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- PDB-9fo7: Cryo-EM structure of the BcsE2F2 regulatory subcomplex from the E... -

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Basic information

Entry
Database: PDB / ID: 9fo7
TitleCryo-EM structure of the BcsE2F2 regulatory subcomplex from the E. coli Bcs macrocomplex for cellulose secretion (local refinement)
Components
  • Cellulose biosynthesis protein BcsF
  • Cyclic di-GMP binding protein BcsE
KeywordsMEMBRANE PROTEIN / Bacterial cellulose secretion
Function / homologyChem-C2E
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 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 11, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Cyclic di-GMP binding protein BcsE
U: Cyclic di-GMP binding protein BcsE
F: Cellulose biosynthesis protein BcsF
S: Cellulose biosynthesis protein BcsF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,4558
Polymers136,6934
Non-polymers2,7624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cyclic di-GMP binding protein BcsE


Mass: 60967.539 Da / Num. of mol.: 2 / Mutation: N-terminal Strep-tag
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsE, yhjS, b3536, JW3504 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#2: Protein Cellulose biosynthesis protein BcsF


Mass: 7378.975 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094 / Gene: bcsF, Z4953, ECs4417 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#3: Chemical
ChemComp-C2E / 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one) / c-di-GMP / Cyclic diguanosine monophosphate


Mass: 690.411 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C20H24N10O14P2
Has ligand of interestY
Has protein modificationN

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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 BcsE2F2 regulatory subcomplex in saturating c-di-GMP
Type: COMPLEX
Details: Locally refined BcsE2F2 subcomplex in the context of the c-di-GMP-saturated Bcs macrocomplex for cellulose secretion in E. coli
Entity ID: #1-#2 / 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: Purification from solubilized membrane fraction using an anti-FLAG M2 affinity resin
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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 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
7Cootmodel fitting
8PHENIXmodel fitting
10Cootmodel refinement
11PHENIXmodel refinement
13cryoSPARCv4final Euler assignment
15cryoSPARCv43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1359795
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 260501 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDDetailsSource nameTypeAccession codeInitial refinement model-ID
1ColabFold model of the E. coli BcsE2-BcsF2 complexOtherin silico model
26ybu

6ybu

PDBexperimental model6ybu2
36ybb

6ybb

PDBexperimental model6ybb3

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