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- PDB-8cm0: Rhs2-CT endonuclease toxin in complex with cognate immunity prote... -

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Basic information

Entry
Database: PDB / ID: 8cm0
TitleRhs2-CT endonuclease toxin in complex with cognate immunity protein RhsI2
Components
  • Rhs2CT
  • RhsI2
KeywordsANTIMICROBIAL PROTEIN / T6SS / effector-immunity compex / HNH endonuclease / DNAse / S. marcescens DB10
Function / homologyACETATE ION
Function and homology information
Biological speciesSerratia marcescens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.17 Å
AuthorsPankov, G. / Hunter, W.N. / Coulthurst, S.J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust215599/Z/19/Z United Kingdom
Wellcome Trust220321/Z/20/Z United Kingdom
CitationJournal: To Be Published
Title: Rhs2-CT endonuclease toxin in complex with cognate immunity protein RhsI2
Authors: Pankov, G. / Coulthurst, C. / Hunter, W.N.
History
DepositionFeb 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
BBB: RhsI2
AAA: Rhs2CT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,7196
Polymers36,4792
Non-polymers2394
Water2,360131
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Rhs2CT-RhsI2 copurify together in SEC chromatography with the elutionvolume consistent with the mass of the heterodimer, RhsI2 protects susceptible cells from Rhs2CT ...Evidence: gel filtration, Rhs2CT-RhsI2 copurify together in SEC chromatography with the elutionvolume consistent with the mass of the heterodimer, RhsI2 protects susceptible cells from Rhs2CT intoxication in competition assays
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2210 Å2
ΔGint-4 kcal/mol
Surface area14500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)37.006, 81.931, 101.371
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein RhsI2


Mass: 18682.303 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Serratia marcescens (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein Rhs2CT


Mass: 17797.018 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Serratia marcescens (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H3O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 131 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.13 Å3/Da / Density % sol: 42.41 %
Crystal growTemperature: 296 K / Method: vapor diffusion, sitting drop
Details: Crystals were grown in a condition containing 0.2 uL of 6.5 mg/mL Rhs2CT-RhsI2 (in 20 mM MES & 20 mM imidazole pH 6.0, 150 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP) and 0.2 uL of reservoir solution ...Details: Crystals were grown in a condition containing 0.2 uL of 6.5 mg/mL Rhs2CT-RhsI2 (in 20 mM MES & 20 mM imidazole pH 6.0, 150 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP) and 0.2 uL of reservoir solution (0.2 M ammonium acetate, 0.1 M sodium acetate pH 5.0, 20% PEG4K).

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.8731 Å
DetectorType: DECTRIS PILATUS3 X 2M / Detector: PIXEL / Date: Jun 9, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8731 Å / Relative weight: 1
ReflectionResolution: 2.17→37.98 Å / Num. obs: 17007 / % possible obs: 100 % / Redundancy: 6 % / CC1/2: 0.993 / Rmerge(I) obs: 0.157 / Rpim(I) all: 0.07 / Net I/σ(I): 8.7
Reflection shellResolution: 2.17→2.24 Å / Rmerge(I) obs: 1.511 / Num. unique obs: 1408 / CC1/2: 0.542 / Rpim(I) all: 0.661 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
XDSdata reduction
xia2data scaling
PHASESphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.17→37.98 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.916 / SU B: 6.765 / SU ML: 0.171 / Cross valid method: FREE R-VALUE / ESU R: 0.253 / ESU R Free: 0.212
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2526 842 4.968 %
Rwork0.195 16107 -
all0.198 --
obs-16949 99.906 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 34.743 Å2
Baniso -1Baniso -2Baniso -3
1-0.916 Å2-0 Å2-0 Å2
2---1.073 Å2-0 Å2
3---0.157 Å2
Refinement stepCycle: LAST / Resolution: 2.17→37.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2185 0 16 131 2332
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0132251
X-RAY DIFFRACTIONr_bond_other_d0.0010.0152129
X-RAY DIFFRACTIONr_angle_refined_deg1.4771.6453044
X-RAY DIFFRACTIONr_angle_other_deg1.3091.5834886
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9345270
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.42721.545123
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.86815378
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.4421517
X-RAY DIFFRACTIONr_chiral_restr0.0750.2285
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.022543
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02547
X-RAY DIFFRACTIONr_nbd_refined0.2060.2440
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1960.21844
X-RAY DIFFRACTIONr_nbtor_refined0.1680.21071
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.080.21054
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1460.2105
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1390.215
X-RAY DIFFRACTIONr_nbd_other0.2170.239
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1380.25
X-RAY DIFFRACTIONr_mcbond_it2.7923.3451092
X-RAY DIFFRACTIONr_mcbond_other2.793.3391088
X-RAY DIFFRACTIONr_mcangle_it4.3494.9891356
X-RAY DIFFRACTIONr_mcangle_other4.3474.9931357
X-RAY DIFFRACTIONr_scbond_it3.6763.8681159
X-RAY DIFFRACTIONr_scbond_other3.6753.8691160
X-RAY DIFFRACTIONr_scangle_it6.0255.5791688
X-RAY DIFFRACTIONr_scangle_other6.0245.581689
X-RAY DIFFRACTIONr_lrange_it8.12638.562520
X-RAY DIFFRACTIONr_lrange_other8.12538.5682521
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.17-2.2260.329610.271141X-RAY DIFFRACTION100
2.226-2.2870.352660.2581136X-RAY DIFFRACTION99.6683
2.287-2.3530.278380.2331123X-RAY DIFFRACTION99.6567
2.353-2.4260.303590.2081074X-RAY DIFFRACTION100
2.426-2.5050.277610.1981051X-RAY DIFFRACTION99.9101
2.505-2.5930.287450.1981001X-RAY DIFFRACTION100
2.593-2.6910.287470.208983X-RAY DIFFRACTION100
2.691-2.80.32520.203938X-RAY DIFFRACTION99.8991
2.8-2.9250.299460.202930X-RAY DIFFRACTION100
2.925-3.0670.315430.195884X-RAY DIFFRACTION100
3.067-3.2330.238470.199812X-RAY DIFFRACTION100
3.233-3.4280.246410.194792X-RAY DIFFRACTION99.8801
3.428-3.6640.198410.17749X-RAY DIFFRACTION99.8736
3.664-3.9570.245440.174685X-RAY DIFFRACTION100
3.957-4.3320.235260.168650X-RAY DIFFRACTION100
4.332-4.8410.242300.181602X-RAY DIFFRACTION100
4.841-5.5850.181260.181526X-RAY DIFFRACTION100
5.585-6.8270.282290.2450X-RAY DIFFRACTION100
6.827-9.5990.167240.157359X-RAY DIFFRACTION100
9.599-37.980.187160.231221X-RAY DIFFRACTION98.75

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