 10 min centrifugation at 10,000 g, the supernatants were spun at 100,000 g for 60 min. The final pellets were resuspended in 100 ml/g of 20 mM Tris-HCl, pH 7.4, 50 mM NaCl.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMass spectrometry was performed as described [63]. Sarkosyl-insoluble pellets were resuspended in 200 ml hexafluoroisopropanol. Following a 3 min sonication at 50% amplitude (QSonica), they were incubated at 37\u0026deg; C for 2h and centrifuged at 3,000 g for 5 min; the supernatants were transferred to new tubes before being dried by vacuum centrifugation. They were resuspended in 4M urea and 50 mM ammonium bicarbonate (ambic), before being reduced with 5 mM dithiothreitol at 37\u0026deg; C for 30 min and alkylated in the dark at room temperature for 30 min with 10 mM iodoacetamide. LysC (Promega) was then added to the samples for 2h at 25\u0026deg;C. They were diluted to 1.5 M urea with 50 mM ambic and incubated with trypsin (Promega) at 30\u0026deg;C overnight. Trypsin digestion was stopped by the addition of formic acid to 0.5%, followed by centrifugation at 16,000 g for 5 min. The supernatants were desalted using C18 stage tips (3M Empore) packed with Poros oligo R3 (Thermo Scientific) resin that were produced in-house. Bound peptides were eluted stepwise with 30, 50 and 80% acetonitrile in 0.5% formic acid and partially dried in a SpeedVac (Savant). The peptide mixtures were analysed by LC-MS/MS using a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer, as described [64].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-EM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHoley carbon grids (Quantifoil AuR1.2/1.3, 300 mesh) were glow-discharged with an Edwards (S150B) sputter coater at 30 mA for 30 s. Three ml aliquots were applied to the grids and blotted for 3-5 s with filter paper at 100% humidity and 4\u0026deg; C using a Thermo Fisher Vitrobot Mark IV. Datasets of the G51D case 1 and the H50Q case were acquired on a Thermo Fisher G4 microscope, equipped with a Falcon-4i detector and a Selectris-X energy filter (Thermo Fisher Scientific) with a slit width of 10 eV to remove inelastically scattered electrons. The G51D case 2 dataset was collected on a Titan Krios G3 microscope with a Gatan K3 detector in superresolution counting mode, using a Bioquantum energy filter with a slid width of 20 eV. Images were recorded with a total dose of 40 electrons per \u0026Aring;\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll super-resolution frames were gain-corrected, binned by a factor of 2, aligned, dose-weighted and then summed into a single micrograph using RELION\u0026rsquo;s own implementation of MotionCor2 [65]. Contrast transfer function (CTF) parameters were estimated using CTFFIND-4.1 [66]. Subsequent image-processing steps were performed using helical reconstruction methods in RELION [67,68]. Filaments were picked manually. Reference-free 2D classification was performed to identify homogeneous segments for further processing. Initial 3D reference models were reconstructed de novo from 2D class averages [69] using an estimated rise of 4.75 \u0026Aring; and helical twists according to the observed cross-over distances of filaments in the micrographs. To increase the resolution of the reconstructions, Bayesian polishing and CTF refinement were performed [70]. Blush regularisation was applied to improve the quality of the reconstructions [71]. Final 3D reconstructions, after auto-refinement, were sharpened using the standard post-processing procedures in RELION, and resolutions calculated from Fourier shell correlations at 0.143 between the two independently refined half-maps, using phase-randomisation to correct for convolution effects of a generous, soft-edged solvent mask. Further details of the data acquisition and processing are given in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAtomic models were built in Coot [72] using shared substructures of the Lewy fold (PDB: 8A9L) as template. Coordinate refinements were performed using \u003cem\u003eServalcat\u003c/em\u003e [73]. Final models were obtained using refinement of only the asymmetric unit against the half-maps in \u003cem\u003eServalcat\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e54. \u0026nbsp; \u0026nbsp;Regier, A.A. et al. Functional equivalence of genome sequencing analysis pipelines enables harmonized variant calling across human Genetics projects. Nature Commun. 9, 4038 (2018).\u003c/p\u003e\n\u003cp\u003e55. \u0026nbsp; \u0026nbsp;Poplin, R. et al. A universal SNP and small-indel variant caller using deep neural networks. Nature Biotechnol. 36, 983-987 (2018).\u003c/p\u003e\n\u003cp\u003e56. \u0026nbsp; \u0026nbsp;Yun, T. et al. Accurate, scalable cohort variant calls using DeepVariant and GLnexus. Bioinformatics 36, 5582-5589 (2021).