{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T14:56:57Z","timestamp":1787237817662,"version":"build-2736575974"},"posted":{"date-parts":[[2025,7,4]]},"group-title":"Neuroscience","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T00:00:00Z","timestamp":1751587200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["U24 GM139168"],"award-info":[{"award-number":["U24 GM139168"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["P41 GM136463"],"award-info":[{"award-number":["P41 GM136463"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RF1 NS103873"],"award-info":[{"award-number":["RF1 NS103873"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["U19 NS110456"],"award-info":[{"award-number":["U19 NS110456"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RF1 NS110436"],"award-info":[{"award-number":["RF1 NS110436"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["T32 GM135066"],"award-info":[{"award-number":["T32 GM135066"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["F32 GM149118"],"award-info":[{"award-number":["F32 GM149118"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["NIH T32 GM133398"],"award-info":[{"award-number":["NIH T32 GM133398"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["NIH S10 OD030460"],"award-info":[{"award-number":["NIH S10 OD030460"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"award":["NSF CHE 1827457"],"award-info":[{"award-number":["NSF CHE 1827457"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Fibrillar aggregation of \u03b1-synuclein (\u03b1Syn) is a hallmark of Parkinson\u2019s disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in \u03b1Syn fibril structural characterization, the relevance of\n                  <jats:italic>in vitro<\/jats:italic>\n                  and\n                  <jats:italic>ex vivo<\/jats:italic>\n                  structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of \u03b1Syn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize \u03b1Syn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. F\u00f6rster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [\n                  <jats:sup>3<\/jats:sup>\n                  H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.\n                <\/jats:p>","DOI":"10.1101\/2025.06.30.662461","type":"posted-content","created":{"date-parts":[[2025,7,4]],"date-time":"2025-07-04T17:41:26Z","timestamp":1751650886000},"source":"Crossref","is-referenced-by-count":6,"title":["Complementary Structural and Chemical Biology Methods Reveal the Basis for Selective Radioligand Binding to \u03b1-Synuclein in MSA Tissue"],"prefix":"10.64898","author":[{"given":"Juan C.","family":"Sanchez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryann M.","family":"Perez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dinahlee Saturnino","family":"Guarino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dhruva","family":"Dhavala","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph K.","family":"Baumgardt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyle D.","family":"Shaffer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Kotzbauer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert H.","family":"Mach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"E. James","family":"Petersson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0712-9475","authenticated-orcid":false,"given":"Elizabeth R.","family":"Wright","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025070714410108000_2025.06.30.662461v1.1","doi-asserted-by":"publisher","DOI":"10.1186\/s13024-021-00501-z"},{"key":"2025070714410108000_2025.06.30.662461v1.2","first-page":"e414","article-title":"Clinical Diagnostic Accuracy of Early\/Advanced Parkinson Disease","volume":"11","year":"2021","journal-title":"Neurology Clinical