A strategy for determining the orientations of refractory particles for reconstruction from cryo-electron micrographs with particular reference to round, smooth-surfaced, icosahedral viruses.
Cryo-electron microscopy and three-dimensional image reconstruction are powerful tools for analyzing icosahedral virus capsids at resolutions that now extend below 1 nm. However, the validity of such density maps depends critically on correct identification of the viewing geometry of each particle in the data set. In some cases-for example, round capsids with low surface relief-it is difficult to identify orientations by conventional application of the two most widely used approaches-"common lines" and model-based iterative refinement. We describe here a strategy for determining the orientations of such refractory specimens. The key step is to determine reliable orientations for a base set of particles. For each particle, a list of candidate orientations is generated by common lines: correct orientations are then identified by computing a single-particle reconstruction for each candidate and then systematically matching their reprojections with the original images by visual criteria and cross-correlation analysis. This base set yields a first-generation reconstruction that is fed into the model-based procedure. This strategy has led to the structural determination of two viruses that, in our hands, resisted solution by other means.
Castón
J R
JR
Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892-5624, USA.
Belnap
D M
DM
Steven
A C
AC
Trus
B L
BL
D000465:Algorithms; D002213:Capsid / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D010942:Plant Viruses / Q000648:ultrastructure*; D012328:RNA Viruses / Q000648:ultrastructure*; D014771:Virion / Q000648:ultrastructure*