Abstract：
:While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.
journal_name
J Struct Bioljournal_title
Journal of structural biologyauthors
Schaffer M,Mahamid J,Engel BD,Laugks T,Baumeister W,Plitzko JMdoi
10.1016/j.jsb.2016.07.010subject
Has Abstractpub_date
2017-02-01 00:00:00pages
73-82issue
2eissn
1047-8477issn
1095-8657pii
S1047-8477(16)30151-4journal_volume
197pub_type
杂志文章abstract：:Hitherto, the packing arrangement of the aquaporin-1 (AQP1) tetramer in 2-dimensional (2-D) crystals (two-sided plane group p42(1)2) was observed to be largely similar (canonical crystal form) despite the difference in the source of the protein, the glycosylation state of the protein, the type of lipids, and the ratio...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.2000.4211
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abstract：:Chaperonins mediate protein folding in an ATP-dependent manner. ATP binding and hydrolysis by chaperonins are subject to both homotropic and heterotropic allosteric regulation. In the case of GroEL and CCT, homotropic regulation by ATP is manifested in nested cooperativity, which involves positive intra-ring cooperati...
journal_title：Journal of structural biology
pub_type： 杂志文章,评审
doi：10.1006/jsbi.2001.4377
更新日期：2001-08-01 00:00:00
abstract：:Third harmonic generation microscopy is shown to be a robust method for obtaining structural information on a variety of biological specimens. Its nature allows depth-resolved imaging of inhomogeneities with virtually no background from surrounding homogeneous media. With an appropriate illumination geometry, third ha...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/S1047-8477(03)00125-4
更新日期：2004-07-01 00:00:00
abstract：:Antifreeze proteins have the ability to bind to ice with high affinity and inhibit further crystal growth. The insect antifreeze protein from spruce budworm exhibits very high thermal hysteresis activity and is implicated in the protection of overwintering larvae from freezing. This protein has been crystallized in 20...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.1999.4087
更新日期：1999-06-01 00:00:00
abstract：:Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins. The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins. GroEL uses a nonspecific mechanism by which any conformation of practically an...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.2001.4359
更新日期：2001-08-01 00:00:00
abstract：:It has been postulated that phosphophoryn (PP) molecules bind specifically to type I collagen fibrils as the key event in inducing matrix mineralization in dentin. The nature and specificity of the collagen molecule-PP interaction has been examined by rotary shadowing-electron microscopy of mixtures of native, monomer...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.1998.4025
更新日期：1998-10-01 00:00:00
abstract：:We have studied two members of the family of morphogenetic factors or chaperonins, the GroEL-like factors from Escherichia coli and Bacillus subtilis, in order to determine the possible structural basis of their related function in promoting the correct and efficient assembly of biological oligomers. The main objectiv...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/1047-8477(91)90071-4
更新日期：1991-06-01 00:00:00
abstract：:Gap junctions containing extensive, highly ordered crystalline arrays of hexagonally packed connexons have been isolated from the hepatopancreas of the arthropod, Homarus americanus (American lobster). The structure of such junctions has been studied to a resolution of approximately 25 A in three dimensions by electro...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/1047-8477(91)90075-8
更新日期：1991-06-01 00:00:00
abstract：:The Omp21 protein from the proteobacterium Comamonas (Delftia) acidovorans belongs to the recently described beta8 family of outer membrane proteins, characterized by eight antiparallel beta-strands which form a beta-barrel. This family includes virulence proteins, OmpA and OmpX from Escherichia coli, and other relate...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.2000.4261
更新日期：2000-08-01 00:00:00
abstract：:Gap junction proteins, termed connexins, constitute a multigene family of polytopic membrane channel proteins that have four hydrophobic transmembrane domains with the N- and C-termini located on the cytoplasmic membrane face. The principal gap junction protein in the heart, alpha 1 connexin (also designated Cx43), me...
