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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 dataMain map non-uniformly refined. It represents two BcsB octamers (6YG8) linked by a central miscellaneous structure.
Sample
  • Complex: Self-assembly of the BcsB protein
    • Protein or peptide: Bacterial cellulose synthase regulator protein 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: 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.
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

emd_11356.png

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)
AnnotationMain map non-uniformly refined. It represents two BcsB octamers (6YG8) linked by a central miscellaneous structure.
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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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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

Histogram

Histogram (log scale)

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Sample components

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Entire : Self-assembly of the BcsB protein

EntireName: Self-assembly of the BcsB protein
Components
  • Complex: Self-assembly of the BcsB protein
    • Protein or peptide: Bacterial cellulose synthase regulator protein BcsB

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Supramolecule #1: Self-assembly of the BcsB protein

SupramoleculeName: Self-assembly of the BcsB protein / 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: pET-21b
Molecular weightTheoretical: 1.378 MDa

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Macromolecule #1: Bacterial cellulose synthase regulator protein BcsB

MacromoleculeName: Bacterial cellulose synthase regulator protein BcsB / type: protein_or_peptide / ID: 1
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...
Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPS PSLL PVQSQLKVYL NDELMGVLPV TKEQLGKKTL AQMPINPLFI TDFNRVRLEF VGHYQD VCE NPASTTLWLD ...String:
TPATQPLINA EPAVAAQTEQ NPQVGQVMPG VQGADAPVVA QNGPSRDVKL TFAQIAPPPG SMVLRGINPN GSIEFGMRSD EVVTKAMLNL EYTPS PSLL PVQSQLKVYL NDELMGVLPV TKEQLGKKTL AQMPINPLFI TDFNRVRLEF VGHYQD VCE NPASTTLWLD VGRSSGLDLT YQTLNVKNDL SHFPVPFFDP RDNRTNTLPM VFAGAPD VG LQQASAIVAS WFGSRSGWRG QNFPVLYNQL PDRNAIVFAT NDKRPDFLRD HPAVKAPV I EMINHPQNPY VKLLVVFGRD DKDLLQAAKG IAQGNILFRG ESVVVNEVKP LLPRKPYDA PNWVRTDRPV TFGELKTYEE QLQSSGLEPA AINVSLNLPP DLYLMRSTGI DMDINYRYTM PPVKDSSRM DISLNNQFLQ SFNLSSKQEA NRLLLRIPVL QGLLDGKTDV SIPALKLGAT N QLRFDFEY MNPMPGGSVD NCITFQPVQN HVVIGDDSTI DFSKYYHFIP MPDLRAFANA GF PFSRMAD LSQTITVMPK APNEAQMETL LNTVGFIGAQ TGFPAINLTV TDDGSTIQGK DAD IMIIGG IPDKLKDDKQ IDLLVQATES WVKTPMRQTP FPGIVPDESD RAAETRSTLT SSGA MAAVI GFQSPYNDQR SVIALLADSP RGYEMLNDAV NDSGKRATMF GSVAVIRESG INSLR VGDV YYVGHLPWFE RLWYALANHP ILLAVLAAIS VILLAWVLWR LLRIISRRRL NPDNEAAALE HHHHHH

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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

BufferpH: 8
Component:
ConcentrationFormula
0.02 MHEPES
0.5 MNaClSodium chloride
0.2 MImidazol

Details: 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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 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: 36000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 0.74 e/Å2

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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: non-uniform refinement in cryoSPARC v2
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. V2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v2) / Number images used: 46807

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