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

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Basic information

Entry
Database: PDB / ID: 7zzq
TitleBcsH-BcsD 'beads-on-a-string' filament, local refine
Components
  • BcsH fragment
  • Cellulose biosynthesis protein
KeywordsSTRUCTURAL PROTEIN / Bacterial cytoskeleton / cellulose secretion
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 / Uncharacterized protein / Cellulose biosynthesis protein
Function and homology information
Biological speciesKomagataeibacter hansenii ATCC 23769 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKrasteva, P.V. / Abidi, W. / Decossas, M.
Funding supportEuropean Union, France, 2items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cellulose biosynthesis protein
B: Cellulose biosynthesis protein
C: Cellulose biosynthesis protein
D: Cellulose biosynthesis protein
E: Cellulose biosynthesis protein
F: Cellulose biosynthesis protein
G: Cellulose biosynthesis protein
H: Cellulose biosynthesis protein
I: Cellulose biosynthesis protein
J: Cellulose biosynthesis protein
K: Cellulose biosynthesis protein
L: Cellulose biosynthesis protein
M: Cellulose biosynthesis protein
N: Cellulose biosynthesis protein
O: Cellulose biosynthesis protein
P: Cellulose biosynthesis protein
Q: BcsH fragment
R: BcsH fragment
S: BcsH fragment
T: BcsH fragment
U: BcsH fragment
V: BcsH fragment
W: Cellulose biosynthesis protein
X: BcsH fragment
Y: BcsH fragment
Z: BcsH fragment
a: BcsH fragment
b: BcsH fragment
c: Cellulose biosynthesis protein
d: BcsH fragment


Theoretical massNumber of molelcules
Total (without water)434,63230
Polymers434,63230
Non-polymers00
Water1,892105
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cellulose biosynthesis protein / Cellulose synthase subunit D


Mass: 17532.963 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Details: BcsD from Gluconacetobacter hansenii ATCC 23769 expressed recombinantly in BL21 Star DE3 cells
Source: (gene. exp.) Komagataeibacter hansenii ATCC 23769 (bacteria)
Gene: acsD, GXY_04292 / Plasmid: pRSFDuet1-BcsD / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Star DE3 / References: UniProt: Q76KJ6
#2: Protein
BcsH fragment


Mass: 9919.921 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Details: BcsH C-terminal domain from G. hansenii expressed from a pProEx-Htb vector with a HRV3c-cleavable hexahistidine tag
Source: (gene. exp.) Komagataeibacter hansenii ATCC 23769 (bacteria)
Gene: GXY_04267 / Plasmid: pProEx-Htb / Details (production host): pProEx-BcsH / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Star DE3 / References: UniProt: D5QCK0
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 105 / 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: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BcsHD 'beads-on-a-string' cis-filaments / Type: COMPLEX
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
Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Komagataeibacter hansenii ATCC 23769 (bacteria) / Strain: ATCC 23769
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: Star DE3 / Plasmid: pRSF-Duet1 and pProEx-Htb
Buffer solutionpH: 8 / Details: 20 mM NaCl, 100 mM NaCL
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Filaments of various sizes eluting in the void volume of a Superdex 200 Increase size-exclusion column.
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: 293 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 FIELD / Nominal defocus max: 2750 nm / Nominal defocus min: 480 nm / Cs: 2.7 mm
Image recordingElectron dose: 49.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
4GctfCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARC3classification
12cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1425195 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Details: Model building with Coot, refinement with Phenix and Namdinator
Atomic model buildingPDB-ID: 3A8E

3a8e


Pdb chain-ID: A / Accession code: 3A8E / Source name: PDB / Type: experimental model

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