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- PDB-6zxj: Fully-loaded anthrax lethal toxin in its heptameric pre-pore stat... -

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Basic information

Entry
Database: PDB / ID: 6zxj
TitleFully-loaded anthrax lethal toxin in its heptameric pre-pore state, in which the third lethal factor is masked out (PA7LF3-masked)
Components
  • Lethal factor
  • Protective antigen
KeywordsTOXIN / anthrax lethal toxin / fully-loaded pre-pore state / membrane translocase / cytotoxic substrate
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / positive regulation of apoptotic process in another organism / 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 / positive regulation of apoptotic process in another organism / 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
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. ...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.
Similarity search - Domain/homology
Protective antigen / Lethal factor / Protective antigen
Similarity search - Component
Biological speciesBacillus anthracis (anthrax bacterium)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsQuentin, D. / Antoni, C. / Gatsogiannis, C. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
European Research Council (ERC)615984 Germany
CitationJournal: PLoS Pathog / Year: 2020
Title: Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state.
Authors: Claudia Antoni / Dennis Quentin / Alexander E Lang / Klaus Aktories / Christos Gatsogiannis / Stefan Raunser /
Abstract: Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA) ...Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.
History
DepositionJul 29, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • EMDB-11522
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Protective antigen
B: Protective antigen
C: Protective antigen
D: Protective antigen
E: Protective antigen
F: Protective antigen
G: Protective antigen
H: Lethal factor
I: Lethal factor


Theoretical massNumber of molelcules
Total (without water)787,5689
Polymers787,5689
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23360 Å2
ΔGint-68 kcal/mol
Surface area241700 Å2
MethodPISA

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Components

#1: Protein
Protective antigen


Mass: 85679.930 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus anthracis (anthrax bacterium) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q68GS1, UniProt: P13423*PLUS
#2: Protein Lethal factor / LF / Anthrax lethal toxin endopeptidase component


Mass: 93904.211 Da / Num. of mol.: 2
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: Bacillus anthracis (anthrax bacterium)
References: UniProt: P15917, anthrax lethal factor endopeptidase

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Fully-loaded anthrax lethal toxin in its heptameric pre-pore stateCOMPLEXHeptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The flexible third LF is masked out in the final reconstruction to boost the resolution of the rest of the complex.all0MULTIPLE SOURCES
2Protective antigenCOMPLEXThe trypsin-activated 63 kDa fragments assemble into a hepatameric pre-pore#11RECOMBINANT
3Lethal factorCOMPLEXThree LF molecules crown the heptameric PA ring. The density corresponding to the third LF is masked out in the reconstruction.#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.72 MDaNO
210.063 MDaNO
310.093 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Bacillus anthracis (anthrax bacterium)1392
33Bacillus anthracis (anthrax bacterium)1392
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33Bacillus anthracis (anthrax bacterium)1392
Buffer solutionpH: 8.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 0.06 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 286 K
Details: 4 uL sample was applied to grid (with 2 nm additional carbon layer) and incubated for 45 s prior blotting.

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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 / Nominal magnification: 130000 X / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 74.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5238
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPU1.1image acquisition
4SPHIRECTF correction
7iMODFITmodel fitting
10SPHIREfinal Euler assignment
11SPHIREclassification
12SPHIRE3D reconstruction
13PHENIX1.18.2model refinement
14Coot0.8.9.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 382000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 210000 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: The protective antigen (PA) monomer from 3HVD was placed seven times into the density corresponding to the heptameric PA ring using the rigid-body fit in Chimera. Two copies of the monomeric ...Details: The protective antigen (PA) monomer from 3HVD was placed seven times into the density corresponding to the heptameric PA ring using the rigid-body fit in Chimera. Two copies of the monomeric lethal factor (LF) from 1J7N were fitted similarly into the corresponding LF density located atop of the PA7 ring. The resulting model was then flexibly fitted using iMODFIT. The model was further refined in iterative rounds of phenix and coot. Unresolved regions were deleted and side chain information was removed for less well-defined regions.
Atomic model building

3D fitting-ID: 1 / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-ID
13HVD

3hvd

3HVD1
21J7N

1j7n

1J7N2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01638867
ELECTRON MICROSCOPYf_angle_d1.28752895
ELECTRON MICROSCOPYf_dihedral_angle_d5.4755428
ELECTRON MICROSCOPYf_chiral_restr0.076183
ELECTRON MICROSCOPYf_plane_restr0.0086947

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