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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 datacomposite map made from two experimental sharpened maps of the same sample data
Sample
  • Complex: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
    • Complex: Homopentameric assembly of the BcsB protein
      • Protein or peptide: Bacterial cellulose secretion regulator BcsB
Function / homology
Function and homology information


cellulose biosynthetic process / UDP-glucose metabolic process / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Cyclic di-GMP-binding protein / Cyclic di-GMP-binding protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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.
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
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6yg8
  • Surface level: 10
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10799.png

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)
Annotationcomposite map made from two experimental sharpened maps of the same sample data
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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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
Components
  • Complex: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
    • Complex: Homopentameric assembly of the BcsB protein
      • Protein or peptide: Bacterial cellulose secretion regulator BcsB

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Supramolecule #1: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the ...

SupramoleculeName: Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assembly
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094 / Location in cell: periplasm
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pCDF-duet
Molecular weightTheoretical: 734 KDa

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Supramolecule #2: Homopentameric assembly of the BcsB protein

SupramoleculeName: Homopentameric assembly of the BcsB protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094 / Location in cell: periplasm
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pCDF-duet

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Macromolecule #1: Bacterial cellulose secretion regulator BcsB

MacromoleculeName: Bacterial cellulose secretion regulator BcsB / type: protein_or_peptide / ID: 1 / Details: Bacterial cellulose synthesis subunit B / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Molecular weightTheoretical: 86.184383 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVL RGINPNGSIE FGMRSDEVVT KAMLNLEYTP SPSLLPVQSQ LKVYLNDELM GVLPVTKEQL GKKTLAQMPI N PLFITDFN ...String:
MKRKLFWICA VAMGMSAFPS FMTQATPATQ PLINAEPAVA AQTEQNPQVG QVMPGVQGAD APVVAQNGPS RDVKLTFAQI APPPGSMVL RGINPNGSIE FGMRSDEVVT KAMLNLEYTP SPSLLPVQSQ LKVYLNDELM GVLPVTKEQL GKKTLAQMPI N PLFITDFN RVRLEFVGHY QDVCENPAST TLWLDVGRSS GLDLTYQTLN VKNDLSHFPV PFFDPRDNRT NTLPMVFAGA PD VGLQQAS AIVASWFGSR SGWRGQNFPV LYNQLPDRNA IVFATNDKRP DFLRDHPAVK APVIEMINHP QNPYVKLLVV FGR DDKDLL QAAKGIAQGN ILFRGESVVV NEVKPLLPRK PYDAPNWVRT DRPVTFGELK TYEEQLQSSG LEPAAINVSL NLPP DLYLM RSTGIDMDIN YRYTMPPVKD SSRMDISLNN QFLQSFNLSS KQEANRLLLR IPVLQGLLDG KTDVSIPALK LGATN QLRF DFEYMNPMPG GSVDNCITFQ PVQNHVVIGD DSTIDFSKYY HFIPMPDLRA FANAGFPFSR MADLSQTITV MPKAPN EAQ METLLNTVGF IGAQTGFPAI NLTVTDDGST IQGKDADIMI IGGIPDKLKD DKQIDLLVQA TESWVKTPMR QTPFPGI VP DESDRAAETR STLTSSGAMA AVIGFQSPYN DQRSVIALLA DSPRGYEMLN DAVNDSGKRA TMFGSVAVIR ESGINSLR V GDVYYVGHLP WFERLWYALA NHPILLAVLA AISVILLAWV LWRLLRIISR RRLNPDNE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
0.02 MHEPES
0.12 MNaClSodium chloride
0.005 MMgCl2
2e-06 MAppCp
2e-06 Mc-di-GMPCyclic di-GMP
0.008 %LM.NPG
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf / Details: Gctf through the cryoSPARC v2 interface.
Startup modelType of model: INSILICO MODEL / In silico model: Ab Initio model generation in cryosparc V2
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. V2)
Details: All 2D classification and particle curation was performed in cryoSPARC v2
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. V2)
Details: Final refinement using non-uniform refinement in cryoSPARC v2, followed by density segmation and mask creation in UCSF Chimera, particle subtraction for removal of noisy/unresolved regions ...Details: Final refinement using non-uniform refinement in cryoSPARC v2, followed by density segmation and mask creation in UCSF Chimera, particle subtraction for removal of noisy/unresolved regions and local refinement of a BcsB pentamer in cryoSPARC v2.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. V2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v2) / Number images used: 576455
DetailsA 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

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6yg8:
Cryo-EM structure of a BcsB pentamer in the context of an assembled Bcs macrocomplex

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