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- EMDB-17735: Cryo-EM structure of Orrella dioscoreae BcsD -

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Basic information

Entry
Database: EMDB / ID: EMD-17735
TitleCryo-EM structure of Orrella dioscoreae BcsD
Map dataSharpened cryo-EM map of Orrella dioscoreae BcsD
Sample
  • Complex: Orrella dioscoreae BcsD
    • Protein or peptide: Cellulose synthase operon protein D
  • Ligand: water
KeywordsBacterial cytoskeleton / bacterial cellulose / bacterial secretion / bacterial biofilms / STRUCTURAL PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsPuygrenier L / Decossas M / Krasteva PV
Funding supportEuropean Union, 1 items
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, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17735.png

Downloads & links

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Map

FileDownload / File: emd_17735.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map of Orrella dioscoreae BcsD
Voxel sizeX=Y=Z: 0.657 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.9246595 - 3.3822882
Average (Standard dev.)0.00019461827 (±0.0834315)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 197.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened cryo-EM map of Orrella dioscoreae BcsD

Fileemd_17735_additional_1.map
AnnotationUnsharpened cryo-EM map of Orrella dioscoreae BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Deep EMhancer sharpened cryo-EM map of Orrella dioscoreae BcsD

Fileemd_17735_additional_2.map
AnnotationDeep EMhancer sharpened cryo-EM map of Orrella dioscoreae BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half-map for O. dioscoreae BcsD

Fileemd_17735_half_map_1.map
AnnotationEM half-map for O. dioscoreae BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half-map for O. dioscoreae BcsD

Fileemd_17735_half_map_2.map
AnnotationEM half-map for O. dioscoreae BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Orrella dioscoreae BcsD

EntireName: Orrella dioscoreae BcsD
Components
  • Complex: Orrella dioscoreae BcsD
    • Protein or peptide: Cellulose synthase operon protein D
  • Ligand: water

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Supramolecule #1: Orrella dioscoreae BcsD

SupramoleculeName: Orrella dioscoreae BcsD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Orrella dioscoreae (bacteria)
Molecular weightTheoretical: 69.7 KDa

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Macromolecule #1: Cellulose synthase operon protein D

MacromoleculeName: Cellulose synthase operon protein D / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Orrella dioscoreae (bacteria)
Molecular weightTheoretical: 17.448371 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGSMENPLFD YYRNRQAPLQ WRGALGALAQ SLTNHFSPEQ LRTLLREAGQ HFASQHPVQA AETVQSMQDA MNGVWTTQDW GWVDIHDLD SFLTLTHYAA PLESAFGAQN LAWSAAFLEG VYEQWFRQLG ASDALHVRQS EESDVRKAIV LRLGR

UniProtKB: Cellulose synthase operon protein D

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 16 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8 / Details: 100 mM NaCl, 20 mM HEPES pH 8.0
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was monodispersed, size-exclusion purified protein.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.4 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 19118 / Average electron dose: 51.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6563631
Startup modelType of model: INSILICO MODEL
In silico model: cryoSPARC Ab initio (map) Alphafold (atomic model)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: cryoSPARC Ab initio (map)
Final 3D classificationNumber classes: 3
Details: Ab initio with three models, dominant class used for final reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Heterogeneous refinement Non-uniform refinement
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: All refinements (heterorefinement and non-uniform refinement performed in cryoSPARC)
Number images used: 2032604
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsIterative model building and refinement in Coot, Namdinator and Phenix.
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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