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- EMDB-44346: Cryo-EM structure of the E. coli cellulose synthase BcsB-BcsC fus... -

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Basic information

Entry
Database: EMDB / ID: EMD-44346
TitleCryo-EM structure of the E. coli cellulose synthase BcsB-BcsC fusion protein
Map dataBcsB_BcsC_sharpened map
Sample
  • Complex: Cellulose synthase BcsB-BcsC fusion protein
    • Protein or peptide: Cellulose synthase BcsB-BcsC fusion protein
Keywordsbacterial cellulose synthesis / cellulose export / outer membrane porin / periplasmic protein / Structural 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsVerma P / Zimmer J
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44346.png

Downloads & links

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Map

FileDownload / File: emd_44346.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBcsB_BcsC_sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.0913513 - 2.3502991
Average (Standard dev.)-0.00032217687 (±0.05255529)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: BcsB BcsC halfmap A

Fileemd_44346_half_map_1.map
AnnotationBcsB_BcsC_halfmap_A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BcsB BcsC halfmap B

Fileemd_44346_half_map_2.map
AnnotationBcsB_BcsC_halfmap_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 : Cellulose synthase BcsB-BcsC fusion protein

EntireName: Cellulose synthase BcsB-BcsC fusion protein
Components
  • Complex: Cellulose synthase BcsB-BcsC fusion protein
    • Protein or peptide: Cellulose synthase BcsB-BcsC fusion protein

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Supramolecule #1: Cellulose synthase BcsB-BcsC fusion protein

SupramoleculeName: Cellulose synthase BcsB-BcsC fusion protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Cellulose synthase BcsB-BcsC fusion protein

MacromoleculeName: Cellulose synthase BcsB-BcsC fusion protein / type: protein_or_peptide / ID: 1
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.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 99.242461 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AWSHPQFEKV EAAPTAQQQL LEQVRLGEAT HREDLVQQSL YRLELIDPNN PDVVAARFRS LLRQGDIDGA QKQLDRLSQL APSSNAYKS SRTTMLLSTP DGRQALQQAR LQATTGHAEE AVASYNKLFN GAPPEGDIAV EYWSTVAKIP ARRGEAINQL K RINADAPG ...String:
AWSHPQFEKV EAAPTAQQQL LEQVRLGEAT HREDLVQQSL YRLELIDPNN PDVVAARFRS LLRQGDIDGA QKQLDRLSQL APSSNAYKS SRTTMLLSTP DGRQALQQAR LQATTGHAEE AVASYNKLFN GAPPEGDIAV EYWSTVAKIP ARRGEAINQL K RINADAPG GSGSGSGVQG ADAPVVAQNG PSRDVKLTFA QIAPPPGSMV LRGINPNGSI EFGMRSDEVV TKAMLNLEYT PS PSLLPVQ SQLKVYLNDE LMGVLPVTKE QLGKKTLAQM PINPLFISDF NRVRLEFVGH YQDVCEKPAS TTLWLDVGRS SGL DLTYQT LNVKNDLSHF PVPFFDPSDN RTNTLPMVFA GAPDVGLQQA SAIVASWFGS RSGWRGQNFP VLYNQLPDRN AIVF ATNDK RPDFLRDHPA VKAPVIEMIN HPQNPYVKLL VVFGRDDKDL LQAAKGIAQG NILFRGESVV VNEVKPLLPR KPYDA PNWV RTDRPVTFGE LKTYEEQLQS SGLEPAAINV SLNLPPDLYL MRSTGIDMDI NYRYTMPPVK DSSRMDISLN NQFLQS FNL SSKQEANRLL LRIPVLQGLL DGKTDVSIPA LKLGATNQLR FDFEYMNPMP GGSVDNCITF QPVQNHVVIG DDSTIDF SK YYHFIPMPDL RAFANAGFPF SRMADLSQTI TVMPKAPNEA QMETLLNTVG FIGAQTGFPA INLTVTDDGS TIQGKDAD I MIIGGIPDKL KDDKQIDLLV QATESWVKTP MRQTPFPGIV PDESDRAAET RSTLTSSGAM AAVIGFQSPY NDQRSVIAL LADSPRGYEM LNDAVNDSGK RATMFGSVAV IRESGINSLR VGDVYYVGHL PWFERVWYAL ANHPILLAVL AAISVILLAW VLWRLLRII SRRRLNPDNE

UniProtKB: Cellulose synthase operon protein C, Cyclic di-GMP-binding protein

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: AlphaFold2 predicted model of complex between BcsB and BcsC's N-terminal TPR #1-4.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 41060
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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