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- PDB-9b8i: Cryo-EM structure of the E. coli cellulose synthase BcsB-BcsC fus... -

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Entry
Database: PDB / ID: 9b8i
TitleCryo-EM structure of the E. coli cellulose synthase BcsB-BcsC fusion protein
ComponentsCellulose synthase BcsB-BcsC fusion protein
KeywordsSTRUCTURAL PROTEIN / bacterial cellulose synthesis / cellulose export / outer membrane porin / periplasmic protein
Function / homology
Function and homology information


cellulose biosynthetic process / UDP-alpha-D-glucose metabolic process / cell outer membrane / plasma membrane
Similarity search - Function
Cellulose synthase operon C, C-terminal / Cellulose synthase operon protein C C-terminus (BCSC_C) / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. ...Cellulose synthase operon C, C-terminal / Cellulose synthase operon protein C C-terminus (BCSC_C) / : / Cellulose synthase, subunit B / Cellulose synthase BcsB, bacterial / Bacterial cellulose synthase subunit / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
Cellulose synthase operon protein C / Cyclic di-GMP-binding protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 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
DepositionMar 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Cellulose synthase BcsB-BcsC fusion protein


Theoretical massNumber of molelcules
Total (without water)99,2421
Polymers99,2421
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cellulose synthase BcsB-BcsC fusion protein


Mass: 99242.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal domain of BcsC (including its first 4 TPRs) was fused to the N-terminus of BcsB via a Gly-Ser linker. The BcsC is at the amino-terminus of the construct, so the order is BcsC-linker-BcsB.
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: bcsC, yhjL, b3530, JW5942, bcsB, yhjN, b3532, JW3500 / Production host: Escherichia coli (E. coli) / References: UniProt: P37650, UniProt: P37652
Has protein modificationN

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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: Cellulose synthase BcsB-BcsC fusion protein / Type: COMPLEX
Details: Proteins: -Bacterial cellulose synthase subunit B (BcsB) -Bacterial cellulose synthase subunit C (BcsC) The BcsC is at the amino-terminus of the construct, so the order is BcsC-linker-BcsB.
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: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41060 / Symmetry type: POINT

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