Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination

Several factors, including spatial and temporal coherence of the electron m icroscope, specimen movement, recording medium, and scanner optics, contrib ute to the decay of the measured Fourier amplitude in electron image intens ities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimenta l B-factor, We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering a nd numerical fitting to the average power spectrum of particle images. A st atistical estimation from over 200 micrographs of herpes simplex virus type -1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the o bjective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles req uired to determine the structure at a lower resolution can be used to estim ate the minimum fold increase in the number of particles that would be requ ired to extend a single particle reconstruction to a specified higher resol ution. We conclude that microscope and imaging improvements to reduce the e xperimental B-factor will be critical for obtaining an atomic resolution st ructure. (C) 2001 Academic Press.