Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM
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.
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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TL;DR: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.
Abstract: MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.
5,491 citations
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.
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.
3,520 citations
Cites background from "Electron counting and beam-induced ..."
...Some new programs exploited specific opportunities provided by the new detectors, like the correction of beam-induced motions through movie processing (Brilot et al., 2012; Li et al., 2013; Rubinstein and Brubaker, 2015; Abrishami et al., 2015; McLeod et al., 2017; Zheng et al., 2017)....
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01 Oct 2019
TL;DR: Recent developments in the Phenix software package are described in the context of macromolecular structure determination using X-rays, neutrons and electrons.
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
3,268 citations
Cites methods from "Electron counting and beam-induced ..."
...Technological advances, such as the development of direct electron detectors (Li et al., 2013) and improvements in image processing (Bai et al., 2015), have transformed the method, leading to cryo-EM maps with greatly improved resolution....
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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.
2,919 citations
Cites background from "Electron counting and beam-induced ..."
...Beam induced movement correction using movies has greatly improved the resolution of the final reconstruction (Bai et al., 2013; Li et al., 2013)....
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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.
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.
1,419 citations
References
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TL;DR: Two unusual extensions are presented: Multiscale, which adds the ability to visualize large‐scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales.
Abstract: The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
35,698 citations
TL;DR: The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy.
1,959 citations
TL;DR: The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging.
Abstract: The three-dimensional structure of the proteasome from the archaebacterium Thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging. The atomic model was built and refined to a crystallographic R factor of 22.1 percent. The 673-kilodalton protease complex consists of 14 copies of two different subunits, alpha and beta, forming a barrel-shaped structure of four stacked rings. The two inner rings consist of seven beta subunits each, and the two outer rings consist of seven alpha subunits each. A narrow channel controls access to the three inner compartments. The alpha 7 beta 7 beta 7 alpha 7 subunit assembly has 72-point group symmetry. The structures of the alpha and beta subunits are similar, consisting of a core of two antiparallel beta sheets that is flanked by alpha helices on both sides. The binding of a peptide aldehyde inhibitor marks the active site in the central cavity at the amino termini of the beta subunits and suggests a novel proteolytic mechanism.
1,532 citations
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.
1,480 citations
TL;DR: Analysis of simulated data with realistic signal-to-noise ratios indicates that the accuracy of the orientation determination is not affected by the exclusion of high-frequency terms, nor by the use of a model that is reconstructed from only half of the particles, as expected.
Abstract: In the field of single-particle analysis of electron cryo-microscopy (cryo-EM) data, a growing concern that some resolution claims might not be substantiated by the data has been one of the instigators of community-wide efforts to develop new validation tools1. A known issue with commonly used cryo-EM structure determination procedures is their liability to overfit the data. Most procedures counter overfitting by low-pass filtering, but the effective frequencies for these filters are often based on suboptimal Fourier Shell Correlation2 (FSC) procedures. In the suboptimal procedure, FSC curves are calculated between reconstructions from two halves of the data, while a single model is used to determine the relative orientations of all particles. It is well known that bias towards noise in the single model may inflate the resulting resolution estimates. To illustrate this, we applied the suboptimal procedure to a simulated cryo-EM data set of 20,212 GroEL particles. Whereas the reported resolution was 4.6 A, the true resolution of the map was only 7.8 A. Also the presence of expected density features in the map does not necessarily provide sufficient evidence for a resolution claim: we could make convincingly looking figures of apparent side-chain density that in reality corresponded to overfitted noise (Supplementary Figure 1). Consequently, overfitting may remain undetected and interpretation of cryo-EM maps may be subject to errors.
The dangers of overfitting have been recognized, and refinement procedures with resolution-dependent weighting schemes to reduce overfitting have been proposed3,4. However, two known solutions to prevent it are not in common use. By refining two models independently (one for each half of the data), so-called gold-standard1 FSC curves may be calculated that are free from spurious correlations. Alternatively, the data used for the orientation determination may be limited to a user-specified frequency, so that model bias beyond that frequency may be avoided. However, the argument that withholding part of the data from the refinement would substantially deteriorate the orientations and thereby the quality of the structure has prevented the wide-spread use of either of these solutions. In what follows, we prove this thesis to be false.
Analysis of simulated data with realistic signal-to-noise ratios (SNRs) indicates that the accuracy of the orientation determination is not affected by the exclusion of high-frequency terms, nor by the use of a model that is reconstructed from only half of the particles (Supplementary Figure 2). These simulations illustrate that only the low-medium frequency terms in the individual particles contain sufficiently high SNRs to contribute significantly to the orientation determination, which is in good agreement with experimental evidence that cryo-EM particles may be aligned accurately using only low-frequency data5. Because in most cryo-EM studies the low-medium frequencies of reconstructions from half of the particles are not expected to be significantly worse than those of reconstructions from all particles, we hypothesize that overfitting may be prevented without a notable loss of resolution using either frequency-limited refinement or refinement based on gold-standard FSCs. Since the former involves a decision by the user, i.e. choosing the frequency at which to limit the refinement, we favour gold-standard FSCs and implemented a procedure to independently refine two models as a script on top of the conventional projection matching protocol in the XMIPP package6 (Supplementary Figure 3 & Supplementary Software).
We tested our hypothesis using three cryo-EM data sets: 5,053 GroEL particles that are distributed by the National Center for Macromolecular Imaging; an in-house collected data set of 50,330 β-galactosidase particles (Supplementary Methods); and 5,403 hepatitis B capsid particles from a previously published study7. High-resolution crystal structures are available for all three data sets, and these were used to assess the “true” resolution obtained using refinements based on either gold-standard or conventional FSC procedures (Figure 1). For all three cases, the conventional procedure reported apparently better FSC curves than the gold-standard procedure, but in no case did the gold-standard procedure actually result in a lower resolution map compared to the crystal structure. On the contrary, for the β-galactosidase data the gold-standard procedure yielded a structure that correlated up to higher frequencies with the crystal structure than the conventional procedure, which suffered from severe overfitting and gave rise to strong artefacts in the map. We also note that, in the absence of overfitting, the frequency at which the gold-standard FSC drops below 0.143 is a good indicator of the true resolution of the map (Supplementary Table 1), which is as expected from theory8. Finally, in the limit of very small data sets, division of the data into two halves might affect resolution. However, calculations with subsets of the GroEL particles suggest that this only becomes an issue for data sets that are much smaller than those typically used in cryo-EM reconstructions (Supplementary Figure 4).
Figure 1
The prevention of overfitting
The principal conclusion is therefore that overfitting of noise using suboptimal FSCs causes worse orientations and leads to a worse structure. In contrast, the use of gold-standard FSCs provides a realistic estimate of the true signal, which ultimately leads to a better map. The procedures proposed here are straightforward to implement in existing programs, and their application will eradicate the hazards of overfitting from cryo-EM structure determination procedures.
1,099 citations