Antonio Martinez-Sanchez / Zdravko Kochovski / Ulrike Laugks / Johannes Meyer Zum Alten Borgloh / Saikat Chakraborty / Stefan Pfeffer / Wolfgang Baumeister / Vladan Lučić /
PubMed Abstract
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.
EMDB-0074: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 18.0 Å
EMDB-0075: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 21.0 Å
EMDB-0084: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 22.0 Å
EMDB-0085: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 22.0 Å
EMDB-0086: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 14.0 Å
EMDB-0087: Template-free detection and classification of microsomal membrane bound complexes Method: EM (subtomogram averaging) / Resolution: 16.0 Å
EMDB-10432: In situ ER membrane bound ribosome Method: EM (subtomogram averaging) / Resolution: 30.0 Å
EMDB-10433: In situ ER membrane bound translocon-ribosome Method: EM (subtomogram averaging) / Resolution: 36.48 Å
EMDB-10434: In situ ER membrane bound ribosome-free translocon Method: EM (subtomogram averaging) / Resolution: 39.08 Å
EMDB-10435: Putative in situ ER membrane bound major histocompatibility peptide loading complex (PLC) Method: EM (subtomogram averaging) / Resolution: 34.2 Å
EMDB-10436: Putative in situ ER membrane bound major histocompatibility peptide loading complex (PLC) Method: EM (subtomogram averaging) / Resolution: 32.18 Å
EMDB-10437: Putative in situ ER membrane bound inositol trisphosphate receptor complex (IP3R) Method: EM (subtomogram averaging) / Resolution: 34.2 Å
EMDB-10439: Example of tomogram used for in situ template-free membrane bound complexes determination Method: EM (tomography)
EMDB-10449: ER Microsome in Fig. 3 (Martinez et al., Nature Methods) Method: EM (tomography)
EMDB-10450: Endoplasmic Reticulum microsome used for Videos in (Martinez-Sanchez et al., Nature Methods) Method: EM (tomography)
EMDB-10451: ER microsome released with the code in (Martinez-Sanchez et al., Nature Methods) Method: EM (tomography)
Source
Canis lupus familiaris (dog)
Mus musculus (house mouse)
+
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