National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM085043
United States
Citation
Journal: Nat Methods / Year: 2018 Title: emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. Authors: Benjamin A Himes / Peijun Zhang / Abstract: Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by ...Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software.
History
Deposition
Jun 30, 2017
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Header (metadata) release
Oct 10, 2018
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Map release
Oct 10, 2018
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Update
Jan 29, 2020
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Current status
Jan 29, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity (ver. 1.0) Software - details: anisotropic "single-particle wiener filter" for final weighting/reconstruction Details: Independent beyond 3.2 nm as determined by the FSC at the time of dividing the half-sets Number subtomograms used: 3120
Extraction
Number tomograms: 4 / Number images used: 3120 / Reference model: EMD-3228 / Method: template matching in emClarity Details: Template matching using EMD-3228 as an initial reference low-pass filtered to 4.0nm.
CTF correction
Software - Name: emClarity (ver. 1.0) Software - details: included astigmatism and per tilt ctf estimation Details: Phases were corrected on the projections by multiplication of the astigmatic-CTF determined in emClarity in tiles of 320 sq pix with a strip replacement that depended on the tilt angle.
Final angle assignment
Type: OTHER / Software - Name: emClarity (ver. 1.0) Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.2 nanometers. Reference and subtomograms filtered dynamically according to the FSC ...Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.2 nanometers. Reference and subtomograms filtered dynamically according to the FSC at each cycle using the FOM approach.
FSC plot (resolution estimation)
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