Zernike Phase Plate Cryoelectron Microscopy Facilitates Single Particle Analysis of Unstained Asymmetric Protein Complexes

被引:25
作者
Chang, Wei-Hau [1 ,2 ]
Chiu, Michael T. -K. [1 ]
Chen, Chin-Yu [1 ]
Yen, Chi-Fu [1 ]
Lin, Yen-Cheng [1 ]
Weng, Yi-Ping [1 ]
Chang, Ji-Chau [1 ,2 ]
Wu, Yi-Min [1 ]
Cheng, Holland [3 ]
Fu, Jianhua [4 ]
Tu, I-Ping [5 ]
机构
[1] Acad Sinica, Inst Chem, Taipei 115, Taiwan
[2] Natl Taiwan Univ, Dept Biochem Sci & Technol, Taipei 106, Taiwan
[3] Univ Calif Davis, Dept Mol & Cellular Biol, Davis, CA 95616 USA
[4] Med Coll Wisconsin, Dept Biochem, Milwaukee, WI 53226 USA
[5] Acad Sinica, Inst Stat Sci, Taipei 115, Taiwan
关键词
RNA-POLYMERASE-II; TRANSMISSION ELECTRON-MICROSCOPY; CONFORMATIONAL FLEXIBILITY; CRYO-EM; DARK-FIELD; RESOLUTION; CONTRAST; CLASSIFICATION; IMAGES; MACROMOLECULES;
D O I
10.1016/j.str.2009.12.001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:17 / 27
页数:11
相关论文
共 65 条
[51]   Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization [J].
Scheres, Sjors H. W. ;
Gao, Haixiao ;
Valle, Mikel ;
Herman, Gabor T. ;
Eggermont, Paul P. B. ;
Frank, Joachim ;
Carazo, Jose-Maria .
NATURE METHODS, 2007, 4 (01) :27-29
[52]   Maximum likelihood refinement of electron microscopy data with normalization errors [J].
Scheres, Sjors H. W. ;
Valle, Mikel ;
Grob, Patricia ;
Nogales, Eva ;
Carazo, Jose-Maria .
JOURNAL OF STRUCTURAL BIOLOGY, 2009, 166 (02) :234-240
[53]   Fabrication of a Boersch phase plate for phase contrast imaging in a transmission electron microscope [J].
Schultheiss, K ;
Pérez-Willard, F ;
Barton, B ;
Gerthsen, D ;
Schröder, RR .
REVIEW OF SCIENTIFIC INSTRUMENTS, 2006, 77 (03)
[54]   Phase TEM for biological imaging utilizing a Boersch electrostatic phase plate: theory and practice [J].
Shiue, Jessie ;
Chang, Chia-Seng ;
Huang, Sen-Hui ;
Hsu, Chih-Hao ;
Tsai, Jin-Sheng ;
Chang, Wei-Hau ;
Wu, Yi-Min ;
Lin, Yen-Chen ;
Kuo, Pai-Chia ;
Huang, Yang-Shan ;
Hwu, Yeukuang ;
Kai, Ji-Jung ;
Tseng, Fan-Gang ;
Chen, Fu-Rong .
JOURNAL OF ELECTRON MICROSCOPY, 2009, 58 (03) :137-145
[55]   Automated cryoEM data acquisition and analysis of 284742 particles of GroEL [J].
Stagg, Scott M. ;
Lander, Gabriel C. ;
Pulokas, James ;
Fellmann, Denis ;
Cheng, Anchi ;
Quispe, Joel D. ;
Mallick, Satya P. ;
Avila, Radomir M. ;
Carragher, Bridget ;
Potter, Clinton S. .
JOURNAL OF STRUCTURAL BIOLOGY, 2006, 155 (03) :470-481
[56]   Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle [J].
Stark, H ;
Dube, P ;
Lüehrmann, R ;
Kastner, B .
NATURE, 2001, 409 (6819) :539-542
[57]   Complex observation in electron microscopy: III. Inverse theory of observation-scheme dependent information transfer [J].
Sugitani, S ;
Nagayama, K .
JOURNAL OF THE PHYSICAL SOCIETY OF JAPAN, 2002, 71 (03) :744-756
[58]   ELECTRON-DIFFRACTION OF FROZEN, HYDRATED PROTEIN CRYSTALS [J].
TAYLOR, KA ;
GLAESER, RM .
SCIENCE, 1974, 186 (4168) :1036-1037
[59]   ZUR DEFOKUSSIERUNGSABHANGIGKEIT DES PHASENKONTRASTES BEI DER ELEKTRONENMIKROSKOPISCHEN ABBILDUNG [J].
THON, F .
ZEITSCHRIFT FUR NATURFORSCHUNG PART A-ASTROPHYSIK PHYSIK UND PHYSIKALISCHE CHEMIE, 1966, A 21 (04) :476-&
[60]   Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution [J].
Unwin, N .
JOURNAL OF MOLECULAR BIOLOGY, 2005, 346 (04) :967-989