Formation of filaments by the Ure2 protein constitutes the molecular mechanism of the [URE3] prion in yeast. According to the &DPRIME; amyloid backbone&DPRIME; model, the N-terminal asparagine-rich domains of Ure2p polymerize to form an amyloid core fibril that is surrounded by C-terminal domains in their native conformation. Protease resistance and Congo Red binding as well as β-sheet content detected by spectroscopy-all markers for amyloid-have supported this model, as has the close resemblance between 40 &ANGS; N-domain fibrils and the fibrillar core of intact Ure2p filaments visualized by cryo-electron microscopy and scanning transmission electron microscopy. Here, we present electron diffraction and X-ray diffraction data from filaments of Ure2p, of N-domains alone, of fragments thereof, and of an N-domain-containing fusion protein that demonstrate in each case the 4.7 &ANGS; reflection that is typical for cross-β structure and highly indicative of amyloid. This reflection was observed for specimens prepared by air-drying with and without sucrose embedding. To confirm that the corresponding Structure is not an artifact of air-drying, the reflection was also demonstrated for specimens preserved in vitreous ice. Local area electron diffraction and X-ray diffraction from partially aligned specimens showed that the 4.7 &ANGS; reflection is meridional and therefore the underlying structure is cross-β. Published by Elsevier Inc.