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- PDB-8poc: Cryo-EM structure of Dickeya dadantii BcsD -

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Basic information

Entry
Database: PDB / ID: 8poc
TitleCryo-EM structure of Dickeya dadantii BcsD
ComponentsCellulose synthase operon protein D
KeywordsCYTOSOLIC PROTEIN / Cellulose secretion / bacterial biofilms / cytoskeleton
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 speciesDickeya dadantii 3937 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsNotopoulou, A. / 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
DepositionJul 4, 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)71,7214
Polymers71,7214
Non-polymers00
Water0
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
Cellulose synthase operon protein D


Mass: 17930.289 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Dickeya dadantii 3937 (bacteria) / Gene: bcsD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: E0SES7

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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: Tetrameric BcsD of Dickeya dadantii / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.07 MDa
Source (natural)Organism: Dickeya dadantii 3937 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 20 mM HEPES pH 8.0, 120 mM NaCl
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm
Image recordingElectron dose: 52.9 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 0-50

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7PHENIXmodel fitting
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
13PHENIXmodel refinement
14Cootmodel refinement
Image processingDetails: 3168 out of 3379 movies retained for processing
CTF correctionDetails: Gctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1488279
Details: After first round of 2D classification, 406652 particles retained for Ab-initio model generation and downstream processing
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 295199 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Reiterative refinement in Phenix, Coot and Namdinator
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035036
ELECTRON MICROSCOPYf_angle_d0.6116832
ELECTRON MICROSCOPYf_dihedral_angle_d3.461692
ELECTRON MICROSCOPYf_chiral_restr0.034728
ELECTRON MICROSCOPYf_plane_restr0.004896

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