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- PDB-7t4d: Pore structure of pore-forming toxin Epx4 -

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Basic information

Entry
Database: PDB / ID: 7t4d
TitlePore structure of pore-forming toxin Epx4
ComponentsEpx4
KeywordsTOXIN / pore-forming toxin
Function / homologyLeukocidin/porin MspA / Leukocidin-like / Distorted Sandwich / Mainly Beta
Function and homology information
Biological speciesEnterococcus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsXiong, X.Z. / Dong, M. / Yang, P. / Abraham, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR) United States
CitationJournal: Cell / Year: 2022
Title: Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.
Authors: Xiaozhe Xiong / Songhai Tian / Pan Yang / Francois Lebreton / Huan Bao / Kuanwei Sheng / Linxiang Yin / Pengsheng Chen / Jie Zhang / Wanshu Qi / Jianbin Ruan / Hao Wu / Hong Chen / David T ...Authors: Xiaozhe Xiong / Songhai Tian / Pan Yang / Francois Lebreton / Huan Bao / Kuanwei Sheng / Linxiang Yin / Pengsheng Chen / Jie Zhang / Wanshu Qi / Jianbin Ruan / Hao Wu / Hong Chen / David T Breault / Hao Wu / Ashlee M Earl / Michael S Gilmore / Jonathan Abraham / Min Dong /
Abstract: Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, ...Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of β-barrel pore-forming toxins with a β-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
History
DepositionDec 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 23, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 13, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Oct 18, 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
B: Epx4
A: Epx4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,3714
Polymers71,1352
Non-polymers2362
Water00
1
B: Epx4
A: Epx4
hetero molecules

B: Epx4
A: Epx4
hetero molecules

B: Epx4
A: Epx4
hetero molecules

B: Epx4
A: Epx4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)285,48416
Polymers284,5398
Non-polymers9458
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_465-x-1,-y+1,z1
crystal symmetry operation3_565-y,x+1,z1
crystal symmetry operation4_455y-1,-x,z1
Buried area46390 Å2
ΔGint-142 kcal/mol
Surface area105180 Å2
MethodPISA
Unit cell
Length a, b, c (Å)122.301, 122.301, 128.419
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number79
Space group name H-MI4
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A and segid AA
21chain B and segid BA

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain A and segid AAA0
211chain B and segid BAB0

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Components

#1: Protein Epx4


Mass: 35567.336 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus (bacteria)
Production host: Escherichia coli-Pichia pastoris shuttle vector pPpARG4 (others)
#2: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: precipitant*YM
Has ligand of interestY

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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: 3.38 Å3/Da / Density % sol: 63.56 %
Crystal growTemperature: 277.15 K / Method: evaporation
Details: 5% (w/v) polyethylene glycol 8000, 40% (v/v) MPD, 0.1 M Sodium Cacodylate, pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 12, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 3→64.21 Å / Num. obs: 18919 / % possible obs: 99.97 % / Redundancy: 6.9 % / Rmerge(I) obs: 0.2037 / Net I/σ(I): 8.94
Reflection shellResolution: 3→3.1074 Å / Rmerge(I) obs: 0.9269 / Num. unique obs: 1855

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2ygt

2ygt


Resolution: 3→64.21 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 23.44 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2439 992 5.24 %
Rwork0.2019 17927 -
obs0.204 18919 99.87 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 105.54 Å2 / Biso mean: 49.9564 Å2 / Biso min: 22.8 Å2
Refinement stepCycle: final / Resolution: 3→64.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4818 0 44 0 4862
Biso mean--36.68 --
Num. residues----602
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0024940
X-RAY DIFFRACTIONf_angle_d0.6636716
X-RAY DIFFRACTIONf_chiral_restr0.025744
X-RAY DIFFRACTIONf_plane_restr0.003862
X-RAY DIFFRACTIONf_dihedral_angle_d14.5131804
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2748X-RAY DIFFRACTION6.22TORSIONAL
12B2748X-RAY DIFFRACTION6.22TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
3.0003-3.15840.34012210.27692449
3.1584-3.35630.27061310.24042568
3.3563-3.61540.26451430.22062548
3.6154-3.97920.2541150.20322587
3.9792-4.55490.2179950.17542607
4.5549-5.73810.22691430.18552558
5.7381-64.210.20061440.19332610

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