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 micros...

Full description

Saved in:
Bibliographic Details
Published in:Structure (London) 2010-01, Vol.18 (1), p.17-27
Main Authors: Chang, Wei-Hau, Chiu, Michael T.-K., Chen, Chin-Yu, Yen, Chi-Fu, Lin, Yen-Cheng, Weng, Yi-Ping, Chang, Ji-Chau, Wu, Yi-Min, Cheng, Holland, Fu, Jianhua, Tu, I-Ping
Format: Article
Language:English
Subjects:
Bacteriophage T7
Cryoelectron Microscopy - methods
Muramidase - analysis
Muramidase - ultrastructure
PROTEINS
Receptors, Transferrin - analysis
Receptors, Transferrin - ultrastructure
RNA Polymerase II - analysis
RNA Polymerase II - ultrastructure
Saccharomyces cerevisiae
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0969-2126
1878-4186
DOI:10.1016/j.str.2009.12.001