ジャーナル: Nat Methods / 年: 2020 タイトル: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms. 著者: Antonio Martinez-Sanchez / Zdravko Kochovski / Ulrike Laugks / Johannes Meyer Zum Alten Borgloh / Saikat Chakraborty / Stefan Pfeffer / Wolfgang Baumeister / Vladan Lučić / 要旨: With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, ...With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.
C2レンズ絞り径: 70.0 µm / 照射モード: OTHER / 撮影モード: OTHER / Cs: 2.7 mm / 最大 デフォーカス(公称値): 0.25 µm / 最小 デフォーカス(公称値): 0.2 µm / 倍率(公称値): 42000
試料ステージ
ホルダー冷却材: NITROGEN
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
最終 再構成
アルゴリズム: BACK PROJECTION / ソフトウェア - 名称: RELION (ver. 2.1) 詳細: Original reconstructed pixel size is 3.42 Angstroms, however due to size restriction a 4-times binned version with 13.68 Angstroms per pixel is provided here 使用した粒子像数: 60