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- EMDB-6225: CryoEM single particle reconstruction of anthrax toxin protective... -

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Basic information

Entry
Database: EMDB / ID: EMD-6225
TitleCryoEM single particle reconstruction of anthrax toxin protective antigen pore (lacking the membrane-spanning beta barrel) at 3.6 Angstrom resolution
Map dataSingle particle reconstruction of anthrax toxin protective antigen pore lacking the membrane-spanning beta barrel
Sample
  • Sample: Heptamer of C-terminal 63-kDa fragment of anthrax toxin protective antigen, pore conformation
  • Protein or peptide: Anthrax toxin protective antigen
Keywordsbacterial toxin / anthrax toxin / protective antigen / protein translocation channel
Function / homology
Function and homology information


positive regulation of apoptotic process in another organism / host cell cytosol / negative regulation of MAPK cascade / Uptake and function of anthrax toxins / host cell endosome membrane / protein homooligomerization / toxin activity / host cell plasma membrane / extracellular region / membrane ...positive regulation of apoptotic process in another organism / host cell cytosol / negative regulation of MAPK cascade / Uptake and function of anthrax toxins / host cell endosome membrane / protein homooligomerization / toxin activity / host cell plasma membrane / extracellular region / membrane / metal ion binding / identical protein binding
Similarity search - Function
Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / 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 ...Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / 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/GLEYA domain / PA14 domain profile. / PA14 domain / PA14 / PA14 domain
Similarity search - Domain/homology
Biological speciesBacillus anthracis (anthrax bacterium)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsJiang J / Pentelute BL / Collier RJ / Zhou ZH
CitationJournal: Nature / Year: 2015
Title: Atomic structure of anthrax protective antigen pore elucidates toxin translocation.
Authors: Jiansen Jiang / Bradley L Pentelute / R John Collier / Z Hong Zhou /
Abstract: Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. ...Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed.
History
DepositionDec 25, 2014-
Header (metadata) releaseMar 11, 2015-
Map releaseMar 11, 2015-
UpdateJun 10, 2015-
Current statusJun 10, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_6225.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction of anthrax toxin protective antigen pore lacking the membrane-spanning beta barrel
Voxel sizeX=Y=Z: 1.28 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.04
Minimum - Maximum-0.08884162 - 0.16128567
Average (Standard dev.)0.00123628 (±0.00982956)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 204.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z204.800204.800204.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-0.0890.1610.001

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Supplemental data

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Sample components

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Entire : Heptamer of C-terminal 63-kDa fragment of anthrax toxin protectiv...

EntireName: Heptamer of C-terminal 63-kDa fragment of anthrax toxin protective antigen, pore conformation
Components
  • Sample: Heptamer of C-terminal 63-kDa fragment of anthrax toxin protective antigen, pore conformation
  • Protein or peptide: Anthrax toxin protective antigen

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Supramolecule #1000: Heptamer of C-terminal 63-kDa fragment of anthrax toxin protectiv...

SupramoleculeName: Heptamer of C-terminal 63-kDa fragment of anthrax toxin protective antigen, pore conformation
type: sample / ID: 1000 / Oligomeric state: Heptamer / Number unique components: 1
Molecular weightTheoretical: 440 KDa

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Macromolecule #1: Anthrax toxin protective antigen

MacromoleculeName: Anthrax toxin protective antigen / type: protein_or_peptide / ID: 1 / Name.synonym: PA / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Molecular weightTheoretical: 63 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET22b
SequenceUniProtKB: Protective antigen

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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

Concentration0.05 mg/mL
BufferpH: 5 / Details: 50 mM NaOAc, 0.05% Igepal CA-630
GridDetails: Quantifoil R1.2/1.3 grid with thin carbon support, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39062 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.1 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 22500
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJan 8, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 7062 / Average electron dose: 30 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 21632

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