National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM085043
米国
引用
ジャーナル: Nat Methods / 年: 2018 タイトル: emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging. 著者: Benjamin A Himes / Peijun Zhang / 要旨: 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.
使用したクラス数: 1 / 想定した対称性 - 点群: C1 (非対称) / アルゴリズム: BACK PROJECTION / 解像度のタイプ: BY AUTHOR / 解像度: 7.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: emClarity (ver. 1.0) ソフトウェア - 詳細: anisotropic "single-particle wiener filter" for final weighting/reconstruction 詳細: Independent beyond 3.2 nm as determined by the FSC at the time of dividing the half-sets 使用したサブトモグラム数: 3120
抽出
トモグラム数: 4 / 使用した粒子像数: 3120 / 参照モデル: EMD-3228 / 手法: template matching in emClarity 詳細: Template matching using EMD-3228 as an initial reference low-pass filtered to 4.0nm.
CTF補正
ソフトウェア - 名称: emClarity (ver. 1.0) ソフトウェア - 詳細: included astigmatism and per tilt ctf estimation 詳細: 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.
最終 角度割当
タイプ: OTHER / ソフトウェア - 名称: emClarity (ver. 1.0) 詳細: 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 ...詳細: 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.