\u003c/p\u003e\n\u003cp\u003e57. \u0026nbsp; \u0026nbsp;Iwaki, H. et al. Accelerating Medicines Partnership: Parkinson\u0026rsquo;s disease. Genetic Resource. Mov. Disord. 36, 1795-1804 (2021).\u003c/p\u003e\n\u003cp\u003e58. \u0026nbsp; \u0026nbsp;McLaren, W. et al. The Ensembl Variant Effect Predictor. Genome Biol. 17, 122 (2016).\u003c/p\u003e\n\u003cp\u003e59. \u0026nbsp; \u0026nbsp;Bandres-Ciga, S. et al. NeuroBooster array: A genome-wide genotyping platform to study neurological disorders across diverse populations. Mov. Disord. 39, 2039-2048 (2024).\u003c/p\u003e\n\u003cp\u003e60. \u0026nbsp; \u0026nbsp;Braak, H. et al. Silver impregnation of Alzheimer\u0026rsquo;s neurofibrillary changes counterstained for basophilic material and lipofuscin pigment. Stain Technol. 63, 197-200 (1988).\u003c/p\u003e\n\u003cp\u003e61. \u0026nbsp; \u0026nbsp;Gallyas, F. Silver staining of Alzheimer\u0026rsquo;s neurofibrillary changes by means of physical development. Acta Morphol. Acad. Sci. Hung. 19, 1-8 (1971).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e62. \u0026nbsp; \u0026nbsp;Tarutani, A. et al. Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods. Acta Neuropathol. Commun. 6, 29 (2018).\u003c/p\u003e\n\u003cp\u003e63. \u0026nbsp; \u0026nbsp;N\u0026auml;slund, J. et al. Relative abundance of Alzheimer Aβ amyloid peptide variants in Alzheimer disease and normal aging. Proc. Natl. Acad. Sci. U.S.A. 91, 8378-8382 (1994).\u003c/p\u003e\n\u003cp\u003e64. \u0026nbsp; \u0026nbsp;Yang, Y. et al. Cryo-EM structures of amyloid-β filaments from human brains. Science 357, 167-172 (2022).\u003c/p\u003e\n\u003cp\u003e65. \u0026nbsp; \u0026nbsp;Zheng, S.Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nature Meth. 14, 331-332 (2017).\u003c/p\u003e\n\u003cp\u003e66. \u0026nbsp; \u0026nbsp;Rohou, A. \u0026amp; Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216-221 (2015).\u003c/p\u003e\n\u003cp\u003e67. \u0026nbsp; \u0026nbsp;He, S. \u0026amp; Scheres, S.H.W. Helical reconstruction in RELION. J. Struct. Biol. 193, 163-176 (2017).\u003c/p\u003e\n\u003cp\u003e68. \u0026nbsp; \u0026nbsp;Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, 42166 (2018).\u003c/p\u003e\n\u003cp\u003e69. \u0026nbsp; \u0026nbsp;Scheres, S.H.W. Amyloid structure determination in RELION 3.1. Acta Crystallogr. D 76, 94-101 (2020).\u003c/p\u003e\n\u003cp\u003e70. \u0026nbsp; \u0026nbsp;Zivanov, J., Nakane, T. \u0026amp; Scheres, S.H.W. A Bayesian approach to beam-induced motion correction in cryo-EM single particle analysis. IUCrJ 6, 5-17 (2019).\u003c/p\u003e\n\u003cp\u003e71. \u0026nbsp; \u0026nbsp;Kimanius, D. et al. Data-driven regularization lowers the size barrier of cryo-EM structure determination. Nature Meth. 21, 1216-1221 (2024).\u003c/p\u003e\n\u003cp\u003e72. \u0026nbsp; \u0026nbsp;Casa\u0026ntilde;al, A., Lohkamp, B. \u0026amp; Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1055-1064 (2020).\u003c/p\u003e\n\u003cp\u003e73. \u0026nbsp; \u0026nbsp;Yamashita, K. et al. Cryo-EM single particle structure refinement and map calculation using Servalcat. Acta Crystallogr. D 77, 1282-1291 (2021).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM maps were deposited to the EM Data Bank under accession codes EMD-70252 and EMD-70253. Corresponding refined atomic models were deposited to the PDB under accession codes PDB:9O9J and PDB: 9O9K. Please address requests for materials to the corresponding authors. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients and their families without whose generous donations this study would not have been possible. We thank the staff of the Queen Square Brain Bank for their help with material preparation and Sebastian Brandner for facilitating the digitisation of histology slides. We are grateful to Max Jacobsen and Kelsey Cox for help with neuropathology. We also thank the Electron Microscopy Facility of the Medical Research Council (MRC) Laboratory of Molecular Biology for help with cryo-EM; and Jake Grimmett, Toby Darling and Ivan Clayson for support with high-performance computing. This work was financed by the MRC as part of U.K. Research and Innovation (UKRI) (MC_UP_A025_1013 to S.H.W.S. and MC_1051284291 to M.G.). It was also supported by the Indiana University School of Medicine (B.G.). Genetic data were obtained from the Global Parkinson\u0026rsquo;s Genetics Program, which is funded through the Aligning Science across Parkinson\u0026rsquo;s (ASAP) initiative and implemented by the Michael J. Fox Foundation for Parkinson\u0026rsquo;s Research (https://gp2.org). For a list of members, see https://gp2.org\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.W.C., R.R., H.M., H.H., B.G. and Z.J. identified participants and performed neuropathology and DNA sequencing. Y.Y., S.P.C. and C.F. performed mass spectrometry. Y.Y. and T.H. collected cryo-EM data. Y.Y., A.G.M. and T.H. analysed cryo-EM data. S.H.W.S. and M.G. supervised the project. All authors contributed to the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScheres, S.H.W., Ryskeldi-Falcon, B. \u0026amp; Goedert M. Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids. Nature 621, 701\u0026ndash;710 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y. et al. Structures of a-synuclein filaments from human brains with Lewy pathology. Nature 610, 791\u0026ndash;795 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrismer, F. et al. Multiple system atrophy: advances in pathophysiology, diagnosis, and treatment. Lancet Neurol. 23, 1252\u0026ndash;1266 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweighauser, M. et al. Structures of a-synuclein filaments from multiple system atrophy. Nature 585, 464\u0026ndash;469 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, Y. et al. New \u003cem\u003eSNCA\u003c/em\u003e mutation and structures of a-synuclein filaments from juvenile-onset synucleinopathy. Acta Neuropathol. 145, 561\u0026ndash;572 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolymeropoulos, M. et al. Mutation in the alpha-synuclein gene identified in families with Parkinson\u0026rsquo;s disease. Science 276, 2045\u0026ndash;2047 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKr\u0026uuml;ger, R. et al. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson\u0026rsquo;s disease. Nature Genet. 18, 106\u0026ndash;108 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZarranz, J.J. et al. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164\u0026ndash;173 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProukakis, C. et al. A novel alpha-synuclein missense mutation in Parkinson\u0026rsquo;s disease. Neurology 80, 1062\u0026ndash;1064 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAppel-Cresswell, S. et al. Alpha-synuclein H50Q, a novel pathogenic mutation for Parkinson\u0026rsquo;s disease. Mov. Disord. 28, 811\u0026ndash;813 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLesage, S. et al. G51D a-synuclein mutation causes a novel Parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459\u0026ndash;471 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiely, A.P. et al. a-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson\u0026rsquo;s disease and multiple system atrophy? Acta Neuropathol. 125, 753\u0026ndash;769 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasanen, P. et al. A novel a-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson\u0026rsquo;s disease-type pathology. Neurobiol. Aging 35, 2180.e1-e5 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokutake, T. et al. Clinical and neuroimaging features of patient with early-onset Parkinson\u0026rsquo;s disease with dementia carrying SNCA p.G51D mutation. Parkinsonism Relat. Disord. 20, 262\u0026ndash;264 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiely, A.P. et al. Distinct clinical and neuropathological features of G51D \u003cem\u003eSNCA\u003c/em\u003e mutation cases compared with \u003cem\u003eSNCA\u003c/em\u003e duplication and H50Q mutation. Mol. Neurodegeneration 10, 41 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshino, H. et al. Homozygous alpha-synuclein p.A53V in familial Parkinson\u0026rsquo;s disease. Neurobiol. Aging 57, 248.e7-248.e12 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, H. et al. A novel SNCA A30G mutation causes familial Parkinson\u0026rsquo;s disease. Mov. Disord. 36, 1624\u0026ndash;1633 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingleton, A.B. et al. a-Synuclein locus triplication causes Parkinson\u0026rsquo;s disease. Science 302, 841 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChartier-Harlin, M.C. et al. Alpha-synuclein locus duplication as a cause of familial Parkinson\u0026rsquo;s disease. Lancet 364, 1167\u0026ndash;1169 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIb\u0026aacute;\u0026ntilde;ez, P. et al. Causal relation between alpha-synuclein gene duplication and familial Parkinson\u0026rsquo;s disease. Lancet 364, 1169\u0026ndash;1171 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNalls, M.A. et al. Identification of novel risk loci, causal insights, and heritable risk for Parkinson\u0026rsquo;s disease: a meta-analysis of genome-wide association studies. Lancet Neurol. 18, 1091\u0026ndash;1102 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan, H. et al. Genome-wide association study using whole-genome sequencing identifies risk loci for Parkinson\u0026rsquo;s disease in Chinese population. N.P.J. Parkinson\u0026rsquo;s Dis. 9, 22 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrews, S.V. et al. The genetic drivers of juvenile, young, and early-onset Parkinson\u0026rsquo;s disease in India. Mov. Disord. 