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The pathogenic agent, prion, is a protease\u2010resistant, \u03b2\u2010sheet\u2010rich protein aggregate, converted from a membrane protein called PrP<jats:sup>C<\/jats:sup>. PrP<jats:sup>Sc<\/jats:sup> is the misfolded form of PrP<jats:sup>C<\/jats:sup> and undergoes self\u2010propagation to form the infectious amyloids. Since the key hallmark of prion disease is amyloid formation, identifying and studying which segments are involved in the amyloid core can provide molecular details about prion diseases. It has been known that the prion protein could also form non\u2010infectious fibrils in the presence of denaturants. In this study, we employed a combination of site\u2010directed nitroxide spin\u2010labeling, fibril seeding, and electron spin resonance (ESR) spectroscopy to identify the structure of the in vitro\u2010prepared full\u2010length mouse prion fibrils. It is shown that in the in vitro amyloidogenesis, the formation of the amyloid core is linked to an \u03b1\u2010to\u2010\u03b2 structural transformation involving the segment 160\u2010224, which contains strand 2, helix 2, and helix 3. This method is particularly suitable for examining the hetero\u2010seeded amyloid fibril structure, as the unlabeled seeds are invisible by ESR spectroscopy. It can be applied to study the structures of different strains of infectious prions or other amyloid fibrils in the future.<\/jats:p>","DOI":"10.1002\/pro.4326","type":"journal-article","created":{"date-parts":[[2022,5,17]],"date-time":"2022-05-17T10:57:05Z","timestamp":1652785025000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Location of the cross\u2010\u03b2 structure in prion fibrils: A search by seeding and electron spin resonance spectroscopy"],"prefix":"10.1002","volume":"31","author":[{"given":"Brett K.\u2010Y.","family":"Chu","sequence":"first","affiliation":[{"name":"Institute of Biological Chemistry Academia Sinica  Taipei Taiwan"},{"name":"Department of Chemistry National Taiwan University  Taipei Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruei\u2010Fong","family":"Tsai","sequence":"additional","affiliation":[{"name":"Department of Chemistry National Tsing Hua University 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