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Yorodumi- PDB-6zxk: Fully-loaded anthrax lethal toxin in its heptameric pre-pore stat... -
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-Basic information
Entry | Database: PDB / ID: 6zxk | ||||||
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Title | Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1B) arrangement | ||||||
Components |
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Keywords | TOXIN / 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 / 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 / identical protein binding / membrane / metal ion binding Similarity search - Function | ||||||
Biological species | Bacillus anthracis (anthrax bacterium) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
Authors | Quentin, D. / Antoni, C. / Gatsogiannis, C. / Raunser, S. | ||||||
Funding support | Germany, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6zxk.cif.gz | 967.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6zxk.ent.gz | 774.7 KB | Display | PDB format |
PDBx/mmJSON format | 6zxk.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6zxk_validation.pdf.gz | 915.6 KB | Display | wwPDB validaton report |
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Full document | 6zxk_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 6zxk_validation.xml.gz | 156.8 KB | Display | |
Data in CIF | 6zxk_validation.cif.gz | 238.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/zx/6zxk ftp://data.pdbj.org/pub/pdb/validation_reports/zx/6zxk | HTTPS FTP |
-Related structure data
Related structure data | 11523MC 6zxjC 6zxlC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 | Mass: 93904.211 Da / Num. of mol.: 3 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 BL21(DE3) (bacteria) References: UniProt: P15917, anthrax lethal factor endopeptidase |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1B) arrangement Type: COMPLEX Details: Heptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The LFs form a continuous chain of head-to-tail ...Details: Heptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1B) arrangement, the third LF binds with its C-terminal region to the N-terminal domain of the 1st LF. Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 0.72 MDa / Experimental value: NO | |||||||||||||||
Source (natural) | Organism: Bacillus anthracis (anthrax bacterium) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) | |||||||||||||||
Buffer solution | pH: 8.5 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.06 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||
Vitrification | Instrument: 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 382000 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99000 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Details: The previously generated PA7LF3-masked model (PDB:6ZXJ) served as starting point and was placed into the density using rigid-body fit in Chimera. From this model, chain H was duplicated and ...Details: The previously generated PA7LF3-masked model (PDB:6ZXJ) served as starting point and was placed into the density using rigid-body fit in Chimera. From this model, chain H was duplicated and fitted into the density corresponding to the third LF. For the entire model a restrained refinement in phenix was performed. The resulting model was further refined with a combination of phenix and coot. Unresolved regions were deleted and side chain information was removed for less well-resolved regions. | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6ZXJ Accession code: 6ZXJ / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||
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