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- EMDB-11356: Structure of detergent-extracted full-length E.coli BcsB -

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Basic information

Entry
Database: EMDB / ID: EMD-11356
TitleStructure of detergent-extracted full-length E.coli BcsB
Map data
SampleSelf-assembly of the BcsB protein
  • Bacterial cellulose synthase regulator protein BcsB
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsAbidi W / Zouhir S / Krasteva P
Funding support5 items
OrganizationGrant numberCountry
ATIP-Avenir
European Research Council (ERC)Biomatrix
Centre National de la Recherche Scientifique (CNRS)
Institute for Integrative Biology of the Cell (I2BC)
European Institute of Chemistry and Biology (IECB)
CitationJournal: Sci Adv / Year: 2021
Title: Architecture and regulation of an enterobacterial cellulose secretion system.
Authors: Wiem Abidi / Samira Zouhir / Meryem Caleechurn / Stéphane Roche / Petya Violinova Krasteva /
Abstract: Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c- ...Many free-living and pathogenic enterobacteria secrete biofilm-promoting cellulose using a multicomponent, envelope-embedded Bcs secretion system under the control of intracellular second messenger c-di-GMP. The molecular understanding of system assembly and cellulose secretion has been largely limited to the crystallographic studies of a distantly homologous BcsAB synthase tandem and a low-resolution reconstruction of an assembled macrocomplex that encompasses most of the inner membrane and cytosolic subunits and features an atypical layered architecture. Here, we present cryo-EM structures of the assembled Bcs macrocomplex, as well as multiple crystallographic snapshots of regulatory Bcs subcomplexes. The structural and functional data uncover the mechanism of asymmetric secretion system assembly and periplasmic crown polymerization and reveal unexpected subunit stoichiometry, multisite c-di-GMP recognition, and ATP-dependent regulation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJul 9, 2020-
Header (metadata) releaseFeb 24, 2021-
Map releaseFeb 24, 2021-
UpdateFeb 24, 2021-
Current statusFeb 24, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11356.map.gz / Format: CCP4 / Size: 262.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.13 Å/pix.
x 410 pix.
= 463.3 Å
1.13 Å/pix.
x 410 pix.
= 463.3 Å
1.13 Å/pix.
x 410 pix.
= 463.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.13 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.21540874 - 0.8016243
Average (Standard dev.)0.00063505815 (±0.04355908)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions410410410
Spacing410410410
CellA=B=C: 463.3 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.131.131.13
M x/y/z410410410
origin x/y/z0.0000.0000.000
length x/y/z463.300463.300463.300
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS410410410
D min/max/mean-0.2150.8020.001

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Supplemental data

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Additional map: Main map non-uniformly refined and sharpened.

Fileemd_11356_additional_1.map
AnnotationMain map non-uniformly refined and sharpened.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: The volumes corresponding to the octamers were extracted...

Fileemd_11356_additional_2.map
AnnotationThe volumes corresponding to the octamers were extracted and locally refined to be finally combined resulting in a map without the central miscellaneous structure.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: The volumes corresponding to the octamers were extracted...

Fileemd_11356_additional_3.map
AnnotationThe volumes corresponding to the octamers were extracted and locally refined to be finally combined and sharpened resulting in a map without the central miscellaneous structure.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Self-assembly of the BcsB protein

EntireName: Self-assembly of the BcsB protein / Number of components: 2

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Component #1: protein, Self-assembly of the BcsB protein

ProteinName: Self-assembly of the BcsB protein / Recombinant expression: No
MassTheoretical: 1.378 MDa
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pET-21b
Source (natural)Location in cell: periplasm

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Component #2: protein, Bacterial cellulose synthase regulator protein BcsB

ProteinName: Bacterial cellulose synthase regulator protein BcsB
Details: BcsB harbors a signal sequence to be addressed to the periplasm where according the ServerP4.1 Server prediction would result in mature form starting with the residue #26 TPATQ...
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionBuffer solution: BcsB was incubated with a mix of detergents: 0.4% DDM, 0.4 % digitonin, 0.4% DM-NPG, 0.2 % GDN-101, and 0.2% LM-NPG and then purified on IMAC using free-detergents buffers.
pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

ImagingMicroscope: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.74 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 750.0 - 2750.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 46807
3D reconstructionSoftware: cryoSPARC / CTF correction: Gctf through the cryoSPARC v2 interface. / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF

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