biostudies-literatureUnknownMirzakhanyan YHHS | National Institutes of HealthNIH HHSNIGMS NIH HHSVaccinia virus, the prototypical poxvirus and smallpox/monkeypox vaccine, has proven a challenging entity for structural biology, defying many of the approaches leading to molecular and atomic models for other viruses. Via a combination of deep learning and cross-linking mass spectrometry, we have developed an atomic-level model and an integrated processing/assembly pathway for a structural component of the vaccinia virion, protein P4a. Within the pathway, proteolytic separation of the C-terminal P4a-3 segment of P4a triggers a massive conformational rotation within the N-terminal P4a-1 segment that becomes fixed by disulfide-locking while removing a steric block to trimerization of the processing intermediate P4a-1+2. These events trigger the proteolytic separation of P4a-2, allowing the mBioe0113523https://www.ebi.ac.uk/biostudies/studies/S-EPMC10653903biostudies-literatureCombination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion.PMC106539031R01GM134144-01S10 OD016328R01 GM134144Mirzakhanyan YJankevics AGershon PDScheltema RAfalseCombination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion.Vaccinia virus, the prototypical poxvirus and smallpox/monkeypox vaccine, has proven a challenging entity for structural biology, defying many of the approaches leading to molecular and atomic models for other viruses. Via a combination of deep learning and cross-linking mass spectrometry, we have developed an atomic-level model and an integrated processing/assembly pathway for a structural component of the vaccinia virion, protein P4a. Within the pathway, proteolytic separation of the C-terminal P4a-3 segment of P4a triggers a massive conformational rotation within the N-terminal P4a-1 segment that becomes fixed by disulfide-locking while removing a steric block to trimerization of the processing intermediate P4a-1+2. These events trigger the proteolytic separation of P4a-2, allowing the 2023-01-01T00:00:00Z2023 Aug2025-04-03T22:05:45.707Z2024-10-15T11:03:09.773ZS-EPMC106539033764653110.1128/mbio.01135-23