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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 dataCDTb Long Form Composite Map
Sample
  • Complex: CDTb
    • Protein or peptide: ADP-ribosyltransferase binding component
KeywordsCDTb / Clostridium / Toxin / Binary / difficil / TRANSFERASE
Function / homology
Function and homology information


protein homooligomerization / transferase activity / extracellular region / metal ion binding / identical protein binding
Similarity search - Function
Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA domain 2 / Clostridial binary toxin B/anthrax toxin PA domain 3 / PA14 domain / PA14 / PA14 domain
Similarity search - Domain/homology
ADP-ribosyltransferase binding component
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsLacy DB / Sheedlo MJ
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.
History
DepositionFeb 24, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseOct 30, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing 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
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6o2m
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0608.png

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)
AnnotationCDTb Long Form Composite Map
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-10.3016615 - 20.780972999999999
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
Components
  • Complex: CDTb
    • Protein or peptide: ADP-ribosyltransferase binding component

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Supramolecule #1: CDTb

SupramoleculeName: CDTb / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridioides difficile (bacteria)

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Macromolecule #1: ADP-ribosyltransferase binding component

MacromoleculeName: ADP-ribosyltransferase binding component / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 98.916828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIQMRNKKV LSFLTLTAIV SQALVYPVYA QTSTSNHSNK KKEIVNEDIL PNNGLMGYYF TDEHFKDLKL MAPIKDGNLK FEEKKVDKL LDKDKSDVKS IRWTGRIIPS KDGEYTLSTD RDDVLMQVNT ESTISNTLKV NMKKGKEYKV RIELQDKNLG S IDNLSSPN ...String:
MKIQMRNKKV LSFLTLTAIV SQALVYPVYA QTSTSNHSNK KKEIVNEDIL PNNGLMGYYF TDEHFKDLKL MAPIKDGNLK FEEKKVDKL LDKDKSDVKS IRWTGRIIPS KDGEYTLSTD RDDVLMQVNT ESTISNTLKV NMKKGKEYKV RIELQDKNLG S IDNLSSPN LYWELDGMKK IIPEENLFLR DYSNIEKDDP FIPNNNFFDP KLMSDWEDED LDTDNDNIPD SYERNGYTIK DL IAVKWED SFAEQGYKKY VSNYLESNTA GDPYTDYEKA SGSFDKAIKT EARDPLVAAY PIVGVGMEKL IISTNEHAST DQG KTVSRA TTNSKTESNT AGVSVNVGYQ NGFTANVTTN YSHTTDNSTA VQDSNGESWN TGLSINKGES AYINANVRYY NTGT APMYK VTPTTNLVLD GDTLSTIKAQ ENQIGNNLSP GDTYPKKGLS PLALNTMDQF SSRLIPINYD QLKKLDAGKQ IKLET TQVS GNFGTKNSSG QIVTEGNSWS DYISQIDSIS ASIILDTENE SYERRVTAKN LQDPEDKTPE LTIGEAIEKA FGATKK DGL LYFNDIPIDE SCVELIFDDN TANKIKDSLK TLSDKKIYNV KLERGMNILI KTPTYFTNFD DYNNYPSTWS NVNTTNQ DG LQGSANKLNG ETKIKIPMSE LKPYKRYVFS GYSKDPLTSN SIIVKIKAKE EKTDYLVPEQ GYTKFSYEFE TTEKDSSN I EITLIGSGTT YLDNLSITEL NSTPEILDEP EVKIPTDQEI MDAHKIYFAD LNFNPSTGNT YINGMYFAPT QTNKEALDY IQKYRVEATL QYSGFKDIGT KDKEMRNYLG DPNQPKTNYV NLRSYFTGGE NIMTYKKLRI YAITPDDREL LVLSVD

UniProtKB: ADP-ribosyltransferase binding component

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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
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 2 / Number real images: 4914 / Average exposure time: 9.6 sec. / Average electron dose: 110.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 13266
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6o2m:
CDTb Double Heptamer Long Form Modeled from Cryo-EM Map Reconstructed using C7 Symmetry

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