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- PDB-6uji: Low resolution crystal structure (5.5 A) of the anthrax toxin pro... -

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Basic information

Entry
Database: PDB / ID: 6uji
TitleLow resolution crystal structure (5.5 A) of the anthrax toxin protective antigen heptamer prepore D425A mutant
ComponentsProtective antigen PA-63
KeywordsTOXIN / Anthrax Toxin / PA63 heptamer
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
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 ...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
Similarity search - Domain/homology
Biological speciesBacillus anthracis (anthrax bacterium)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 5.5 Å
AuthorsLovell, S. / Mehzabeen, N. / Battaile, K.P. / Bann, J.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30 GM110761 United States
CitationJournal: J Mol Biol / Year: 2022
Title: Structure of the Anthrax Protective Antigen D425A Dominant Negative Mutant Reveals a Stalled Intermediate State of Pore Maturation.
Authors: Harry Scott / Wei Huang / Kiran Andra / Sireesha Mamillapalli / Srinivas Gonti / Alexander Day / Kaiming Zhang / Nurjahan Mehzabeen / Kevin P Battaile / Anjali Raju / Scott Lovell / James G ...Authors: Harry Scott / Wei Huang / Kiran Andra / Sireesha Mamillapalli / Srinivas Gonti / Alexander Day / Kaiming Zhang / Nurjahan Mehzabeen / Kevin P Battaile / Anjali Raju / Scott Lovell / James G Bann / Derek J Taylor /
Abstract: The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of β-barrel pore-forming toxins. The protective antigen (PA) component forms an ...The tripartite protein complex produced by anthrax bacteria (Bacillus anthracis) is a member of the AB family of β-barrel pore-forming toxins. The protective antigen (PA) component forms an oligomeric prepore that assembles on the host cell surface and serves as a scaffold for binding of lethal and edema factors. Following endocytosis, the acidic environment of the late endosome triggers a pH-induced conformational rearrangement to promote maturation of the PA prepore to a functional, membrane spanning pore that facilitates delivery of lethal and edema factors to the cytosol of the infected host. Here, we show that the dominant-negative D425A mutant of PA stalls anthrax pore maturation in an intermediate state at acidic pH. Our 2.7 Å cryo-EM structure of the intermediate state reveals structural rearrangements that involve constriction of the oligomeric pore combined with an intramolecular dissociation of the pore-forming module. In addition to defining the early stages of anthrax pore maturation, the structure identifies asymmetric conformational changes in the oligomeric pore that are influenced by the precise configuration of adjacent protomers.
History
DepositionOct 3, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 30, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 11, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protective antigen PA-63
B: Protective antigen PA-63
C: Protective antigen PA-63
D: Protective antigen PA-63
E: Protective antigen PA-63
F: Protective antigen PA-63
G: Protective antigen PA-63
H: Protective antigen PA-63
I: Protective antigen PA-63
J: Protective antigen PA-63
K: Protective antigen PA-63
L: Protective antigen PA-63
M: Protective antigen PA-63
N: Protective antigen PA-63


Theoretical massNumber of molelcules
Total (without water)888,65714
Polymers888,65714
Non-polymers00
Water0
1
A: Protective antigen PA-63
B: Protective antigen PA-63
C: Protective antigen PA-63
D: Protective antigen PA-63
E: Protective antigen PA-63
F: Protective antigen PA-63
G: Protective antigen PA-63


Theoretical massNumber of molelcules
Total (without water)444,3297
Polymers444,3297
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24500 Å2
ΔGint-64 kcal/mol
Surface area145080 Å2
MethodPISA
2
H: Protective antigen PA-63
I: Protective antigen PA-63
J: Protective antigen PA-63
K: Protective antigen PA-63
L: Protective antigen PA-63
M: Protective antigen PA-63
N: Protective antigen PA-63


Theoretical massNumber of molelcules
Total (without water)444,3297
Polymers444,3297
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area24420 Å2
ΔGint-65 kcal/mol
Surface area149930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)166.180, 144.247, 304.825
Angle α, β, γ (deg.)90.000, 102.410, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Protective antigen PA-63 / PA / Anthrax toxins translocating protein / PA-63 / PA63


Mass: 63475.500 Da / Num. of mol.: 14 / Mutation: D425A
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: pQE80 / Production host: Escherichia coli (E. coli) / Strain (production host): UTH780 / References: UniProt: P13423

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.02 Å3/Da / Density % sol: 69.37 % / Mosaicity: 0.26 °
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 10% (v/v) ethanol, 0.1 M Tris

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 17-ID / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 1, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 5.5→49.78 Å / Num. obs: 45836 / % possible obs: 99.2 % / Redundancy: 3.4 % / Biso Wilson estimate: 257.2 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.091 / Net I/σ(I): 9 / Num. measured all: 155920 / Scaling rejects: 4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Net I/σ(I) obs% possible all
5.5-5.693.60.6121596544870.7722.299.8
21.3-49.782.80.03317866410.99726.975.1

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation9.25 Å47.61 Å
Translation9.25 Å47.61 Å

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
Aimless0.7.4data scaling
PHASER2.5.6phasing
BUSTERrefinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1TZO

1tzo


Resolution: 5.5→44.83 Å / Cor.coef. Fo:Fc: 0.898 / Cor.coef. Fo:Fc free: 0.873 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 1.587
Details: Rigid body refinement with B-factors set to the Wilson B-factor
RfactorNum. reflection% reflectionSelection details
Rfree0.278 2250 4.91 %RANDOM
Rwork0.251 ---
obs0.253 45809 99.3 %-
Displacement parametersBiso max: 257 Å2 / Biso mean: 257 Å2 / Biso min: 257 Å2
Baniso -1Baniso -2Baniso -3
1--33.6954 Å20 Å2-23.2205 Å2
2--61.7408 Å20 Å2
3----28.0454 Å2
Refine analyzeLuzzati coordinate error obs: 0.9 Å
Refinement stepCycle: final / Resolution: 5.5→44.83 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms53490 0 0 0 53490
Num. residues----6757
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d19287SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes9288HARMONIC5
X-RAY DIFFRACTIONt_it54349HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion7585SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact60588SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d54349HARMONIC20.007
X-RAY DIFFRACTIONt_angle_deg73658HARMONIC20.98
X-RAY DIFFRACTIONt_omega_torsion1.7
X-RAY DIFFRACTIONt_other_torsion22.8
LS refinement shellResolution: 5.5→5.54 Å / Rfactor Rfree error: 0 / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.2812 45 4.91 %
Rwork0.2929 872 -
all0.2923 917 -
obs--99.67 %

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