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- PDB-7kxr: Protective antigen pore translocating lethal factor N-terminal domain -

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Basic information

Entry
Database: PDB / ID: 7kxr
TitleProtective antigen pore translocating lethal factor N-terminal domain
Components
  • Lethal factor
  • Protective antigen
KeywordsTOXIN / translocation / complex / anthrax / refolding
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / : / host cell cytosol / Uptake and function of anthrax toxins / negative regulation of MAPK cascade / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity ...anthrax lethal factor endopeptidase / : / host cell cytosol / Uptake and function of anthrax toxins / negative regulation of MAPK cascade / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / identical protein binding / membrane / metal ion binding
Similarity search - Function
Immunoglobulin-like - #810 / 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/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain ...Immunoglobulin-like - #810 / 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/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain / Anthrax toxin lethal factor (ATLF)-like domain profile. / 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. / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Protective antigen / Lethal factor
Similarity search - Component
Biological speciesBacillus anthracis (anthrax bacterium)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMachen, A.J. / Freudenthal, B.D.
Funding support United States, 1items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
L: Lethal factor
A: Protective antigen
B: Protective antigen
C: Protective antigen
D: Protective antigen
E: Protective antigen
F: Protective antigen
G: Protective antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)472,21522
Polymers471,6538
Non-polymers56114
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, surface plasmon resonance, biolayer interferometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58020 Å2
ΔGint-224 kcal/mol
Surface area201610 Å2

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Components

#1: Protein Lethal factor / LF / Anthrax lethal toxin endopeptidase component


Mass: 30520.408 Da / Num. of mol.: 1 / Fragment: UNP Residues 34-296 / Mutation: E126C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus anthracis (anthrax bacterium) / Gene: lef, pXO1-107, BXA0172, GBAA_pXO1_0172 / Production host: Escherichia coli (E. coli)
References: UniProt: P15917, anthrax lethal factor endopeptidase
#2: Protein
Protective antigen / PA / Anthrax toxins translocating protein / PA-83 / PA83


Mass: 63019.012 Da / Num. of mol.: 7 / Fragment: UNP Residues 203-764
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus anthracis (anthrax bacterium) / Gene: pagA, pag, pXO1-110, BXA0164, GBAA_pXO1_0164 / Production host: Escherichia coli (E. coli) / References: UniProt: P13423
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Anthrax toxin protective antigen translocating lethal factor N-terminal domain
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
10cryoSPARC2.15initial Euler assignment
11cryoSPARC3.0.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122651 / Symmetry type: POINT

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