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- EMDB-0074: Template-free detection and classification of microsomal membrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-0074
TitleTemplate-free detection and classification of microsomal membrane bound complexes
Map dataRibosome bound to fully assembled translocon complex
Sample
  • Complex: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
Biological speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 18.0 Å
AuthorsMartinez-Sanchez A / Lucic V
Funding support Germany, 1 items
OrganizationGrant numberCountry
Seneca Foundation Germany
CitationJournal: Nat Methods / Year: 2020
Title: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.
Authors: Antonio Martinez-Sanchez / Zdravko Kochovski / Ulrike Laugks / Johannes Meyer Zum Alten Borgloh / Saikat Chakraborty / Stefan Pfeffer / Wolfgang Baumeister / Vladan Lučić /
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.
History
DepositionJun 22, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseOct 2, 2019-
UpdateFeb 26, 2020-
Current statusFeb 26, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.358
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.358
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0074.png

Downloads & links

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Map

FileDownload / File: emd_0074.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome bound to fully assembled translocon complex
Voxel sizeX=Y=Z: 5.24 Å
Density
Contour LevelBy AUTHOR: 0.358 / Movie #1: 0.358
Minimum - Maximum-1.5920615 - 1.9050354
Average (Standard dev.)0.006569184 (±0.13416108)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 628.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.245.245.24
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z628.800628.800628.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-1.5921.9050.007

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Supplemental data

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Half map: Ribosome bound to fully assembled translocon complex, 2nd half map

Fileemd_0074_half_map_1.map
AnnotationRibosome bound to fully assembled translocon complex, 2nd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Ribosome bound to fully assembled translocon complex, 1st half map

Fileemd_0074_half_map_2.map
AnnotationRibosome bound to fully assembled translocon complex, 1st half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mammalian ribosome bound to the native protein translocon on cani...

EntireName: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
Components
  • Complex: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles

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Supramolecule #1: Mammalian ribosome bound to the native protein translocon on cani...

SupramoleculeName: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Canis lupus familiaris (dog)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 7.6
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: Blot 3 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 55 / Number images used: 64000
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 1064
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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