Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy

Rosenthal, Peter B.; Henderson, Richard A.

doi:10.1016/j.jmb.2003.07.013

Cited by 2,136 publications

(2,122 citation statements)

References 43 publications

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“…The mouse TRPM4 was purified and reconstituted into nanodiscs and its structures in the apo and ATP-bound states were determined by cryo-EM to a resolution of 3.1 and 2.9 Å, respectively, using the gold-standard Fourier shell correlation (FSC) = 0.143 criteria 28 (Extended Data Fig. 2–6 and Methods).…”

Section: Resultsmentioning

confidence: 99%

Structures of the calcium-activated, non-selective cation channel TRPM4

Guo

She

Zeng

et al. 2017

Nature

164

196

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TRPM4 is a calcium-activated, phosphatidylinositol bisphosphate (PtdInsP2) modulated, non-selective cation channel, and belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the cryo-EM structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide binding domain (NBD) and the C-terminal coiled coil participate in the tetrameric assembly of the channel; ATP binds at NBD and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. S1-S4 domain and post-S6 TRP domain form the central gating apparatus that likely house the Ca2+ and PtdInsP2 binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and also reveal the molecular architecture of the TRPM family for the first time.

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Section: Resultsmentioning

confidence: 99%

Structures of the calcium-activated, non-selective cation channel TRPM4

Guo

She

Zeng

et al. 2017

Nature

164

196

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“…a, Fourier-shell correlation (FSC) curve (black) for the 70S ribosome cryo-EM reconstruction computed between the masked independent half-maps (half1 and half2) that were obtained by so-called 'gold-standard' refinement in RELION 37 . The resolution of the cryo-EM reconstruction is ,2.9 Å according to the 0.143 criterion 63 (black dashed line). b, FSC curves computed between cryo-EM maps and model maps generated from refined atomic coordinates.…”

Section: Extended Datamentioning

confidence: 99%

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

Fischer

Neumann

Konevega

et al. 2015

Nature

353

443

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Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution 1,2 and all structures available to date have been limited to resolutions above 3 Å 3,4 . Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (C s )-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome 5 (2.8 Å ), but provides more detailed information (2.65 Å ) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.Determining the structure of large, dynamic biological macromolecules at a uniformly high resolution provides a challenge both for X-ray crystallography and cryo-EM. Here we have used aberration-corrected cryo-EM in combination with extensive computational sorting to solve *These authors contributed equally to this work.

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“…In our case, we populate the first half with P ref (f) and the second half with P test (f). The resolution is determined by applying the threshold T = 0.5 on the spectral correlation curve which typically decays monotonically 66 .…”

Section: Computation Of the Image Resolution Using The Fourier Ring Cmentioning

confidence: 99%

Quantitative evaluation of software packages for single-molecule localization microscopy

et al. 2015

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the quality of super-resolution images obtained by singlemolecule localization microscopy (smlm) depends largely on the software used to detect and accurately localize point sources. in this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. these metrics reflect the various tradeoffs of smlm software packages and help users to choose the software that fits their needs.We have conducted a large-scale comparative study of software packages developed in the context of SMLM, including recently developed algorithms. We designed realistic data that are generic and cover a broad range of experimental conditions and compared the software packages using a multiple-criterion quantitative assessment that is based on a known ground truth.Our study is based on the active participation of developers of SMLM software. More than 30 groups have participated so far, and the study is still under way. We provide participants access to our benchmark data as an ongoing public challenge. Participants run their own software on our data and report their list of localized particles for evaluation. The results of the challenge are accessible online and updated regularly.SMLM was demonstrated in 2006, independently by three research groups 1-3 , and has enabled subsequent breakthroughs in diverse fields 4,5 . SMLM can resolve biological structures at the nanometer scale (typically 20 nm lateral resolution), circumventing Abbe's diffraction limit. At the cost of a relatively simple setup 6,7 , it has opened exciting new opportunities in life science research 8,9 .The underlying principle of SMLM is the sequential imaging of sparse subsets of fluorophores distributed over thousands of frames, to populate a high-density map of fluorophore positions. Such large data sets require automated image-analysis algorithms to detect and precisely infer the position of individual fluorophore, taking advantage of their separation in space and time.The acquired data cannot be visualized directly; further computerized image-reconstruction methods are required. These typically comprise four steps: preprocessing, detection, localization and rendering. Preprocessing reduces the effects of the background and noise; detection identifies potential molecule candidates in each frame; localization performs a subpixel refine...

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Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy

Cited by 2,136 publications

References 43 publications

Structures of the calcium-activated, non-selective cation channel TRPM4

Structures of the calcium-activated, non-selective cation channel TRPM4

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

Quantitative evaluation of software packages for single-molecule localization microscopy

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