Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry.
During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement. Here we analyze how errors in the subjective assignment of this position affect the determination of symmetry, and the accuracy of calculating Euler angles for each raw image. In this study we have optimized the method and applied it to study the membrane-attachment complex of Semliki Forest virus (SFV), a model system for enveloped virus fusion. The resulting reconstruction of the SFV-membrane complex with a fiducial provides the first experimental evidence that this pre-fusion cell entry intermediate approaches the membrane along the viral 5-fold axis. The analysis reported here, and its subsequent application to enveloped virus fusion, indicate that this is a robust tool for solving the structures of mixed-symmetry complexes.
Bubeck
Doryen
D
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK.
Filman
David J
DJ
Kuzmin
Mikhail
M
Fuller
Stephen D
SD
Hogle
James M
JM
D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D008854:Microscopy, Electron / Q000379:methods; D011991:Receptors, Virus / Q000378:metabolism; D015203:Reproducibility of Results; D012672:Semliki forest virus / Q000378:metabolism; D012984:Software; D014759:Viral Envelope Proteins / Q000737:chemistry*
D008081:Liposomes; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; C066100:poliovirus receptor