39, 339\u0026ndash;349 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRutherford, N.J. et al. Divergent effects of the H50Q and G51D \u003cem\u003eSNCA\u003c/em\u003e mutations on the aggregation of a-synuclein. J Neurochem 131, 859\u0026ndash;867 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFares, M.B. et al. The novel Parkinson\u0026rsquo;s disease linked mutation G51D attenuates \u003cem\u003ein vitro\u003c/em\u003e aggregation and membrane binding of a-synuclein and enhances its secretion and nuclear localization in cells. Hum. Mol. Genet. 23, 4491\u0026ndash;4509 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlagmeier, P. et al. Mutations associated with familial Parkinson\u0026rsquo;s disease alter the initiation and amplification steps of alpha-synuclein aggregation. Proc. Natl. Acad. Sci. U.S.A. 113, 10328\u0026ndash;10333 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStefanovic, A.N. et al. Oligomers of Parkinson\u0026rsquo;s disease-related alpha-synuclein mutants have similar structures but distinctive membrane permeabilization properties. Biochemistry 54, 3142\u0026ndash;3150 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, Y. et al. The hereditary mutation G51D unlocks a distinct fibril strain transmissible to wild-type a-synuclein. Nature Commun. 12, 6252 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan, Y. et al. Pathogenic mutations differentially regulate cell-to-cell transmission of alpha-synuclein. Front. Cell. Neurosci. 14, 159 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalaf, O. et al. The H50Q mutation enhances a-synuclein aggregation, secretion and toxicity. J. Biol. Chem. 289, 21856\u0026ndash;21876 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorcari, R. et al. The H50Q mutation induces a 10-fold decrease in the solubility of a-synuclein. J. Biol. Chem. 290, 2395\u0026ndash;2404 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlauwendraat, C. et al. Insufficient evidence for pathogenicity of SNCA His50Gln (H50Q) in Parkinson\u0026rsquo;s disease. Neurobiol. Aging 64, 159.e5-e8 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerrero-Ferreira, R. et al. Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy. eLife 8, e48907 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLonghena, F. et al. Synapsin III is a key component of a-synuclein fibrils in Lewy bodies of PD brains. Brain Pathol. 28, 875\u0026ndash;888 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyer, D.R. et al. Structures of fibrils formed by a-synuclein hereditary disease mutation H50Q reveal new polymorphs. Nature Struct. Mol. Biol. 26, 1044\u0026ndash;1052 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, K. et al. Parkinson\u0026rsquo;s disease associated mutation E46K of alpha-synuclein triggers the formation of a distinct fibril structure. Nature Commun. 11, 2643 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyer, D.R. et al. The alpha-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure. Proc. Natl. Acad. Sci. USA 117, 3592\u0026ndash;3602 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, Y. et al. Cryo-EM structure of full-length a-synuclein amyloid fibril with Parkinson\u0026rsquo;s disease familial A53T mutation. Cell Res. 30, 360\u0026ndash;362 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, C. et al. Cryo-EM structure of amyloid fibril formed by a-synuclein hereditary A53E mutation reveals a distinct protofilament interface. J. Biol. Chem. 299, 104566 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorley, V. et al. \u003cem\u003eIn vivo\u003c/em\u003e \u003csup\u003e18\u003c/sup\u003eF-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D a-synuclein mutation. Front. Neurosci. 17, 1095761 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuber, S. et al. Generation of G51D and 3D mice reveals decreased a-synuclein tetramer-monomer ratios promote Parkinson\u0026rsquo;s disease phenotypes. N.P.J. Parkinson\u0026rsquo;s Dis. 10, 47 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, Y. et al. Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice. Proc. Natl. Acad. Sci. U.S.A. 121, e2406479121 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan, N.L. et al. Cryo-EM structure of a novel a-synuclein filament subtype from multiple system atrophy. FEBS Lett. 599, 33\u0026ndash;40 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCullinane, P.W. et al. Identical seeding characteristics and cryo-EM filament structures in FTLD-synuclein and typical multiple system atrophy. Neuropathol. Appl. Neurobiol. 