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- EMDB-10799: Cryo-EM structure of a BcsB pentamer in the context of an assembl... -

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Basic information

Entry
Database: EMDB / ID: EMD-10799
TitleCryo-EM structure of a BcsB pentamer in the context of an assembled Bcs macrocomplex
Map data
SampleCo-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
  • Homopentameric assembly of the BcsB protein
  • Bacterial cellulose secretion regulator BcsB
Function / homologyCellulose synthase, subunit B / Galactose-binding-like domain superfamily / Cellulose synthase BcsB, bacterial / cellulose biosynthetic process / UDP-glucose metabolic process / integral component of membrane / plasma membrane / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsZouhir S / Krasteva PV
Funding support5 items
OrganizationGrant numberCountry
ATIP-Avenir
European Research Council (ERC)BioMatrix, ERC StG #757507
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
DepositionMar 27, 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: 10
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yg8
  • Surface level: 10
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10799.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
AxesX (Sec.)Y (Row.)Z (Col.)
1.05 Å/pix.
x 410 pix.
= 431.517 Å
1.05 Å/pix.
x 410 pix.
= 431.517 Å
1.05 Å/pix.
x 410 pix.
= 431.517 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05248 Å
Density
Contour LevelBy AUTHOR: 4.5 / Movie #1: 10
Minimum - Maximum-31.928823 - 55.8326
Average (Standard dev.)0.0006355236 (±0.9885274)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions410410410
Spacing410410410
CellA=B=C: 431.51678 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.05248048780491.05248048780491.0524804878049
M x/y/z410410410
origin x/y/z0.0000.0000.000
length x/y/z431.517431.517431.517
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ410410410
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS410410410
D min/max/mean-31.92955.8330.001

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Supplemental data

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Additional map: 2.9A locally refined and sharpened map used for...

Fileemd_10799_additional_1.map
Annotation2.9A locally refined and sharpened map used for the main composite map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: 3.1A average resolution sharpened map used for the main composite map

Fileemd_10799_additional_2.map
Annotation3.1A average resolution sharpened map used for the main composite map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Non sharpened map

Fileemd_10799_additional_3.map
AnnotationNon sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Non sharpened map

Fileemd_10799_additional_4.map
AnnotationNon sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the ...

EntireName: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
Number of components: 3

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Component #1: protein, Co-expression of Bcs subunits R, Q, A, B, E, F and G lea...

ProteinName: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
Recombinant expression: No
MassTheoretical: 734 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pCDF-duet
Source (natural)Location in cell: periplasm

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Component #2: protein, Homopentameric assembly of the BcsB protein

ProteinName: Homopentameric assembly of the BcsB protein / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pCDF-duet
Source (natural)Location in cell: periplasm

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Component #3: protein, Bacterial cellulose secretion regulator BcsB

ProteinName: Bacterial cellulose secretion regulator BcsB / Details: Bacterial cellulose synthesis subunit B / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 86.184383 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
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 solutionSpecimen conc.: 0.8 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.2 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 750.0 - 2750.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 576455
Details: A total of 9,129 movies recorded on the CM01 Titan Krios transmission electron microscope (Thermo Fisher Scientific) at the ESRF Grenoble operated at 300 kV and equipped with a Gatan K2 ...Details: A total of 9,129 movies recorded on the CM01 Titan Krios transmission electron microscope (Thermo Fisher Scientific) at the ESRF Grenoble operated at 300 kV and equipped with a Gatan K2 Summit direct electron detector and a GIF Quantum LS imaging filter
3D reconstructionSoftware: cryoSPARC / CTF correction: Gctf through the cryoSPARC v2 interface. / Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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