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- PDB-8f43: HNH Nuclease Domain from G. stearothermophilus Cas9, K597A mutant -

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Basic information

Entry
Database: PDB / ID: 8f43
TitleHNH Nuclease Domain from G. stearothermophilus Cas9, K597A mutant
ComponentsCRISPR-associated endonuclease Cas9
KeywordsHYDROLASE / nuclease domain / CRISPR Cas9 / DNA binding protein
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Cas9, alpha-helical lobe domain / Cas9 alpha-helical lobe domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas9
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.37 Å
AuthorsD'Ordine, A.M. / Belato, H.B. / Lisi, G.P. / Jogl, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136815 United States
Citation
Journal: J.Chem.Phys. / Year: 2022
Title: Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.
Authors: Belato, H.B. / Norbrun, C. / Luo, J. / Pindi, C. / Sinha, S. / D'Ordine, A.M. / Jogl, G. / Palermo, G. / Lisi, G.P.
History
DepositionNov 10, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9


Theoretical massNumber of molelcules
Total (without water)13,0211
Polymers13,0211
Non-polymers00
Water2,036113
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)45.415, 85.751, 26.921
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Space group name HallP22ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x+1/2,y+1/2,-z
#4: -x,-y,z
Components on special symmetry positions
IDModelComponents
11A-249-

HOH

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 13020.691 Da / Num. of mol.: 1 / Fragment: Nuclease Domain / Mutation: K597A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Gene: cas9, GS458_0313 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A250DVH8, Hydrolases; Acting on ester bonds
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 113 / Source method: isolated from a natural source / Formula: H2O

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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: 2.01 Å3/Da / Density % sol: 38.9 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 0.2M calcium chloride hexahydrate, 0.1M HEPES pH 7.0, 20% polyethylene glycol 6000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-1 / Wavelength: 0.920105 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jul 18, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.920105 Å / Relative weight: 1
ReflectionResolution: 1.37→28.58 Å / Num. obs: 22933 / % possible obs: 100 % / Redundancy: 13.1 % / Biso Wilson estimate: 11.83 Å2 / CC1/2: 0.988 / Net I/σ(I): 7.3
Reflection shellResolution: 1.37→1.39 Å / Num. unique obs: 1146 / CC1/2: 0.878

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7MPZ

7mpz


Resolution: 1.37→26.92 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.1841 --
Rwork0.1678 --
obs-22847 99.83 %
Refinement stepCycle: LAST / Resolution: 1.37→26.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms910 0 0 113 1023
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00541008
X-RAY DIFFRACTIONf_angle_d0.91331371
X-RAY DIFFRACTIONf_chiral_restr0.0694141
X-RAY DIFFRACTIONf_plane_restr0.0086184
X-RAY DIFFRACTIONf_dihedral_angle_d4.8781143
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.37-1.430.22391500.1982648X-RAY DIFFRACTION99.68
1.43-1.510.22221400.18392666X-RAY DIFFRACTION99.72
1.51-1.60.19191350.16752667X-RAY DIFFRACTION99.72
1.6-1.730.20541460.172661X-RAY DIFFRACTION99.86
1.73-1.90.17281310.16762713X-RAY DIFFRACTION99.79
1.9-2.170.19051510.15682685X-RAY DIFFRACTION99.96
2.17-2.740.16761500.16852758X-RAY DIFFRACTION99.97
2.74-26.920.17721430.16562903X-RAY DIFFRACTION99.9
Refinement TLS params.Method: refined / Origin x: 14.6869403231 Å / Origin y: 13.3420374967 Å / Origin z: 4.73755081448 Å
111213212223313233
T0.0785011288471 Å20.002611631407 Å20.000133592611717 Å2-0.0840851441289 Å2-0.00152536311941 Å2--0.0819499762823 Å2
L0.294407186802 °2-0.150860704611 °2-0.113167273069 °2-0.630249503474 °2-0.125466475257 °2--0.481001649538 °2
S-0.00664975076531 Å °-0.00421533638081 Å °-0.0384650140151 Å °-0.00500768722057 Å °-0.0161270170459 Å °-0.00148859037773 Å °0.00907258471569 Å °-0.0260110201911 Å °-0.00266842251713 Å °
Refinement TLS groupSelection details: all

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