51, e70013 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Azzini, M. et al. A novel alpha-synuclein K58 missense variant in a patient with Parkinson\u0026rsquo;s disease. Medrxiv (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFevga, C. et al. A new a-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson\u0026rsquo;s disease. Parkinsonism Relat. Disord. 89, 63\u0026ndash;72 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapasi, A. et al. A novel \u003cem\u003eSNCA\u003c/em\u003e E83Q mutation in a case of dementia with Lewy bodies and atypical frontotemporal lobar degeneration. Neuropathology 40, 620\u0026ndash;626 92020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBr\u0026uuml;cke, C. et al. A novel G14R missense variant is associated with atypical neuropathological features. Medrxiv (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCali, F. et al. Interpreting genetic variants: Hints from a family cluster of Parkinson\u0026rsquo;s disease. J. Parkinson\u0026rsquo;s Dis. 9, 203\u0026ndash;206 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerrin, R.J. et al. Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins. J. Biol. Chem. 289, 21856\u0026ndash;21876 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnead, D. \u0026amp; Eliezer, D. Alpha-synuclein function and dysfunction in cellular membranes. Exp. Neurobiol. 23, 292\u0026ndash;313 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUchihara, T. et al. Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: Comparison between Gallyas-Braak and Campbell-Switzer methods. Acta Neuropathol. 110, 255\u0026ndash;260 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLau, H.H.C. et al. The G51D \u003cem\u003eSNCA\u003c/em\u003e mutation generates a slowly progressive a-synuclein strain in early-onset Parkinson\u0026rsquo;s disease. Acta Neuropathol. Commun. 11, 72 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6490169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6490169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGene dosage and point mutations in \u003cem\u003eSNCA\u003c/em\u003e, the a-synuclein gene, give rise to familial forms of Parkinson\u0026rsquo;s disease and dementia with Lewy bodies; an insertion mutation in \u003cem\u003eSNCA\u003c/em\u003e causes juvenile-onset synucleinopathy. We previously reported the electron cryo-microscopy (cryo-EM) structures of a-synuclein filaments from the brains of individuals with Parkinson\u0026rsquo;s disease, dementia with Lewy bodies and multiple system atrophy, as well as from the brain of an individual with juvenile-onset synucleinopathy. Here we report the cryo-EM structures of a-synuclein filaments from the frontal cortex of two cases with Parkinsonism and mutation G51D in a-synuclein and those from the amygdala of a case with Parkinson\u0026rsquo;s disease and variant H50Q in a-synuclein. The G51D filaments of assembled a-synuclein consist of two identical protofilaments with the Lewy fold and island B, but without the previously identified disconnected density island A. The protofilament interface is made of residues E46, V48 and H50. Filaments with the H50Q variant comprise a single protofilament with the Lewy fold and both islands A and B. Unlike G51D, the pathogenicity of H50Q has been questioned. It remains to be seen if dimerisation of the Lewy fold may also underlie the pathogenicity of other missense mutations in a-synuclein. Moreover, filaments with a single Lewy fold have a right-handed helical twist, while the G51D, multiple system atrophy and juvenile-onset synucleinopathy filaments are left-handed, which may also be significant.\u003c/p\u003e","manuscriptTitle":"Cryo-EM structures of filaments from the brains of individuals with variants G51D and H50Q in α-synuclein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 11:26:48","doi":"10.21203/rs.3.rs-6490169/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-structural-and-molecular-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nsmb","sideBox":"Learn more about [Nature Structural \u0026 Molecular Biology](http://www.nature.com/nsmb/)","snPcode":"","submissionUrl":"","title":"Nature Structural \u0026 Molecular Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4d6c8ba3-9186-4ebf-a72f-87d471fa01ea","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47835520,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease"},{"id":47835521,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":47835522,"name":"Biological sciences/Molecular biology/Protein folding/Protein aggregation"},{"id":47835523,"name":"Health sciences/Diseases/Neurological disorders/Neurodegenerative diseases/Parkinson's disease"}],"tags":[],"updatedAt":"2025-05-09T20:10:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 11:26:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6490169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6490169","identity":"rs-6490169","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}