journal_title：Journal of structural biology
pub_type： 杂志文章,评审
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更新日期：1998-01-01 00:00:00
abstract：:Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for dete...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2007.09.003
更新日期：2007-12-01 00:00:00
abstract：:Accurately determining a cryoEM particle's alignment parameters is crucial to high resolution single particle 3-D reconstruction. We developed Multi-Path Simulated Annealing, a Monte-Carlo type of optimization algorithm, for globally aligning the center and orientation of a particle simultaneously. A consistency crite...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2007.06.009
更新日期：2007-10-01 00:00:00
abstract：:The properties and characteristics shared by amyloid fibrils formed from disease and non-disease associated proteins that are unrelated in sequence and structure offer the prospect that model systems can be used to systematically assess the factors that predispose a native protein to form amyloid fibrils. Based on a d...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2006.01.017
更新日期：2006-08-01 00:00:00
abstract：:Although the fundamental steps in Fourier-based image analysis of electron micrographs of helical structures have not changed significantly since the techniques were developed 30 years ago, there have been developments which aid both the analysis itself and the interpretation of results. Increases in computational res...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.1996.0027
更新日期：1996-01-01 00:00:00
abstract：:Since the foundation for the three-dimensional image reconstruction of helical objects from electron micrographs was laid more than 30 years ago, there have been sustained developments in specimen preparation, data acquisition, image analysis, and interpretation of results. However, the boxing of filaments in large nu...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.2001.4415
更新日期：2001-09-01 00:00:00
abstract：:Structures of biological macromolecules determined by transmission cryoelectron microscopy (cryo-TEM) and three-dimensional image reconstruction are often displayed as surface-shaded representations with depth cueing along the viewed direction (Z cueing). Depth cueing to indicate distance from the center of virus part...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1006/jsbi.1997.3902
更新日期：1997-10-01 00:00:00
abstract：:The 32 kDa hydrophilic and acidic enamelin, the most stable cleavage fragment of the enamel specific glycoprotein, is believed to play vital roles in controlling crystal nucleation or growth during enamel biomineralization. Circular dichroism and Fourier transform infrared spectra demonstrate that the secondary struct...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2008.04.007
更新日期：2008-07-01 00:00:00
abstract：:Virus particle 24 (VP24) is the smallest protein of the Ebola and Marburg virus genomes. Recent experiments show that Ebola VP24 blocks binding of tyrosine-phosphorylated STAT-1 homodimer (PY-STAT1) to the NPI-1 subfamily of importin alpha, thereby preventing nuclear accumulation of this interferon-promoting transcrip...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2009.05.001
更新日期：2009-08-01 00:00:00
abstract：:A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several importa...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2008.01.008
更新日期：2008-05-01 00:00:00
abstract：:Protein tertiary structure modeling is a critical step for the interpretation of three dimensional (3D) election microscopy density. Our group participated the 2015/2016 EM Model Challenge using the MAINMAST software for a de novo main chain modeling. The software generates local dense points using the mean shifting a...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2018.07.013
更新日期：2018-11-01 00:00:00
abstract：:The crystallographic structures of the adducts of salicylate 1,2-dioxygenase (SDO) with substrates salicylate, gentisate and 1-hydroxy-2-naphthoate, obtained under anaerobic conditions, have been solved and analyzed. This ring fission dioxygenase from the naphthalenesulfonate-degrading bacterium Pseudaminobacter salic...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2011.11.026
更新日期：2012-02-01 00:00:00
abstract：:Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must ...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2006.12.002
更新日期：2007-06-01 00:00:00
abstract：:The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2005.01.006
更新日期：2005-04-01 00:00:00
abstract：:Three-dimensional reconstruction of large macromolecules like viruses at resolutions below 10 A requires a large set of projection images. Several automatic and semi-automatic particle detection algorithms have been developed along the years. Here we present a general technique designed to automatically identify the p...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2003.11.028
更新日期：2004-01-01 00:00:00
abstract：:The formation of amyloid is considered an intrinsic ability of most polypeptides. It is a structure adopted by many neuropeptides and neurohormones during the formation of dense core vesicles in secretory cells, yet the mechanisms mediating assembly and disassembly of these amyloids remain unclear. Neurokinin B is a n...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2019.09.011
更新日期：2019-12-01 00:00:00
abstract：:We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen. Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three-dimensional reconstruction of the macromolecular structure. The process...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2003.10.008
更新日期：2003-10-01 00:00:00
abstract：:The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that ca...
journal_title：Journal of structural biology
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doi：10.1016/j.jsb.2020.107531
更新日期：2020-08-01 00:00:00
abstract：:HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occ...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2018.03.011
更新日期：2018-08-01 00:00:00
abstract：:We describe a new implementation for the reconstruction of helical assemblies in the empirical Bayesian framework of RELION. Our approach calculates optimal linear filters for the 3D reconstruction by embedding helical symmetry operators in Fourier-space, and deals with deviations from perfect helical symmetry through...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2017.02.003
更新日期：2017-06-01 00:00:00
abstract：:Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these ...
journal_title：Journal of structural biology
pub_type： 杂志文章
doi：10.1016/j.jsb.2010.11.004
更新日期：2011-03-01 00:00:00
