Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging.
Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.
Zanetti
Giulia
G
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Riches
James D
JD
Fuller
Stephen D
SD
Briggs
John A G
JA
D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods