Accurate determination of local defocus and specimen tilt in electron microscopy

doi:10.1016/S1047-8477(03)00069-8

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Accurate determination of local defocus and specimen tilt in electron microscopy

Journal Article•DOI•

Accurate determination of local defocus and specimen tilt in electron microscopy

Joseph A. Mindell¹, Nikolaus Grigorieff²•Institutions (2)

National Institutes of Health¹, Howard Hughes Medical Institute²

01 Jun 2003-Journal of Structural Biology (J Struct Biol)-Vol. 142, Iss: 3, pp 334-347

TL;DR: Two computer programs are presented, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from image of tilted specimens, respectively, using a simple algorithm.

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About: This article is published in Journal of Structural Biology.The article was published on 2003-06-01. It has received 1480 citations till now. The article focuses on the topics: Tilt (optics) & Contrast transfer function.

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Journal Article•DOI•

New tools for automated high-resolution cryo-EM structure determination in RELION-3.

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Jasenko Zivanov¹, Takanori Nakane¹, Björn O. Forsberg², Dari Kimanius², Wim J. H. Hagen, Erik Lindahl³, Erik Lindahl², Sjors H.W. Scheres¹ - Show less +4 more•Institutions (3)

Laboratory of Molecular Biology¹, Science for Life Laboratory², Royal Institute of Technology³

09 Nov 2018-eLife

TL;DR: CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations in the third major release of RELION.

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Abstract: Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 A compared to previous RELION versions.

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3,520 citations

Cites methods from "Accurate determination of local def..."

...Therefore, in practice it would probably be faster to start from defoci provided by local CTF estimation programs, such as CTFTILT (Mindell and Grigorieff, 2003), Gctf (Zhang, 2016) or Warp (Tegunov and Cramer, 2018)....
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Journal Article•DOI•

CTFFIND4: Fast and accurate defocus estimation from electron micrographs.

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Alexis Rohou¹, Nikolaus Grigorieff¹•Institutions (1)

Howard Hughes Medical Institute¹

01 Nov 2015-Journal of Structural Biology

TL;DR: Modifications to the CTFFIND algorithm are described which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates.

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3,512 citations

Cites background or methods from "Accurate determination of local def..."

...Of those, CTFFIND3 (Mindell and Grigorieff, 2003) is widely used and thought to perform well under a range of circumstances (Marabini et al., 2015)....
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...Of those, CTFFIND3 (Mindell and Grigorieff, 2003) is widely used and thought to perform well under a range of circumstances (Marabini et al....
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...We measured execution times of CTFFIND3 and CTFFIND4 with micrographs of bacteriorhodopsin, which had been used in benchmarking CTFFIND3 (Mindell and Grigorieff, 2003), as inputs....
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...They were also very low (1.75 on average) when processing bacteriorhodopsin micrographs, which have relatively large amounts of astigmatism (12–20% of Df 1; Table 2). d by crystallographic refinement of bacteriorhodopsin (Mindell and Grigorieff, 2003). in nm (Df 1;Df 2) and degrees (aast)....
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...Table 2: Comparison of defocus parameter estimates from CTFFIND3 and CTFFIND4 to those obtained by crystallographic refinement of bacteriorhodopsin (Mindell and Grigorieff, 2003)....
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Journal Article•DOI•

Gctf: Real-time CTF determination and correction

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Kai Zhang¹•Institutions (1)

Laboratory of Molecular Biology¹

01 Jan 2016-Journal of Structural Biology

TL;DR: The main target of Gctf is to maximize the cross-correlation of a simulated CTF with the logarithmic amplitude spectra of observed micrographs after background subtraction to improve CTF parameters of all particles for subsequent image processing.

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2,919 citations

Cites background or methods or result from "Accurate determination of local def..."

...The popular program CTFFIND3 (Mindell and Grigorieff, 2003) shows the most self-consistent results using real datasets in this benchmark test, in spite of a slightly lower rank using simulated micrographs....
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...There are currently several programs available for CTF determination (Ludtke et al., 1999; Mallick et al., 2005; Mindell and Grigorieff, 2003; Penczek et al., 2014; Shaikh et al., 2008; Sorzano et al., 2004; Tang et al., 2007; Vargas et al., 2013; Voortman et al., 2011)....
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...Speed comparison was done with CTFFIND3 (Mindell and Grigorieff, 2003), CTFFIND4 (Rohou and Grigorieff, 2015), FASTDEF (Vargas et al....
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...The estimation of the background uses a box-convolution of LnjFð~sÞj, similar to CTFFIND3 (Mindell and Grigorieff, 2003) but different....
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...The diagnosis file is similar to that of that of CTFFIND3 (Grigorieff, 2007; Mindell and Grigorieff, 2003) but contains additional user defined options....
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Journal Article•DOI•

