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- EMDB-10450: Endoplasmic Reticulum microsome used for Videos in (Martinez-Sanc... -

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Basic information

Entry
Database: EMDB / ID: EMD-10450
TitleEndoplasmic Reticulum microsome used for Videos in (Martinez-Sanchez et al., Nature Methods)
Map dataER microsome used for Videos (Martinez-Sanchez et al., Nature Methods)
Sample
  • Organelle or cellular component: Representative tomogram of a canine pancreatic rough Endoplasmic Reticulum vesicle.
Biological speciesCanis lupus familiaris (dog)
Methodelectron tomography / cryo EM
AuthorsMartinez-Sanchez A / Lucic V / Pfeffer S / Forster F
Funding support Spain, 1 items
OrganizationGrant numberCountry
Seneca Foundation Spain
Citation
Journal: 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.
#1: Journal: Nature Communications / Year: 2015
Title: Structure of the native Sec61 protein-conducting channel
Authors: Pfeffer S / Forster F
History
DepositionNov 4, 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
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_10450.png

Downloads & links

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Map

FileDownload / File: emd_10450.map.gz / Format: CCP4 / Size: 37.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationER microsome used for Videos (Martinez-Sanchez et al., Nature Methods)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.48 Å/pix.
x 127 pix.
= 1330.96 Å		10.48 Å/pix.
x 273 pix.
= 2861.04 Å		10.48 Å/pix.
x 285 pix.
= 2986.8 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.48 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.93009955 - 0.6026039
Average (Standard dev.)-0.00031982694 (±0.031086618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions273285127
Spacing285273127
CellA: 2986.7998 Å / B: 2861.0398 Å / C: 1330.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z10.4810.4810.48
M x/y/z285273127
origin x/y/z0.0000.0000.000
length x/y/z2986.8002861.0401330.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS285273127
D min/max/mean-0.9300.603-0.000

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Supplemental data

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Sample components

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Entire : Representative tomogram of a canine pancreatic rough Endoplasmic ...

EntireName: Representative tomogram of a canine pancreatic rough Endoplasmic Reticulum vesicle.
Components
  • Organelle or cellular component: Representative tomogram of a canine pancreatic rough Endoplasmic Reticulum vesicle.

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Supramolecule #1: Representative tomogram of a canine pancreatic rough Endoplasmic ...

SupramoleculeName: Representative tomogram of a canine pancreatic rough Endoplasmic Reticulum vesicle.
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Canis lupus familiaris (dog) / Tissue: pancreas

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration2.00 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
20.0 mMHepes
50.0 mMKCl
2.0 mMMgCl2
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: Blot 3 seconds before plunging..
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion, Wageningen, The Netherlands / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 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: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 42000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsReconstruction of a single microsome after two binning steps (from 2.62A/pixel to 10.48A/pixel)
Final reconstructionSoftware - Name: PyTom / Number images used: 61
CTF correctionSoftware - Name: PyTom

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