Practice"},{"key":"2025070714410108000_2025.06.30.662461v1.3","doi-asserted-by":"publisher","DOI":"10.1001\/archneur.57.3.369"},{"key":"2025070714410108000_2025.06.30.662461v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.abo1557"},{"key":"2025070714410108000_2025.06.30.662461v1.5","doi-asserted-by":"publisher","DOI":"10.1016\/S1474-4422(23)00109-6"},{"key":"2025070714410108000_2025.06.30.662461v1.6","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2024.0792"},{"key":"2025070714410108000_2025.06.30.662461v1.7","doi-asserted-by":"publisher","DOI":"10.1212\/WNL.54.10.1916"},{"key":"2025070714410108000_2025.06.30.662461v1.8","doi-asserted-by":"publisher","DOI":"10.1002\/acn3.51482"},{"key":"2025070714410108000_2025.06.30.662461v1.9","doi-asserted-by":"publisher","DOI":"10.1002\/mds.21956"},{"key":"2025070714410108000_2025.06.30.662461v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(94)00395-5"},{"key":"2025070714410108000_2025.06.30.662461v1.11","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.16.9443"},{"key":"2025070714410108000_2025.06.30.662461v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/0896-6273(95)90302-X"},{"key":"2025070714410108000_2025.06.30.662461v1.13","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1002\/bip.10179","article-title":"Structural transformation and aggregation of human \u03b1\u2010synuclein in trifluoroethanol: Non\u2010amyloid component sequence is essential and \u03b2\u2010sheet formation is prerequisite to aggregation","volume":"64","year":"2002","journal-title":"Biopolymers: Original Research on Biomolecules"},{"key":"2025070714410108000_2025.06.30.662461v1.14","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M107045200"},{"key":"2025070714410108000_2025.06.30.662461v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(98)01146-6"},{"key":"2025070714410108000_2025.06.30.662461v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2022.167859"},{"key":"2025070714410108000_2025.06.30.662461v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/nrn3406"},{"key":"2025070714410108000_2025.06.30.662461v1.18","doi-asserted-by":"crossref","unstructured":"R. Khounlo et al., Membrane Binding of \u03b1-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore. Frontiers in Cell and Developmental Biology 9 (2021).","DOI":"10.3389\/fcell.2021.663431"},{"key":"2025070714410108000_2025.06.30.662461v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/nature10324"},{"key":"2025070714410108000_2025.06.30.662461v1.20","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1113260108"},{"key":"2025070714410108000_2025.06.30.662461v1.21","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.11.6469"},{"key":"2025070714410108000_2025.06.30.662461v1.22","doi-asserted-by":"crossref","unstructured":"R. Guerrero-Ferreira et al., Cryo-EM structure of alpha-synuclein fibrils. Elife 7 (2018).","DOI":"10.7554\/eLife.36402"},{"key":"2025070714410108000_2025.06.30.662461v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-018-0075-x"},{"key":"2025070714410108000_2025.06.30.662461v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41594-019-0322-y"},{"key":"2025070714410108000_2025.06.30.662461v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-16386-3"},{"key":"2025070714410108000_2025.06.30.662461v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-020-0299-4"},{"key":"2025070714410108000_2025.06.30.662461v1.27","first-page":"879","article-title":"Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson\u2019s disease and dementia with Lewy bodies","volume":"152","year":"1998","journal-title":"Am J Pathol"},{"key":"2025070714410108000_2025.06.30.662461v1.28","doi-asserted-by":"publisher","DOI":"10.1007\/s00401-023-02550-8"},{"key":"2025070714410108000_2025.06.30.662461v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-05319-3"},{"key":"2025070714410108000_2025.06.30.662461v1.30","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-020-2317-6"},{"key":"2025070714410108000_2025.06.30.662461v1.31","doi-asserted-by":"publisher","DOI":"10.1007\/s12272-025-01538-0"},{"key":"2025070714410108000_2025.06.30.662461v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/s41582-024-00942-2"},{"key":"2025070714410108000_2025.06.30.662461v1.33","doi-asserted-by":"publisher","DOI":"10.1039\/d0sc02159h"},{"key":"2025070714410108000_2025.06.30.662461v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.3194"},{"key":"2025070714410108000_2025.06.30.662461v1.35","doi-asserted-by":"publisher","DOI":"10.1101\/2025.04.30.651534"},{"key":"2025070714410108000_2025.06.30.662461v1.36","first-page":"e5171","article-title":"High-resolution Cryo-EM Structure Determination of a-Synuclein\u2014A Prototypical Amyloid