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- PDB-9fmz: Cryo-EM structure of the c-di-GMP-bound synthase:pEtN transferase... -

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Basic information

Entry
Database: PDB / ID: 9fmz
TitleCryo-EM structure of the c-di-GMP-bound synthase:pEtN transferase complex (BcsA-Bct-G3) from the E. coli cellulose secretion macrocomplex
Components
  • Cellulose biosynthesis protein BcsG
  • Cellulose synthase catalytic subunit [UDP-forming]
  • Cyclic 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 / Chem-C2E / Cyclic di-GMP-binding protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Cellulose biosynthesis protein BcsG
C: Cellulose biosynthesis protein BcsG
D: Cellulose biosynthesis protein BcsG
B: Cyclic di-GMP-binding protein
A: Cellulose synthase catalytic subunit [UDP-forming]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)367,8067
Polymers366,4255
Non-polymers1,3812
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 Cellulose biosynthesis protein BcsG


Mass: 59687.984 Da / Num. of mol.: 3
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: bcsG, yhjU, b3538, JW3506 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3)
#2: Protein Cyclic di-GMP-binding protein / Cellulose synthase regulatory subunit


Mass: 83345.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: 1094
Gene: bcsB, ACU57_05345, BK292_24560, E6D34_20685, EIZ93_09345, FJQ40_15525, GP975_08505, GQA06_10940, GQM04_12065, GRW57_12105, NEP60_23610
Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: UniProt: A0A061KLG7
#3: Protein Cellulose synthase catalytic subunit [UDP-forming]


Mass: 104015.688 Da / Num. of mol.: 1 / Mutation: HA-FLAG tagged
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: bcsA, yhjO, yhjP, b3533, JW5665 / Production host: Escherichia coli (E. coli) / Strain (production host): NiCo21(DE3) / References: cellulose synthase (UDP-forming)
#4: 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.: 2 / Source method: obtained synthetically / Formula: C20H24N10O14P2 / Feature type: SUBJECT OF INVESTIGATION
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: Purified Bcs macrocomplex with stoichiometry BcsA-BcsB6-BcsR2-BcsQ2-BcsE2-BcsF2-BcsG3
Type: COMPLEX
Details: Locally refined c-di-GMP-bound synthase:pEtN-transferase complex from the E. coli Bcs cellulose secretion macrocomplex
Entity ID: #1-#3 / 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
11cryoSPARCv4final Euler assignment
14Cootmodel refinement
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1359795
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 259200 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingDetails: ColabFold model of the E> coli BcsA-BcsG2 complex / Source name: Other / Type: in silico model

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