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- PDB-3j9c: CryoEM single particle reconstruction of anthrax toxin protective... -

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Entry
Database: PDB / ID: 3j9c
TitleCryoEM single particle reconstruction of anthrax toxin protective antigen pore at 2.9 Angstrom resolution
ComponentsProtective antigen PA-63
KeywordsTOXIN / TRANSPORT PROTEIN / bacterial 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 / identical protein binding / metal ion binding
Similarity search - Function
Ubiquitin-like (UB roll) - #110 / 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 ...Ubiquitin-like (UB roll) - #110 / 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 / Ubiquitin-like (UB roll) / Roll / Alpha Beta
Similarity search - Domain/homology
Biological speciesBacillus anthracis (anthrax bacterium)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsJiang, J. / Pentelute, B.L. / Collier, R.J. / Zhou, Z.H.
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, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2015Provider: repository / Type: Initial release
Revision 1.1Jun 3, 2015Group: Database references
Revision 1.2Jun 10, 2015Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / pdbx_struct_oper_list / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Assembly

Deposited unit
A: Protective antigen PA-63
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,0993
Polymers63,0191
Non-polymers802
Water0
1
A: Protective antigen PA-63
hetero molecules
x 7


Theoretical massNumber of molelcules
Total (without water)441,69421
Polymers441,1337
Non-polymers56114
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation6
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C7 (7 fold cyclic))

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Components

#1: Protein Protective antigen PA-63 / Anthrax toxin protective antigen


Mass: 63019.012 Da / Num. of mol.: 1 / Fragment: C-terminal 63-kDa 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 / Plasmid: pET22b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P13423
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

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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
IDNameTypeDetailsParent-ID
1Heptamer of C-terminal 63-kDa fragment of anthrax toxin protective antigen, pore conformationCOMPLEXHeptamer0
2Anthrax toxin protective antigen1
Molecular weightValue: 0.44 MDa / Experimental value: NO
Buffer solutionName: 50 mM NaOAc, pH 5.0, 0.05% Igepal CA-630 / pH: 5 / Details: 50 mM NaOAc, pH 5.0, 0.05% Igepal CA-630
SpecimenConc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil R1.2/1.3 grid with thin carbon support, glow discharged
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Details: Plunged into liguid nitrogen (FEI VITROBOT MARK IV).

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Jan 8, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 39062 X / Nominal defocus max: 5100 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen holder type: Liquid nitrogen cooled
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) / Details: Counting mode of the detector was used.
Image scansNum. digital images: 7062
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM softwareName: RELION / Category: 3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionMethod: Maximum a posteriori optimization / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60455 / Nominal pixel size: 1.28 Å / Actual pixel size: 1.28 Å / Details: (Single particle--Applied symmetry: C7) / Symmetry type: POINT
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms3326 0 2 0 3328

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