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- EMDB-10434: In situ ER membrane bound ribosome-free translocon -

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Basic information

Entry
Database: EMDB / ID: EMD-10434
TitleIn situ ER membrane bound ribosome-free translocon
Map dataPutative ribosome-free translocon on ER membrane of intact P19 cells
Sample
  • Cell: Intact P19 cells
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 39.08 Å
AuthorsMartinez-Sanchez A / Lucic V / Chakraborty S
Funding support Spain, 1 items
OrganizationGrant numberCountry
Seneca Foundation Spain
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
DepositionOct 31, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseDec 11, 2019-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.16
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10434.png

Downloads & links

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Map

FileDownload / File: emd_10434.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPutative ribosome-free translocon on ER membrane of intact P19 cells
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 160 pix.
= 547.2 Å		3.42 Å/pix.
x 160 pix.
= 547.2 Å		3.42 Å/pix.
x 160 pix.
= 547.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.51673496 - 0.7207199
Average (Standard dev.)0.0011985324 (±0.062287115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 547.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z547.200547.200547.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.5170.7210.001

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Supplemental data

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Sample components

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Entire : Intact P19 cells

EntireName: Intact P19 cells
Components
  • Cell: Intact P19 cells

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Supramolecule #1: Intact P19 cells

SupramoleculeName: Intact P19 cells / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse) / Tissue: teratocarcinoma

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 310.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 0.25 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 42000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 39.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number subtomograms used: 63
ExtractionNumber tomograms: 12 / Number images used: 172000 / Method: Template-free / Software - Name: IMOD
Software - details: PySeg for particle picking and IMOD for subvolume reconstruction
Details: PySeg software, automated and comprehensive detection of heterogeneous membrane-bound complexes.
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Software - details: Before final classification with RELION, PySeg unsuprevised classification was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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