Zernike phase plate cryoelectron microscopy facilitates single particle analysis of unstained asymmetric protein complexes.
Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa). Our results show Zernike phase plate imaging is more effective as to particle identification and also sorting of orientations, conformations, and compositions. Moreover, our analysis on image alignment indicates that Zernike phase plate can, in principle, reduce the number of particles required to attain near atomic resolution by 10-100 fold for proteins between 100 kDa and 500 kDa.
Chang
Wei-Hau
WH
Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan. weihau40@gmail.com <weihau40@gmail.com>
Chiu
Michael T-K
MT
Chen
Chin-Yu
CY
Yen
Chi-Fu
CF
Lin
Yen-Cheng
YC
Weng
Yi-Ping
YP
Chang
Ji-Chau
JC
Wu
Yi-Min
YM
Cheng
Holland
H
Fu
Jianhua
J
Tu
I-Ping
IP
D017123:Bacteriophage T7; D020285:Cryoelectron Microscopy / Q000379:methods*; D009113:Muramidase / Q000032:analysis* / Q000648:ultrastructure; D012319:RNA Polymerase II / Q000032:analysis* / Q000648:ultrastructure; D011990:Receptors, Transferrin / Q000032:analysis* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae
D011990:Receptors, Transferrin; D012319:RNA Polymerase II; D009113:Muramidase