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- EMDB-23066: Protective antigen pore translocating lethal factor N-terminal domain -

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Basic information

Entry
Database: EMDB / ID: EMD-23066
TitleProtective antigen pore translocating lethal factor N-terminal domain
Map dataAnthrax Toxin Translocation Complex
Sample
  • Complex: Anthrax toxin protective antigen translocating lethal factor N-terminal domain
    • Protein or peptide: Lethal factor
    • Protein or peptide: Protective antigen
  • Ligand: CALCIUM IONCalcium
Keywordstranslocation / complex / anthrax / refolding / TOXIN
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / 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 / metalloendopeptidase activity / metallopeptidase activity / toxin activity ...anthrax lethal factor endopeptidase / 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 / metalloendopeptidase activity / metallopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / membrane / identical protein binding / metal ion binding
Similarity search - Function
Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / Anthrax toxin, lethal/endema factor / : / Anthrax toxin lethal factor (ATLF)-like domain profile. / Anthrax toxin, lethal/endema factor, N-/C-terminal / Anthrax toxin lethal factor, N- and C-terminal domain ...Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / Anthrax toxin, lethal/endema factor / : / Anthrax toxin lethal factor (ATLF)-like domain profile. / Anthrax toxin, lethal/endema factor, N-/C-terminal / Anthrax toxin lethal factor, N- and C-terminal 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 / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Protective antigen / Lethal factor
Similarity search - Component
Biological speciesBacillus anthracis (anthrax bacterium)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMachen AJ / Freudenthal BD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R03-AI142361 United States
CitationJournal: Sci Rep / Year: 2021
Title: Anthrax toxin translocation complex reveals insight into the lethal factor unfolding and refolding mechanism.
Authors: Alexandra J Machen / Mark T Fisher / Bret D Freudenthal /
Abstract: Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema ...Translocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the binding component of this AB toxin, forms an oligomeric pore that translocates lethal factor (LF) or edema factor, the active components of the toxin, into the cell. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PA translocating the N-terminal domain of LF (LF). The resulting 3.3 Å structure of the complex shows density of partially unfolded LF near the canonical PA binding site. Interestingly, we also observe density consistent with an α helix emerging from the 100 Å β barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin β barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins.
History
DepositionDec 4, 2020-
Header (metadata) releaseJul 14, 2021-
Map releaseJul 14, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kxr
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23066.png

Downloads & links

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Map

FileDownload / File: emd_23066.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAnthrax Toxin Translocation Complex
Voxel sizeX=Y=Z: 1.605 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-25.293061999999999 - 40.06006
Average (Standard dev.)0.00060687563 (±0.85370934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 321.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.6051.6051.605
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z321.000321.000321.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-25.29340.0600.001

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Supplemental data

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Sample components

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Entire : Anthrax toxin protective antigen translocating lethal factor N-te...

EntireName: Anthrax toxin protective antigen translocating lethal factor N-terminal domain
Components
  • Complex: Anthrax toxin protective antigen translocating lethal factor N-terminal domain
    • Protein or peptide: Lethal factor
    • Protein or peptide: Protective antigen
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Anthrax toxin protective antigen translocating lethal factor N-te...

SupramoleculeName: Anthrax toxin protective antigen translocating lethal factor N-terminal domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)

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Macromolecule #1: Lethal factor

MacromoleculeName: Lethal factor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: anthrax lethal factor endopeptidase
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Molecular weightTheoretical: 30.520408 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AGGHGDVGMH VKEKEKNKDE NKRKDEERNK TQEEHLKEIM KHIVKIEVKG EEAVKKEAAE KLLEKVPSDV LEMYKAIGGK IYIVDGDIT KHISLEALSE DKKKIKDIYG KDALLHEHYV YAKEGYCPVL VIQSSEDYVE NTEKALNVYY EIGKILSRDI L SKINQPYQ ...String:
AGGHGDVGMH VKEKEKNKDE NKRKDEERNK TQEEHLKEIM KHIVKIEVKG EEAVKKEAAE KLLEKVPSDV LEMYKAIGGK IYIVDGDIT KHISLEALSE DKKKIKDIYG KDALLHEHYV YAKEGYCPVL VIQSSEDYVE NTEKALNVYY EIGKILSRDI L SKINQPYQ KFLDVLNTIK NASDSDGQDL LFTNQLKEHP TDFSVEFLEQ NSNEVQEVFA KAFAYYIEPQ HRDVLQLYAP EA FNYMDKF NEQEINLSLE ELKDQR

UniProtKB: Lethal factor

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Macromolecule #2: Protective antigen

MacromoleculeName: Protective antigen / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Molecular weightTheoretical: 63.019012 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TVPDRDNDGI PDSLEVEGYT VDVKNKRTFL SPWISNIHEK KGLTKYKSSP EKWSTASDPY SDFEKVTGRI DKNVSPEARH PLVAAYPIV HVDMENIILS KNEDQSTQNT DSQTRTISKN TSTSRTHTSE VHGNAEVHAS FFDIGGSVSA GFSNSNSSTV A IDHSLSLA ...String:
TVPDRDNDGI PDSLEVEGYT VDVKNKRTFL SPWISNIHEK KGLTKYKSSP EKWSTASDPY SDFEKVTGRI DKNVSPEARH PLVAAYPIV HVDMENIILS KNEDQSTQNT DSQTRTISKN TSTSRTHTSE VHGNAEVHAS FFDIGGSVSA GFSNSNSSTV A IDHSLSLA GERTWAETMG LNTADTARLN ANIRYVNTGT APIYNVLPTT SLVLGKNQTL ATIKAKENQL SQILAPNNYY PS KNLAPIA LNAQDDFSST PITMNYNQFL ELEKTKQLRL DTDQVYGNIA TYNFENGRVR VDTGSNWSEV LPQIQETTAR IIF NGKDLN LVERRIAAVN PSDPLETTKP DMTLKEALKI AFGFNEPNGN LQYQGKDITE FDFNFDQQTS QNIKNQLAEL NATN IYTVL DKIKLNAKMN ILIRDKRFHY DRNNIAVGAD ESVVKEAHRE VINSSTEGLL LNIDKDIRKI LSGYIVEIED TEGLK EVIN DRYDMLNISS LRQDGKTFID FKKYNDKLPL YISNPNYKVN VYAVTKENTI INPSENGDTS TNGIKKILIF SKKGYE IG

UniProtKB: Protective antigen

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 14 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 5.5
VitrificationCryogen name: ETHANE / 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.15) / Details: ab initio
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.0.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 122651

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