Electron cryotomography sample preparation using the Vitrobot

被引:141
作者
Iancu, Cristina V. [1 ]
Tivol, William F. [1 ]
Schooler, Jordan B. [1 ]
Dias, D. Prabha [1 ]
Henderson, Gregory P. [1 ]
Murphy, Gavin E. [1 ]
Wright, Elizabeth R. [1 ]
Li, Zhuo [1 ]
Yu, Zhiheng [1 ]
Briegel, Ariane [1 ]
Gan, Lu [1 ]
He, Yongning [1 ]
Jensen, Grant J. [1 ]
机构
[1] CALTECH, Div Biol, Pasadena, CA 91125 USA
关键词
D O I
10.1038/nprot.2006.432
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5-2 h.
引用
收藏
页码:2813 / 2819
页数:7
相关论文
共 18 条
[1]   DIRECT VISUALIZATION OF SUPERCOILED DNA-MOLECULES IN SOLUTION [J].
ADRIAN, M ;
TENHEGGELERBORDIER, B ;
WAHLI, W ;
STASIAK, AZ ;
STASIAK, A ;
DUBOCHET, J .
EMBO JOURNAL, 1990, 9 (13) :4551-4554
[2]   CRYO-ELECTRON MICROSCOPY OF VIRUSES [J].
ADRIAN, M ;
DUBOCHET, J ;
LEPAULT, J ;
MCDOWALL, AW .
NATURE, 1984, 308 (5954) :32-36
[3]   Amorphous water [J].
Angell, CA .
ANNUAL REVIEW OF PHYSICAL CHEMISTRY, 2004, 55 :559-583
[4]   Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography [J].
Benjamin, J ;
Ganser-Pornillos, BK ;
Tivol, WF ;
Sundquist, WI ;
Jensen, GJ .
JOURNAL OF MOLECULAR BIOLOGY, 2005, 346 (02) :577-588
[5]   Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography [J].
Briegel, Ariane ;
Dias, D. Prabha ;
Li, Zhuo ;
Jensen, Rasmus B. ;
Frangakis, Achilleas S. ;
Jensen, Grant J. .
MOLECULAR MICROBIOLOGY, 2006, 62 (01) :5-14
[6]  
Dubochet J., 1981, J MICROSC-OXFORD, V124, P3
[7]   Cryoelectron microscopy of liposomes [J].
Frederik, PM ;
Hubert, DHW .
LIPOSOMES, PT E, 2005, 391 :431-+
[8]   Three-dimensional structure of Mycoplasma pneumoniae's attachment organelle and a model for its role in gliding motility [J].
Henderson, GP ;
Jensen, GJ .
MOLECULAR MICROBIOLOGY, 2006, 60 (02) :376-385
[9]   A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography [J].
Iancu, CV ;
Wright, ER ;
Heymann, JB ;
Jensen, GJ .
JOURNAL OF STRUCTURAL BIOLOGY, 2006, 153 (03) :231-240
[10]   A "flip-flop" rotation stage for routine dual-axis electron cryotomography [J].
Iancu, CV ;
Wright, ER ;
Benjamin, J ;
Tivol, WF ;
Dias, DP ;
Murphy, GE ;
Morrison, RC ;
Heymann, JB ;
Jensen, GJ .
JOURNAL OF STRUCTURAL BIOLOGY, 2005, 151 (03) :288-297