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- PDB-9b8v: AlphaFold2 informed cryo-EM model of the E. coli cellulose syntha... -

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Basic information

Entry
Database: PDB / ID: 9b8v
TitleAlphaFold2 informed cryo-EM model of the E. coli cellulose synthase BcsAG3B6 complex
Components
  • Cellulose biosynthesis protein BcsG
  • Cellulose synthase catalytic subunit [UDP-forming]
  • Cellulose synthase operon protein B
KeywordsTRANSFERASE / phosphoethanolamine (pEtN) / catalytic BcsA-B / c-di-GMP binding protein / membrane proteins
Function / homology
Function and homology information


bacterial cellulose biosynthetic process / cellulose synthase (UDP-forming) / cellulose synthase (UDP-forming) activity / cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / hexosyltransferase activity / cyclic-di-GMP binding / plasma membrane
Similarity search - Function
Cellulose biosynthesis protein BcsG / Cellulose biosynthesis protein BcsG / Cellulose synthase, subunit A / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / PilZ domain / PilZ domain / Glycosyltransferase 2-like ...Cellulose biosynthesis protein BcsG / Cellulose biosynthesis protein BcsG / Cellulose synthase, subunit A / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / PilZ domain / PilZ domain / Glycosyltransferase 2-like / Glycosyl transferase family 2 / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Cyclic di-GMP-binding protein / Cellulose synthase catalytic subunit [UDP-forming] / Cellulose biosynthesis protein BcsG
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsVerma, P. / Zimmer, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144130 United States
CitationJournal: Nat Commun / Year: 2024
Title: Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope.
Authors: Preeti Verma / Ruoya Ho / Schuyler A Chambers / Lynette Cegelski / Jochen Zimmer /
Abstract: Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the ...Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle's terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.
History
DepositionApr 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 19, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2025Group: Data collection / Structure summary / Category: em_admin / entity / entity_name_com
Item: _em_admin.last_update / _entity.pdbx_description / _entity_name_com.name
Revision 1.1Feb 26, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Experimental summary / Structure summary / Data content type: EM metadata / EM metadata / Category: em_admin / entity / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _entity.pdbx_description
Revision 1.2May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.2May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Cellulose synthase operon protein B
B: Cellulose biosynthesis protein BcsG
C: Cellulose biosynthesis protein BcsG
D: Cellulose biosynthesis protein BcsG
E: Cellulose synthase catalytic subunit [UDP-forming]
A: Cellulose synthase operon protein B
F: Cellulose synthase operon protein B
H: Cellulose synthase operon protein B
I: Cellulose synthase operon protein B
J: Cellulose synthase operon protein B


Theoretical massNumber of molelcules
Total (without water)797,35710
Polymers797,35710
Non-polymers00
Water00
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 B / Cellulose synthase regulatory subunit / BcsB


Mass: 84375.984 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bcsB, yhjN, b3532, JW3500 / Production host: Escherichia coli (E. coli) / References: UniProt: P37652
#2: Protein Cellulose biosynthesis protein BcsG


Mass: 63079.508 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bcsG, yhjU, b3538, JW3506 / Production host: Escherichia coli (E. coli) / References: UniProt: P37659
#3: Protein Cellulose synthase catalytic subunit [UDP-forming] / Bacterial cellulose synthase subunit A (BcsA)


Mass: 101862.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bcsA, yhjO, yhjP, b3533, JW5665 / Production host: Escherichia coli (E. coli)
References: UniProt: P37653, cellulose synthase (UDP-forming)
Has protein modificationY

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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: Bacterial cellulose synthase BcsA-BcsG3-BcsB6 complex / Type: COMPLEX
Details: Proteins: 1. Bacterial cellulose synthase subunit A (BcsA) 2. Bacterial cellulose synthase subunit B (BcsB) 3. Bacterial cellulose synthase subunit G (BcsG)
Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39522 / Symmetry type: POINT

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