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- EMDB-0608: Reconstruction of CDTb Double Heptamer Long Form using C7 Symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-0608
TitleReconstruction of CDTb Double Heptamer Long Form using C7 Symmetry
Map data
SampleCDTb
  • ADP-ribosyltransferase binding componentPoly (ADP-ribose) polymerase
Function / homology
Function and homology information


protein homooligomerization / transferase activity / pathogenesis / extracellular region
Bacterial exotoxin B / PA14 domain / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily
ADP-ribosyltransferase binding component
Biological speciesClostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsLacy DB / Sheedlo MJ / Anderson DM
Funding support United States, 2 items
OrganizationGrant numberCountry
Other governmentBX002943 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI095755 United States
CitationJournal: Nat Microbiol / Year: 2020
Title: Structural insights into the transition of Clostridioides difficile binary toxin from prepore to pore.
Authors: David M Anderson / Michael J Sheedlo / Jaime L Jensen / D Borden Lacy /
Abstract: Clostridioides (formerly Clostridium) difficile is a Gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in US hospitals. The ...Clostridioides (formerly Clostridium) difficile is a Gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in US hospitals. The disease state is usually preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe diarrhoea. C. difficile infection is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB) and the C. difficile transferase toxin (CDT). Whereas TcdA and TcdB are considered the primary virulence factors, recent studies suggest that CDT increases the severity of C. difficile infection in some of the most problematic clinical strains. To better understand how CDT functions, we used cryo-electron microscopy to define the structure of CDTb, the cell-binding component of CDT. We obtained structures of several oligomeric forms that highlight the conformational changes that enable conversion from a prepore to a β-barrel pore. The structural analysis also reveals a glycan-binding domain and residues involved in binding the host-cell receptor, lipolysis-stimulated lipoprotein receptor. Together, these results provide a framework to understand how CDT functions at the host cell interface.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionFeb 24, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseOct 30, 2019-
UpdateMay 13, 2020-
Current statusMay 13, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o2m
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0608.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.45 Å/pix.
x 250 pix.
= 362.5 Å
1.45 Å/pix.
x 250 pix.
= 362.5 Å
1.45 Å/pix.
x 250 pix.
= 362.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 4 / Movie #1: 4
Minimum - Maximum-10.3016615 - 20.780973
Average (Standard dev.)0.021561196 (±0.8897639)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 362.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z362.500362.500362.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ250250250
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-10.30220.7810.022

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Supplemental data

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Sample components

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Entire CDTb

EntireName: CDTb / Number of components: 2

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Component #1: protein, CDTb

ProteinName: CDTb / Recombinant expression: No
SourceSpecies: Clostridioides difficile (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, ADP-ribosyltransferase binding component

ProteinName: ADP-ribosyltransferase binding componentPoly (ADP-ribose) polymerase
Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 98.916828 kDa
SourceSpecies: Clostridioides difficile (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 110.1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4914

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C7 (7 fold cyclic) / Number of projections: 13266
3D reconstructionSoftware: RELION / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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