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

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Xueming Li¹, Paul Mooney, Shawn Q. Zheng¹, Shawn Q. Zheng², Christopher R. Booth, Michael B. Braunfeld¹, Michael B. Braunfeld², Sander Gubbens, David A. Agard², David A. Agard¹, Yifan Cheng¹ - Show less +7 more•Institutions (2)

University of California, San Francisco¹, Howard Hughes Medical Institute²

01 Jun 2013-Nature Methods

TL;DR: This approach determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density and greatly enhances image quality and data acquisition efficiency.

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Abstract: In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 A). Using this approach, we determined a 3.3-A-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

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1,726 citations

Journal Article•DOI•

Structure of the TRPV1 ion channel determined by electron cryo-microscopy

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Maofu Liao¹, Erhu Cao¹, David Julius¹, Yifan Cheng¹•Institutions (1)

University of California, San Francisco¹

05 Dec 2013-Nature

TL;DR: In this article, a high-resolution electron cryo-microscopy structure of the rat transient receptor potential (TRP) channel in its closed state is presented; the overall structure of this ion channel is found to share some common features with voltage-gated ion channels, although several unique, TRP-specific features are also characterized.

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Abstract: Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 A resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function. A high-resolution electron cryo-microscopy structure of the rat transient receptor potential (TRP) channel TRPV1 in its ‘closed’ state is presented; the overall structure of this ion channel is found to share some common features with voltage-gated ion channels, although several unique, TRP-specific features are also characterized. Transient receptor potential (TRP) channels are sensors for a wide range of physical and chemical stimuli. In the first of a pair of related papers, Maofu Liao et al. solve the high-resolution electron cryo-microscopy structure of rat TRPV1, the receptor for capsaicin (a pungent agent from chili peppers), in a 'closed' state. The overall structure is fairly similar to that of a voltage-gated ion channel, but there are several structural features unique to TRP channels. In the second paper, Erhu Cao et al. present the structures of rat TRPV1 in the presence of a peptide neurotoxin (resiniferatoxin) and in the presence of capsaicin, yielding structures of activated states of the channel. Comparison of the closed and open structures suggests that TRPV1 has a unique two-gate mechanism of channel activation.

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1,419 citations

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References

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Open Access

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Journal Article•DOI•

Three-dimensional model of purple membrane obtained by electron microscopy

[...]

Richard Henderson¹, P. N. T. Unwin¹•Institutions (1)

Laboratory of Molecular Biology¹

04 Sep 1975-Nature

TL;DR: A 7-Å resolution map of the purple membrane has been obtained by electron microscopy of tilted, unstained specimens and shows that Lipid bilayer regions fill the spaces between the protein molecules.

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Abstract: A 7-A resolution map of the purple membrane has been obtained by electron microscopy of tilted, unstained specimens. The protein in the membrane contains seven, closely packed, alpha-helical segments which extend roughly perpendicular to the plane of the membrane for most of its width. Lipid bilayer regions fill the spaces between the protein molecules.

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2,114 citations

"Accurate determination of local def..." refers background in this paper

..., 1997), and to obtain tilt information (Henderson and Unwin, 1975; van Heel et al., 2000)....
[...]

Journal Article•DOI•

Structure of the alpha beta tubulin dimer by electron crystallography.

[...]

Eva Nogales¹, Sharon G. Wolf², Kenneth H. Downing¹•Institutions (2)

Lawrence Berkeley National Laboratory¹, Weizmann Institute of Science²

08 Jan 1998-Nature

TL;DR: An atomic model of the αβ tubulin dimer fitted to a 3.7-Å density map obtained by electron crystallography of zinc-induced tubulin sheets is presented.

...read moreread less

Abstract: The αβ tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is non-exchangeable when it is bound in the α subunit, or N site, and exchangeable when bound in the β subunit, or E site. The α- and β-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of post-translational modifications1. Limited sequence homology has been found with the proteins FtsZ2 and Misato3, which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the αβ tubulin dimer fitted to a 3.7-A density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of α- and β-tubulin are basically identical: each monomer is formed by a core of two β-sheets surrounded by α-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.

