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- PDB-9fp2: Cryo-EM structure of the BcsEFRQ regulatory subcomplex for E. col... -

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Basic information

Entry
Database: PDB / ID: 9fp2
TitleCryo-EM structure of the BcsEFRQ regulatory subcomplex for E. coli cellulose secretion in non-saturating c-di-GMP (local)
Components
  • Cell division protein
  • Cellulose biosynthesis protein BcsF
  • Cyclic di-GMP binding protein BcsE
  • Protein YhjR
KeywordsMEMBRANE PROTEIN / Bacterial cellulose secretion
Function / homology
Function and homology information


negative regulation of cell division / cytoplasmic side of plasma membrane / cell division / ATP hydrolysis activity / ATP binding / cytosol
Similarity search - Function
Cellulose synthase operon protein BcsQ / Cellulose biosynthesis protein BcsQ / : / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Chem-C2E / Cell division protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 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 12, 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
D: Cyclic di-GMP binding protein BcsE
E: Cyclic di-GMP binding protein BcsE
Q: Cell division protein
R: Protein YhjR
V: Protein YhjR
W: Cell division protein
F: Cellulose biosynthesis protein BcsF
S: Cellulose biosynthesis protein BcsF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,73016
Polymers209,9068
Non-polymers3,8258
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

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Protein , 4 types, 8 molecules DEQWRVFS

#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 Cell division protein / Cellulose biosynthesis protein BcsQ / Cellulose synthase operon protein YhjQ / Cellulose synthase / putative


Mass: 27960.832 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: bcsQ, yhjQ, A8C65_00290, ACU57_05335, BMT91_17060, BON75_10030, BvCmsHHP019_01723, BvCmsSINP011_05061, C2U48_15650, D3O91_11725, D9H68_14440, D9I18_15755, D9I97_13990, DAH34_22885, DAH37_19450, ...Gene: bcsQ, yhjQ, A8C65_00290, ACU57_05335, BMT91_17060, BON75_10030, BvCmsHHP019_01723, BvCmsSINP011_05061, C2U48_15650, D3O91_11725, D9H68_14440, D9I18_15755, D9I97_13990, DAH34_22885, DAH37_19450, E2127_16420, E2128_18010, E2129_18145, E2134_17810, EAI42_04085, EC1094V2_71, NCTC10429_00778, NCTC11022_03734, NCTC9058_01652
Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A0A0B1KWQ0
#3: Protein Protein YhjR


Mass: 8645.541 Da / Num. of mol.: 2 / Mutation: N-terminal His-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: yhjR, b3535, JW3503 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#4: 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)

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Non-polymers , 3 types, 8 molecules

#5: 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 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

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 BcsEFRQ regulatory subcomplex in non-saturating c-di-GMP from the E. coli cellulose secretion system
Type: COMPLEX
Details: Local refinement in the context of an assembled macrocomplex
Entity ID: #1-#4 / 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 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
Specimen holderCryogen: NITROGEN
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: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 275132 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
16ybb

6ybb

6ybbcrystal structure of the BcsERQ complex1PDBexperimental model
2ColabFoldColabFold model for full-length BcsE2-BcsF2 complex2Otherin silico model

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