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- EMDB-15039: BcsH-BcsD 'beads-on-a-string' filament, local refine -

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Basic information

Entry
Database: EMDB / ID: EMD-15039
TitleBcsH-BcsD 'beads-on-a-string' filament, local refine
Map dataBcsHD 'beads-on-a-string' filaments : Unsharpened locally refined map of a BcsH-bound dimer of BcsD octamers
Sample
  • Complex: BcsHD 'beads-on-a-string' cis-filaments
    • Protein or peptide: Cellulose biosynthesis protein
    • Protein or peptide: BcsH fragment
  • Ligand: water
Function / homologyCellulose-complementing protein / Cellulose-complementing protein A / Cellulose synthase operon protein D, bacterial / Cellulose synthase subunit D superfamily / Cellulose synthase subunit D / cellulose biosynthetic process / Cellulose-complementing protein / Cellulose biosynthesis protein
Function and homology information
Biological speciesKomagataeibacter hansenii ATCC 23769 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKrasteva PV / Abidi W / Decossas M
Funding supportEuropean Union, France, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)BioMatrix-ERC-2017-StGEuropean Union
Centre National de la Recherche Scientifique (CNRS) France
CitationJournal: Sci Adv / Year: 2022
Title: Bacterial crystalline cellulose secretion via a supramolecular BcsHD scaffold.
Authors: Wiem Abidi / Marion Decossas / Lucía Torres-Sánchez / Lucie Puygrenier / Sylvie Létoffé / Jean-Marc Ghigo / Petya V Krasteva /
Abstract: Cellulose, the most abundant biopolymer on Earth, is not only the predominant constituent of plants but also a key extracellular polysaccharide in the biofilms of many bacterial species. Depending on ...Cellulose, the most abundant biopolymer on Earth, is not only the predominant constituent of plants but also a key extracellular polysaccharide in the biofilms of many bacterial species. Depending on the producers, chemical modifications, and three-dimensional assemblies, bacterial cellulose (BC) can present diverse degrees of crystallinity. Highly ordered, or crystalline, cellulose presents great economical relevance due to its ever-growing number of biotechnological applications. Even if some acetic acid bacteria have long been identified as BC superproducers, the molecular mechanisms determining the secretion of crystalline versus amorphous cellulose remain largely unknown. Here, we present structural and mechanistic insights into the role of the accessory subunits BcsH (CcpAx) and BcsD (CesD) that determine crystalline BC secretion in the lineage. We show that oligomeric BcsH drives the assembly of BcsD into a supramolecular cytoskeletal scaffold that likely stabilizes the cellulose-extruding synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly.
History
DepositionMay 26, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15039.png

Downloads & links

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Map

FileDownload / File: emd_15039.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBcsHD 'beads-on-a-string' filaments : Unsharpened locally refined map of a BcsH-bound dimer of BcsD octamers
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 450 pix.
= 377.55 Å		0.84 Å/pix.
x 450 pix.
= 377.55 Å		0.84 Å/pix.
x 450 pix.
= 377.55 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.23
Minimum - Maximum-0.30165786 - 1.0365735
Average (Standard dev.)0.00065345335 (±0.026039476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 377.55 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15039_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: BcsHD 'beads-on-a-string' filaments : Autosharpened locally refined map...

Fileemd_15039_additional_1.map
AnnotationBcsHD 'beads-on-a-string' filaments : Autosharpened locally refined map of a BcsH-bound dimer of BcsD octamers
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15039_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BcsHD 'beads-on-a-string' filaments : half map B

Fileemd_15039_half_map_2.map
AnnotationBcsHD 'beads-on-a-string' filaments : half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : BcsHD 'beads-on-a-string' cis-filaments

EntireName: BcsHD 'beads-on-a-string' cis-filaments
Components
  • Complex: BcsHD 'beads-on-a-string' cis-filaments
    • Protein or peptide: Cellulose biosynthesis protein
    • Protein or peptide: BcsH fragment
  • Ligand: water

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Supramolecule #1: BcsHD 'beads-on-a-string' cis-filaments

SupramoleculeName: BcsHD 'beads-on-a-string' cis-filaments / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Details: G. hansenii BcsHD complex purified after the co-expression of hexahistidine tagged BcsH C-terminal domain and full-length BcsD. IMAC purification via BcsH, tag and linker removed during purification
Source (natural)Organism: Komagataeibacter hansenii ATCC 23769 (bacteria) / Strain: ATCC 23769

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Macromolecule #1: Cellulose biosynthesis protein

MacromoleculeName: Cellulose biosynthesis protein / type: protein_or_peptide / ID: 1
Details: BcsD from Gluconacetobacter hansenii ATCC 23769 expressed recombinantly in BL21 Star DE3 cells
Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Komagataeibacter hansenii ATCC 23769 (bacteria)
Molecular weightTheoretical: 17.532963 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MGSTIFEKKP DFTLFLQTLS WEIDDQVGIE VRNELLREVG RGMGTRIMPP PCQTVDKLQI ELNALLALIG WGTVTLELLS EDQSLRIVH ENLPQVGSAG EPSGTWLAPV LEGLYGRWVT SQAGAFGDYV VTRDVDAEDL NAVPRQTIIM YMRVRSSAT

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Macromolecule #2: BcsH fragment

MacromoleculeName: BcsH fragment / type: protein_or_peptide / ID: 2
Details: BcsH C-terminal domain from G. hansenii expressed from a pProEx-Htb vector with a HRV3c-cleavable hexahistidine tag
Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Komagataeibacter hansenii ATCC 23769 (bacteria)
Molecular weightTheoretical: 9.919921 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MSYYHHHHHH DYDIPTTLEV LFQGPMGSTK TDTNSSQASR PGSPVASPDG SPTMAEVFMT LGGRATELLS PRPSLREALL RRRENEEES

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 105 / 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 statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8 / Details: 20 mM NaCl, 100 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: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsFilaments of various sizes eluting in the void volume of a Superdex 200 Increase size-exclusion column.

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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: 2.75 µm / Nominal defocus min: 0.48 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2849271
Details: Autopicked particles using cryoSPARC's template picker function. The templates were generated by 2D classification and class selection on ~3000 manually particles.
Startup modelType of model: OTHER / Details: Ab-initio model generated in cryoSPARC
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: Ab-initio model generated in cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: Ab-initio model generated in cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 1425195
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3a8e


Chain - Chain ID: A
DetailsModel building with Coot, refinement with Phenix and Namdinator
RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-7zzq:
BcsH-BcsD 'beads-on-a-string' filament, local refine

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