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- EMDB-17791: Cryo-EM structure of Enterobacter sp. 638 BcsD -

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Basic information

Entry
Database: EMDB / ID: EMD-17791
TitleCryo-EM structure of Enterobacter sp. 638 BcsD
Map dataSharpened cryo-EM map of Enterobacter sp. 638 BcsD
Sample
  • Complex: Tetrameric BcsD of Enterobacter sp. 638
    • Protein or peptide: BcsD of Enterobacter sp. 638
KeywordsCellulose secretion / bacterial biofilms / cytoskeleton / CYTOSOLIC PROTEIN
Function / homologyCellulose synthase operon protein D, bacterial / Cellulose synthase subunit D superfamily / Cellulose synthase subunit D / cellulose biosynthetic process / Cellulose synthase
Function and homology information
Biological speciesEnterobacter sp. 638 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsNotopoulou A / 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
DepositionJul 4, 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_17791.png

Downloads & links

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Map

FileDownload / File: emd_17791.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryo-EM map of Enterobacter sp. 638 BcsD
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 280 pix.
= 260.4 Å		0.93 Å/pix.
x 280 pix.
= 260.4 Å		0.93 Å/pix.
x 280 pix.
= 260.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.98785156 - 1.4415588
Average (Standard dev.)-0.00030290478 (±0.023742514)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 260.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17791_msk_1.map
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 Enterobacter sp. 638 BcsD

Fileemd_17791_additional_1.map
AnnotationDeep EMhancer sharpened cryo-EM map of Enterobacter sp. 638 BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM map of Enterobacter sp. 638 BcsD

Fileemd_17791_additional_2.map
AnnotationUnsharpened cryo-EM map of Enterobacter sp. 638 BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map for Enterobacter sp. 638 BcsD

Fileemd_17791_half_map_1.map
AnnotationCryo-EM half map for Enterobacter sp. 638 BcsD
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map for Enterobacter sp. 638 BcsD

Fileemd_17791_half_map_2.map
AnnotationCryo-EM half map for Enterobacter sp. 638 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 : Tetrameric BcsD of Enterobacter sp. 638

EntireName: Tetrameric BcsD of Enterobacter sp. 638
Components
  • Complex: Tetrameric BcsD of Enterobacter sp. 638
    • Protein or peptide: BcsD of Enterobacter sp. 638

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Supramolecule #1: Tetrameric BcsD of Enterobacter sp. 638

SupramoleculeName: Tetrameric BcsD of Enterobacter sp. 638 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Enterobacter sp. 638 (bacteria)
Molecular weightTheoretical: 70 KDa

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Macromolecule #1: BcsD of Enterobacter sp. 638

MacromoleculeName: BcsD of Enterobacter sp. 638 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Enterobacter sp. 638 (bacteria)
Molecular weightTheoretical: 18.138383 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGSTTTLENP ALLGWFRGQQ TPEGWFDLLA LIVDGMVRNV GELESQPFLR QMGIALADQY PLPASETVGE LEANINAQLA RFGWGCVEI DTSDTDLTLR HQALPVSRVE DQQSRWCHAF CAILEGLYVR WMQGQGGKSH VTVSRERLFS LSDVQFRYHN P Q

UniProtKB: Cellulose synthase

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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: 20 mM HEPES pH 8.0, 120 mM NaCl
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

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 0-50 / Average electron dose: 49.2 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Details5 226 out of 5 949 movies retained for processing
Particle selectionNumber selected: 408125
Details: 408125 aligned particles after 2 rounds of 2D classification
Startup modelType of model: OTHER / Details: Ab initio (cryoSPARC)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Details: Non-uniform refinement (cryoSPARC) / Number images used: 226283
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Details: Ab initio (cryoSPARC)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Details: Ab initio (cryoSPARC) Heterogeneous refinement (cryoSPARC) Non-uniform refinement (cryoSPARC)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC / Details: Heterogeneous refinement (cryoSPARC)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsReiterative refinement in Phenix and Coot
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8pog:
Cryo-EM structure of Enterobacter sp. 638 BcsD

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