Fibril","volume":"15","year":"2025","journal-title":"Bioprotocol"},{"key":"2025070714410108000_2025.06.30.662461v1.37","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4633"},{"key":"2025070714410108000_2025.06.30.662461v1.38","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv314"},{"key":"2025070714410108000_2025.06.30.662461v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.05.022"},{"key":"2025070714410108000_2025.06.30.662461v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2021.08.013"},{"key":"2025070714410108000_2025.06.30.662461v1.41","doi-asserted-by":"publisher","DOI":"10.1039\/C7OB00582B"},{"key":"2025070714410108000_2025.06.30.662461v1.42","doi-asserted-by":"publisher","DOI":"10.1039\/C5OB02329G"},{"key":"2025070714410108000_2025.06.30.662461v1.43","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.1c02509"},{"key":"2025070714410108000_2025.06.30.662461v1.44","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-50315-y"},{"key":"2025070714410108000_2025.06.30.662461v1.45","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-023-01370-x"},{"key":"2025070714410108000_2025.06.30.662461v1.46","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.06.004"},{"key":"2025070714410108000_2025.06.30.662461v1.47","doi-asserted-by":"publisher","DOI":"10.1007\/s00259-024-06806-7"},{"key":"2025070714410108000_2025.06.30.662461v1.48","doi-asserted-by":"publisher","DOI":"10.1021\/acschemneuro.8b00280"},{"key":"2025070714410108000_2025.06.30.662461v1.49","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.3c01749"},{"key":"2025070714410108000_2025.06.30.662461v1.50","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.17.9459"},{"key":"2025070714410108000_2025.06.30.662461v1.51","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.3c00779"},{"key":"2025070714410108000_2025.06.30.662461v1.52","doi-asserted-by":"publisher","DOI":"10.1039\/C9CC09849F"},{"key":"2025070714410108000_2025.06.30.662461v1.53","doi-asserted-by":"publisher","DOI":"10.1002\/2211-5463.13110"},{"key":"2025070714410108000_2025.06.30.662461v1.54","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1810058115"},{"key":"2025070714410108000_2025.06.30.662461v1.55","doi-asserted-by":"publisher","DOI":"10.1006\/jsbi.1996.0013"},{"key":"2025070714410108000_2025.06.30.662461v1.56","doi-asserted-by":"publisher","DOI":"10.1042\/BCJ20210708"},{"key":"2025070714410108000_2025.06.30.662461v1.57","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2015.11.003"},{"key":"2025070714410108000_2025.06.30.662461v1.58","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4193"},{"key":"2025070714410108000_2025.06.30.662461v1.59","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-019-0575-8"},{"key":"2025070714410108000_2025.06.30.662461v1.60","doi-asserted-by":"publisher","DOI":"10.1039\/D2FD00034B"},{"key":"2025070714410108000_2025.06.30.662461v1.61","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2017.02.003"},{"key":"2025070714410108000_2025.06.30.662461v1.62","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1107\/S2059798319016577","article-title":"Amyloid structure determination in RELION-3.1","volume":"76","year":"2020","journal-title":"Acta Crystallogr D Struct Biol"},{"key":"2025070714410108000_2025.06.30.662461v1.63","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"2025070714410108000_2025.06.30.662461v1.64","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444904019158"},{"key":"2025070714410108000_2025.06.30.662461v1.65","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798319011471\/HTTPS:\/\/GOLDBOOK.IUPAC.ORG\/E02002.HTML"},{"key":"2025070714410108000_2025.06.30.662461v1.66","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798318006551"},{"key":"2025070714410108000_2025.06.30.662461v1.67","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909029436"},{"key":"2025070714410108000_2025.06.30.662461v1.68","doi-asserted-by":"publisher","DOI":"10.1002\/PRO.4792"},{"key":"2025070714410108000_2025.06.30.662461v1.69","doi-asserted-by":"publisher","DOI":"10.1002\/PRO.3943"},{"key":"2025070714410108000_2025.06.30.662461v1.70","doi-asserted-by":"publisher","DOI":"10.1021\/ac300540r"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.06.30.662461","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:03:34Z","timestamp":1768467814000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.06.30.662461"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,4]]},"references-count":70,"URL":"https:\/\/doi.org\/10.1101\/2025.06.30.662461","relation":{},"subject":[],"published":{"date-parts":[[2025,7,4]]},"subtype":"preprint"}}