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- PDB-8pkd: Cryo-EM structure of Orrella dioscoreae BcsD -

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Basic information

Entry
Database: PDB / ID: 8pkd
TitleCryo-EM structure of Orrella dioscoreae BcsD
ComponentsCellulose synthase operon protein D
KeywordsSTRUCTURAL PROTEIN / Bacterial cytoskeleton / bacterial cellulose / bacterial secretion / bacterial biofilms
Function / homologyCellulose synthase operon protein D, bacterial / Cellulose synthase subunit D superfamily / Cellulose synthase subunit D / cellulose biosynthetic process / Cellulose synthase operon protein D
Function and homology information
Biological speciesOrrella dioscoreae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsPuygrenier, L. / Decossas, M. / Krasteva, P.V.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)757507European Union
CitationJournal: Curr Biol / Year: 2024
Title: Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.
Authors: Thibault G Sana / Areti Notopoulou / Lucie Puygrenier / Marion Decossas / Sandra Moreau / Aurélien Carlier / Petya V Krasteva /
Abstract: Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. ...Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19 century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing β- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.
History
DepositionJun 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cellulose synthase operon protein D
B: Cellulose synthase operon protein D
C: Cellulose synthase operon protein D
D: Cellulose synthase operon protein D


Theoretical massNumber of molelcules
Total (without water)69,7934
Polymers69,7934
Non-polymers00
Water28816
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 synthase operon protein D


Mass: 17448.371 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Orrella dioscoreae (bacteria) / Gene: ODI_03820 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1C3K6W2
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Formula: H2O

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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: Orrella dioscoreae BcsD / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.0697 MDa / Experimental value: YES
Source (natural)Organism: Orrella dioscoreae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pRSF-Duet1
Buffer solutionpH: 8 / Details: 100 mM NaCl, 20 mM HEPES pH 8.0
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodispersed, size-exclusion purified protein.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil 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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 51.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 19118
EM imaging opticsEnergyfilter name: GIF Quantum LS

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4GctfCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6563631
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2032604
Details: All refinements (heterorefinement and non-uniform refinement performed in cryoSPARC)
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Iterative model building and refinement in Coot, Namdinator and Phenix.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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