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- EMDB-44334: Tetrameric cryo-EM structure of E. coli BcsZ -

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Basic information

Entry
Database: EMDB / ID: EMD-44334
TitleTetrameric cryo-EM structure of E. coli BcsZ
Map dataBcsZ cryo-EM map
Sample
  • Complex: Tetrameric assembly of bacterial cellulose synthase subunit Z
    • Protein or peptide: Endoglucanase
KeywordsCellulase / Endoglucanase / GH-8 family / Carbohydrate-active enzyme / Hydrolase
Function / homology
Function and homology information


cellulase / cellulase activity / cellulose catabolic process / extracellular region
Similarity search - Function
Glycoside hydrolase, family 8, conserved site / Glycosyl hydrolases family 8 signature. / Glycoside hydrolase, family 8 / Glycosyl hydrolases family 8 / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 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 29, 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_44334.png

Downloads & links

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Map

FileDownload / File: emd_44334.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBcsZ cryo-EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 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-2.059886 - 3.0431423
Average (Standard dev.)0.0006976046 (±0.06677103)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 2

Fileemd_44334_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_44334_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrameric assembly of bacterial cellulose synthase subunit Z

EntireName: Tetrameric assembly of bacterial cellulose synthase subunit Z
Components
  • Complex: Tetrameric assembly of bacterial cellulose synthase subunit Z
    • Protein or peptide: Endoglucanase

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Supramolecule #1: Tetrameric assembly of bacterial cellulose synthase subunit Z

SupramoleculeName: Tetrameric assembly of bacterial cellulose synthase subunit Z
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Protein -Endoglucanase
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Endoglucanase

MacromoleculeName: Endoglucanase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: cellulase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 40.730762 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MACTWPAWEQ FKKDYISQEG RVIDPSDARK ITTSEGQSYG MFSALAANDR AAFDNILDWT QNNLAQGSLK ERLPAWLWGK KENSKWEVL DSNSASDGDV WMAWSLLEAG RLWKEQRYTD IGSALLKRIA REEVVTVPGL GSMLLPGKVG FAEDNSWRFN P SYLPPTLA ...String:
MACTWPAWEQ FKKDYISQEG RVIDPSDARK ITTSEGQSYG MFSALAANDR AAFDNILDWT QNNLAQGSLK ERLPAWLWGK KENSKWEVL DSNSASDGDV WMAWSLLEAG RLWKEQRYTD IGSALLKRIA REEVVTVPGL GSMLLPGKVG FAEDNSWRFN P SYLPPTLA QYFTRFGAPW TTLRETNQRL LLETAPKGFS PDWVRYEKDK GWQLKAEKTL ISSYDAIRVY MWVGMMPDSD PQ KARMLNR FKPMATFTEK NGYPPEKVDV ATGKAQGKGP VGFSAAMLPF LQNRDAQAVQ RQRVADNFPG SDAYYNYVLT LFG QGWDQH RFRFSTKGEL LPDWGQECAN SHLEHHHHHH

UniProtKB: Endoglucanase

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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 BIOCONTINUUM (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: 2.2 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: PDB ENTRY
PDB model - PDB ID:

3qxq

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 184800
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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