...read moreread less

1,953 citations

"Accurate determination of local def..." refers background in this paper

...It can be applied to samples of different symmetry and geometry, such as two-dimensional (2D) crystals (Henderson et al., 1990; K€ uhlbrandt andWang, 1991;Mindell et al., 2001;Murata et al., 2000; Nogales et al., 1998), helical particles (Morgan et al....
[...]

Journal Article•DOI•

Structural determinants of water permeation through aquaporin-1.

[...]

Kazuyoshi Murata¹, Kaoru Mitsuoka², Teruhisa Hirai², Thomas Walz³, Peter Agre⁴, J. B. Heymann⁵, Andreas Engel⁵, Yoshinori Fujiyoshi² - Show less +4 more•Institutions (5)

National Institutes of Natural Sciences, Japan¹, Kyoto University², Harvard University³, Johns Hopkins University School of Medicine⁴, University of Basel⁵

05 Oct 2000-Nature

TL;DR: An atomic model of human red cell AQP1 is described, providing a possible molecular explanation to a longstanding puzzle in physiology—how membranes can be freely permeable to water but impermeable to protons.

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Abstract: Human red cell AQP1 is the first functionally defined member of the aquaporin family of membrane water channels. Here we describe an atomic model of AQP1 at 3.8A resolution from electron crystallographic data. Multiple highly conserved amino-acid residues stabilize the novel fold of AQP1. The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport, whereas the water selectivity is due to a constriction of the pore diameter to about 3 A over a span of one residue. The atomic model provides a possible molecular explanation to a longstanding puzzle in physiology-how membranes can be freely permeable to water but impermeable to protons.

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1,662 citations

"Accurate determination of local def..." refers background or methods in this paper

...It can be applied to samples of different symmetry and geometry, such as two-dimensional (2D) crystals (Henderson et al., 1990; K€ uhlbrandt andWang, 1991;Mindell et al., 2001;Murata et al., 2000; Nogales et al., 1998), helical particles (Morgan et al....
[...]
..., 1995) and a high-resolution map of aquaporin (Murata et al., 2000)....
[...]
...To test the performance of the tilt estimation protocol, we used a series of images of 2D crystals of aquaporin (Murata et al., 2000), provided by Dr....
[...]

Journal Article•DOI•

Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.

[...]

Richard J. Kuhn¹, Wei Zhang¹, Michael G. Rossmann¹, Sergei V. Pletnev¹, Jeroen Corver², Edith M. Lenches², Christopher T. Jones¹, Suchetana Mukhopadhyay¹, Paul R. Chipman¹, Ellen G. Strauss², Timothy S. Baker¹, James H. Strauss² - Show less +8 more•Institutions (2)

Purdue University¹, California Institute of Technology²

08 Mar 2002-Cell

TL;DR: The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map, suggesting that flaviviruses employ a fusion mechanism in which the distal beta barrels of domain II of the glycop Protein E are inserted into the cellular membrane.

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1,477 citations

"Accurate determination of local def..." refers background in this paper

..., 2002), highly symmetrical viruses (B€ ottcher et al., 1997; Kuhn et al., 2002; Zhou et al., 2001), and other particles with lower or no symmetry (Frank and Agrawal, 2000; Grigorieff, 1998; Ranson et al....
[...]

Journal Article•DOI•

A ratchet-like inter-subunit reorganization of the ribosome during translocation.

[...]

Joachim Frank¹, Rajendra K. Agrawal¹, Rajendra K. Agrawal²•Institutions (2)

University at Albany, SUNY¹, Wadsworth Center²

20 Jul 2000-Nature

TL;DR: Three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S sub unit relative to the large 50S subunit, indicating a two-step mechanism of translocation.

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Abstract: The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms. It is composed of two unequal subunits: the smaller subunit binds messenger RNA and the anticodon end of transfer RNAs, and helps to decode the mRNA; and the larger subunit interacts with the amino-acid-carrying end of tRNAs and catalyses the formation of the peptide bonds. After peptide-bond formation, elongation factor G (EF-G) binds to the ribosome, triggering the translocation of peptidyl-tRNA from its aminoacyl site to the peptidyl site, and movement of mRNA by one codon. Here we analyse three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states, and show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S subunit relative to the large 50S subunit. Furthermore, our finding indicates a two-step mechanism of translocation: first, relative rotation of the subunits and opening of the mRNA channel following binding of GTP to EF-G; and second, advance of the mRNA/(tRNA)2 complex in the direction of the rotation of the 30S subunit, following GTP hydrolysis.

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825 citations