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- PDB-6yg8: Cryo-EM structure of a BcsB pentamer in the context of an assembl... -

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Basic information

Entry
Database: PDB / ID: 6yg8
TitleCryo-EM structure of a BcsB pentamer in the context of an assembled Bcs macrocomplex
ComponentsBacterial cellulose secretion regulator BcsB
KeywordsSIGNALING PROTEIN / Bacterial biofilms / Bacterial cellulose / Bacterial secretion system / regulator protein
Function / homologyCellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / plasma membrane / Cyclic di-GMP-binding protein / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsZouhir, S. / Krasteva, P.V.
Funding support5items
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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 24, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Bacterial cellulose secretion regulator BcsB
B: Bacterial cellulose secretion regulator BcsB
C: Bacterial cellulose secretion regulator BcsB
D: Bacterial cellulose secretion regulator BcsB
E: Bacterial cellulose secretion regulator BcsB


Theoretical massNumber of molelcules
Total (without water)430,9225
Polymers430,9225
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, The apoBcsB full length protein was purified and used for cryo-EM grid preparation. The 2D projection analysis shows the self polymerisation of BcsB
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26610 Å2
ΔGint-86 kcal/mol
Surface area115430 Å2
MethodPISA

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Components

#1: Protein
Bacterial cellulose secretion regulator BcsB / Bacterial cellulose secretion regulator BcsB


Mass: 86184.383 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: Bacterial cellulose synthesis subunit B / Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094
Gene: bcsB, A8C65_00280, AC789_1c39010, ACU57_05345, AM464_10380, BHS81_21120, BK292_24560, BON72_13325, BON75_10020, BON76_21165, BON94_21155, BZL31_14275, C5P01_24165, C9162_26260, CI641_010915, ...Gene: bcsB, A8C65_00280, AC789_1c39010, ACU57_05345, AM464_10380, BHS81_21120, BK292_24560, BON72_13325, BON75_10020, BON76_21165, BON94_21155, BZL31_14275, C5P01_24165, C9162_26260, CI641_010915, CWS33_18410, D3O91_11715, D9I18_15765, DQF57_09480, E0J34_09745, E0K84_22715, E2127_16410, E2128_18000, E2129_18135, E2134_17800, E5P22_13645, ELV08_08610, F1E03_18495, F1E19_10130, FRV13_18850
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A061KLG7, UniProt: P37652*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Co-expression of Bcs subunits R, Q, A, B, E, F and G lead to the purification of a membrane complex assemblyCOMPLEXall0RECOMBINANT
2Homopentameric assembly of the BcsB proteinCOMPLEXall1RECOMBINANT
Molecular weightValue: 0.734 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellular location
11Escherichia coli (E. coli)5621094periplasm
22Escherichia coli (E. coli)5621094periplasm
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
11Escherichia coli BL21(DE3) (bacteria)469008pCDF-duet
22Escherichia coli BL21(DE3) (bacteria)469008pCDF-duet
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
10.02 MHEPES1
20.120 MNaCl1
30.005 MMgCl21
40.000002 MAppCp1
50.000002 Mc-di-GMP1
60.008 %LM.NPG1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2750 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 1.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCv2particle selectiontemplate picker
2EPUimage acquisition
4GctfCTF correction
9cryoSPARCV2initial Euler assignment
10cryoSPARCV2final Euler assignment
11cryoSPARCV2classification
12cryoSPARCv23D reconstruction
13PHENIX1.16-3549-000model refinement
Image processingDetails: 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
CTF correctionDetails: Gctf through the cryoSPARC v2 interface. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 576455 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424888
ELECTRON MICROSCOPYf_angle_d0.55333879
ELECTRON MICROSCOPYf_dihedral_angle_d5.1515133
ELECTRON MICROSCOPYf_chiral_restr0.0473794
ELECTRON MICROSCOPYf_plane_restr0.0044501

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