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- PDB-9fmt: Cryo-EM structure of the BcsB hexameric crown from the E. coli ce... -

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Basic information

Entry
Database: PDB / ID: 9fmt
TitleCryo-EM structure of the BcsB hexameric crown from the E. coli cellulose secretion macrocomplex
ComponentsCyclic di-GMP-binding protein
KeywordsMEMBRANE PROTEIN / Bacterial cellulose secretion
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
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsAnso, I. / Krasteva, P.V.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)757507European Union
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system.
Authors: Itxaso Anso / Samira Zouhir / Thibault Géry Sana / Petya Violinova Krasteva /
Abstract: Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include ...Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsERQ regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.
History
DepositionJun 7, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cyclic di-GMP-binding protein
B: Cyclic di-GMP-binding protein
D: Cyclic di-GMP-binding protein
E: Cyclic di-GMP-binding protein
F: Cyclic di-GMP-binding protein
C: Cyclic di-GMP-binding protein


Theoretical massNumber of molelcules
Total (without water)500,0756
Polymers500,0756
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Cyclic di-GMP-binding protein / Cellulose synthase regulatory subunit


Mass: 83345.844 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsB, BCB93_004993, BXT93_19170 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A0A193LYZ8
Has protein modificationY

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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

ComponentName: Locally refined hexameric BcsB periplasmic crown from the E. coli cellulose secretion system
Type: COMPLEX
Details: Local refinement in the context of an assembled macrocomplex
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.99 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 1094
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: NiCo21(DE3)
Buffer solutionpH: 8
Details: 120 mM NaCL 20 mM HEPES pH8 5 mM MgCl2 10 uM ApppCp 4 uM c-di-GMP 0.01% LM-NPG
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen 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 defocus max: 2100 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm
Image recordingElectron dose: 49.35 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 20022
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4particle selection
4cryoSPARC4CTF correction
7Cootmodel fitting
8PHENIXmodel fitting
10cryoSPARCV4initial Euler assignment
11cryoSPARCv4final Euler assignment
12cryoSPARCv4classification
13cryoSPARCv43D reconstruction
14PHENIXmodel refinement
15Cootmodel refinement
Image processingDetails: GIF Quantum LS
CTF correctionDetails: cryoSPARC V.4 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1359795
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 834077 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6yg8

6yg8


Pdb chain-ID: C / Accession code: 6yg8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00230860
ELECTRON MICROSCOPYf_angle_d0.44842018
ELECTRON MICROSCOPYf_dihedral_angle_d4.1714159
ELECTRON MICROSCOPYf_chiral_restr0.0444686
ELECTRON MICROSCOPYf_plane_restr0.0045563

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