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- EMDB-3864: Negative stain electron microscopy reconstruction of a cellulose ... -

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Basic information

Entry
Database: EMDB / ID: 3864
TitleNegative stain electron microscopy reconstruction of a cellulose secretion (Bcs) macrocomplex from E. coli
SampleProtein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
SourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Map dataBcs macrocomplex encompassing most inner-membrane and cytosolic components of the E. coli cellulose secretion system. Negative-stain EM
Methodsingle particle reconstruction, at 16.7 Å resolution
AuthorsKrasteva PV / Fronzes R
CitationNat Commun, 2017, 8, 2065-2065

Nat Commun, 2017, 8, 2065-2065 Yorodumi Papers
Insights into the structure and assembly of a bacterial cellulose secretion system.
Petya Violinova Krasteva / Joaquin Bernal-Bayard / Laetitia Travier / Fernando Ariel Martin / Pierre-Alexandre Kaminski / Gouzel Karimova / Rémi Fronzes / Jean-Marc Ghigo

DateDeposition: Sep 11, 2017 / Header (metadata) release: Dec 20, 2017 / Map release: Dec 27, 2017 / Last update: Dec 27, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0104
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by height
  • Surface level: 0.0104
  • Imaged by UCSF CHIMERA
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Supplemental images

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Map

Fileemd_3864.map.gz (map file in CCP4 format, 37045 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
210 pix
1.9 Å/pix.
= 399. Å
210 pix
1.9 Å/pix.
= 399. Å
210 pix
1.9 Å/pix.
= 399. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.9 Å
Density
Contour Level:0.0104 (by author), 0.0104 (movie #1):
Minimum - Maximum-0.07754264 - 0.13978234
Average (Standard dev.)0.00051137165 (0.008628474)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions210210210
Origin000
Limit209209209
Spacing210210210
CellA=B=C: 399 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z399.000399.000399.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.0780.1400.001

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Supplemental data

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Sample components

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Entire Protein macrocomplex encompassing most cytosolic and inner-membra...

EntireName: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
Details: Protein macrocomplex encompassing cytosolic (BcsRQEF) and inner-membrane (BcsAB) components. Isolated from purified and detergent-solubilised E. coli 1094 membranes using FLAG-tagged BcsA as bait. Stabilised by gentle glutaraldehyde cross-linking over a density gradient (GraFix).
Number of components: 1
MassTheoretical: 1.4 MDa

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Component #1: protein, Protein macrocomplex encompassing most cytosolic and inn...

ProteinName: Protein macrocomplex encompassing most cytosolic and inner-membrane components of the E. coli system for cellulose secretion
Details: Protein macrocomplex encompassing cytosolic (BcsRQEF) and inner-membrane (BcsAB) components. Isolated from purified and detergent-solubilised E. coli 1094 membranes using FLAG-tagged BcsA as bait. Stabilised by gentle glutaraldehyde cross-linking over a density gradient (GraFix).
Recombinant expression: No
MassTheoretical: 1.4 MDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Strain: 1094
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Vector: Chromosome-driven expression / Strain: 1094 2K7 BcsA-HA-FLAG

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.1 mg/ml
Buffer solution: 20 mM HEPES pH 8.0 120 mM NaCl, 10%-40% glycerol, 5 mM MgCl2 10 uM AppCp 2 uM cyclic c-di-GMP cOmplete protease inhibitors
pH: 8
Support film7mA, 10 microns
Staining5 micrometers of sample were spotted on glow-discharged carbon-coated copper grids. After 1 minutes the liquid was blotted off and the sample was stained by quick passage through 3 drops of 2% uranyl acetate. The sample was left for 20-30 seconds on the last drop of stain, after which the liquid was blotted off and the sample was allowed to air-dry
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 12 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: - 4000 nm
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 24769
3D reconstructionSoftware: RELION
CTF correction: CTF correction was performed in EMAN2.0 (e2ctf) using the automated fitting function before and after structure factor generation
Resolution: 16.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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  • Richard Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) was determined the first biomolecule structure by EM. The first EM entry in PDB, PDB-1brd is determinedby him.

External links: The 2017 Nobel Prize in Chemistry - Press Release

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