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- EMDB-11836: Cryo-EM density map corresponding to BcsRQAB subcomplex -

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Basic information

Entry
Database: EMDB / ID: EMD-11836
TitleCryo-EM density map corresponding to BcsRQAB subcomplex
Map data
SampleCryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
  • (Bacterial cellulose synthase regulator protein ...) x 3
  • Bacterial cellulose synthase protein BcsA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsZouhir S
Funding support France, 4 items
OrganizationGrant numberCountry
ATIP-Avenir2016 France
European Research Council (ERC)Biomatrix 757507 France
Centre National de la Recherche Scientifique (CNRS) France
European Institute of Chemistry and Biology (IECB) France
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
DepositionOct 13, 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: 7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11836.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: 7.0 / Movie #1: 7
Minimum - Maximum-43.477592 - 123.02881
Average (Standard dev.)-1.1382667e-12 (±1.0)
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-43.478123.029-0.000

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Supplemental data

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Additional map: #1

Fileemd_11836_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #3

Fileemd_11836_additional_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #4

Fileemd_11836_additional_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: #2

Fileemd_11836_additional_4.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtaine...

EntireName: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 ...Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 resin (Sigma). The proposed stoichiometry for the resultant assembly is BcsR2-Q2-E2-F2-A-B(5-6). Densities corresponding to a BcsR2Q2AB subcomplex were improved after particle subtraction and local refinement in cryoSPARC V2. BcsE and BcsF partake in the assembled Bcs macrocomplex, BcsG does not co-purify stably with the macrocomplex.
Number of components: 5

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Component #1: protein, Cryo-EM density map corresponding to BcsR2Q2AB subcomple...

ProteinName: Cryo-EM density map corresponding to BcsR2Q2AB subcomplex obtained after local refinement within the assembled Bcs macrocomplex(BcsRQABEF-BcsMacrocomplex.mrc).
Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 ...Details: Bcs macrocomplex was purified using recombinant co-expression of constructs pRSFDuet1-Bcs(Strep)E-F-G and pCDFDuet1-Bcs(His)R-Q-A(HA-FLAG)-B and affinity pull-down using an anti-FLAG M2 resin (Sigma). The proposed stoichiometry for the resultant assembly is BcsR2-Q2-E2-F2-A-B(5-6). Densities corresponding to a BcsR2Q2AB subcomplex were improved after particle subtraction and local refinement in cryoSPARC V2. BcsE and BcsF partake in the assembled Bcs macrocomplex, BcsG does not co-purify stably with the macrocomplex.
Recombinant expression: No
MassTheoretical: 257 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)
Vector: pRSFDuet1-Bcs(Strep)EFG and pCDFDuet1-Bcs(His)RQA(HA-FLAG)B
Source (natural)Location in cell: Inner membrane

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

ProteinName: Bacterial cellulose synthase regulator protein BcsB
Details: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 ...Details: Genome-encoded BcsB harbors a signal sequence to be addressed to the periplam where according to the ServerP4.1 server prediction would result in a mature form starting with the residue #26 TPATQ BcsB sequence was cloned in a pCDFDuet1 vector along Bcs(His)R, BcsQ and BcsA(HA-FLAG). 1 copy in locally refined BcsR2Q2AB map, up to 6 copies in the assembled Bcs macrocomplex.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, Bacterial cellulose synthase protein BcsA

ProteinName: Bacterial cellulose synthase protein BcsA
Details: BcsA sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsQ and BcsB. BcsA harbours a C-terminus HA-Flag Tag. 1 copy in both the locally refined BcsR2Q2AB complex and the assembled Bcs macrocomplex.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, Bacterial cellulose synthase regulator protein BcsR

ProteinName: Bacterial cellulose synthase regulator protein BcsR
Details: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion ...Details: BcsR sequence was cloned in pCDFDuet1 vector along with BcsQ, BcsA(HA-FLAG) and BcsB. BcsR harbours a N-terminal octahistidine tag (N-His8). Likely two copies in the assembled secretion complexes, however the local resolution does not allow backbone tracing. High-resolution structures solved by X-ray crystallography.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: 1094
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, Bacterial cellulose synthase regulator protein BcsQ

ProteinName: Bacterial cellulose synthase regulator protein BcsQ
Details: BcsQ sequence was cloned in pCDFDuet1 vector along with Bcs(His)R, BcsA(HA-FLAG) and BcsB
Recombinant expression: No
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
Support filmElmo Glow Discharge system
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: 173294
Details: Data collection at the CM01 line in ESRF Grenoble (Titan Krion, GATAN K2 Summit DED and Quantum LS Imaging filter).
3D reconstructionSoftware: cryoSPARC / CTF correction: Gctf through the cryoSPARC v2 interface / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Two rounds of local refinement of the BcsR2Q2AB assembly were performed using different fulcrum placements. The resulting maps gave FSC values of ~3.9 and 4.1A, respectively, as determined ...Details: Two rounds of local refinement of the BcsR2Q2AB assembly were performed using different fulcrum placements. The resulting maps gave FSC values of ~3.9 and 4.1A, respectively, as determined by cryoSPARC using the 0.143 cut-off. The unsharpened maps were combined and conservatively sharpened to 4.5 A resolution. The sharpened map was used for building the C-terminal tail-anchor of BcsB and flexible fitting of a Robetta-derived homology model of BcsA. A BcsR2Q2 dimer solved by X-ray crystallography was rigid-body fitted in the apical density.

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