biostudies-literatureUnknown12(3)Lin CSMembrane fusion is essential for viral entry. Unlike class I-III fusion proteins, vaccinia virus (VACV) uses a multicomponent entry fusion complex (EFC). Using cryo-electron microscopy, we determined the full-length structure of the VACV EFC at near-atomic resolution, revealing a 15-protein asymmetric assembly organized into three layers. The central A16/G9/J5 heterotrimer forms the fusion core, stabilized by conserved PXXCW and Delta motifs, and anchors two A28/H2 adaptor dimers linked to peripheral G3/L5/A21/O3 scaffolds. Structural and evolutionary analyses identify a conserved N-terminal domain in A16 containing a myristoyl-binding pocket and a phenylalanine-rich region that stabilizes the trimer and may regulate lipid engagement. An additional component, F9, binds peripherally to J5, Science advanceseaec0254https://www.ebi.ac.uk/biostudies/studies/S-EPMC12802840biostudies-literatureCryo-EM structure of the vaccinia virus entry fusion complex reveals a multicomponent fusion machinery.PMC12802840Wang CHYeh MCLee HMLin CSGao HDHo MCChiu HJLi CAKao CFChang WfalseCryo-EM structure of the vaccinia virus entry fusion complex reveals a multicomponent fusion machinery.Membrane fusion is essential for viral entry. Unlike class I-III fusion proteins, vaccinia virus (VACV) uses a multicomponent entry fusion complex (EFC). Using cryo-electron microscopy, we determined the full-length structure of the VACV EFC at near-atomic resolution, revealing a 15-protein asymmetric assembly organized into three layers. The central A16/G9/J5 heterotrimer forms the fusion core, stabilized by conserved PXXCW and Delta motifs, and anchors two A28/H2 adaptor dimers linked to peripheral G3/L5/A21/O3 scaffolds. Structural and evolutionary analyses identify a conserved N-terminal domain in A16 containing a myristoyl-binding pocket and a phenylalanine-rich region that stabilizes the trimer and may regulate lipid engagement. An additional component, F9, binds peripherally to J5, 2026-01-01T00:00:00Z2026 Jan2026-06-06T13:16:13.518Z2026-05-31T03:07:09.019ZS-EPMC128028404153378210.1126/sciadv.aec0254