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- PDB-7mpz: HNH Nuclease Domain from G. stearothermophilus Cas9 -

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Basic information

Entry
Database: PDB / ID: 7mpz
TitleHNH Nuclease Domain from G. stearothermophilus Cas9
ComponentsCRISPR-associated endonuclease Cas9
KeywordsDNA BINDING PROTEIN / nuclease domain / CRISPR Cas9
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 / MOLECULAR REPLACEMENT / Resolution: 2.04 Å
AuthorsD'Ordine, A.M. / Belato, H.B. / Lisi, G.P. / Jogl, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136815 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM109035 United States
CitationJournal: J.Struct.Biol. / Year: 2021
Title: Structural and dynamic insights into the HNH nuclease of divergent Cas9 species.
Authors: Belato, H.B. / D'Ordine, A.M. / Nierzwicki, L. / Arantes, P.R. / Jogl, G. / Palermo, G. / Lisi, G.P.
History
DepositionMay 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 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
A: CRISPR-associated endonuclease Cas9


Theoretical massNumber of molelcules
Total (without water)18,5081
Polymers18,5081
Non-polymers00
Water1,20767
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)57.956, 57.956, 119.393
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Space group name HallP4abw2nw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 18507.867 Da / Num. of mol.: 1
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.: 67 / 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: 2.71 Å3/Da / Density % sol: 54.59 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 0.12 M [0.3 M diethylene glycol, 0.3 M triethylene glycol, 0.3 M tetraethylene glycol, 0.3 M pentaethylene glycol], 0.1 M [MES and imidazole] pH 6.5, 30% (v/v) [40% (v/v) glycerol, 20% (w/v) ...Details: 0.12 M [0.3 M diethylene glycol, 0.3 M triethylene glycol, 0.3 M tetraethylene glycol, 0.3 M pentaethylene glycol], 0.1 M [MES and imidazole] pH 6.5, 30% (v/v) [40% (v/v) glycerol, 20% (w/v) polyethylene glycol 4000]

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: RIGAKU MICROMAX-003 / Wavelength: 1.54178 Å
DetectorType: RIGAKU SATURN 944 / Detector: CCD / Date: Sep 5, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 2.04→38.76 Å / Num. obs: 24738 / % possible obs: 99.85 % / Redundancy: 7.3 % / Biso Wilson estimate: 39.68 Å2 / CC1/2: 1 / CC star: 1 / Net I/σ(I): 20.31
Reflection shellResolution: 2.04→2.11 Å / Redundancy: 5.6 % / Num. unique obs: 2458 / CC1/2: 0.539 / CC star: 0.837 / % possible all: 99.39

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Processing

Software
NameVersionClassification
PHENIX1.17rc4_3626refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6J9N

6j9n


Resolution: 2.04→38.76 Å / SU ML: 0.2174 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.2523
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2377 1201 4.86 %
Rwork0.1937 23515 -
obs0.1958 24716 99.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 49.48 Å2
Refinement stepCycle: LAST / Resolution: 2.04→38.76 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1162 0 0 67 1229
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00651184
X-RAY DIFFRACTIONf_angle_d0.81651592
X-RAY DIFFRACTIONf_chiral_restr0.046169
X-RAY DIFFRACTIONf_plane_restr0.0055206
X-RAY DIFFRACTIONf_dihedral_angle_d17.596468
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.04-2.120.28051180.2612621X-RAY DIFFRACTION99.46
2.12-2.220.3221470.24192593X-RAY DIFFRACTION100
2.22-2.330.27471300.23812629X-RAY DIFFRACTION100
2.34-2.480.32411130.23012644X-RAY DIFFRACTION100
2.48-2.670.24771630.21152570X-RAY DIFFRACTION99.93
2.67-2.940.24251480.20972613X-RAY DIFFRACTION99.82
2.94-3.370.22771120.20722618X-RAY DIFFRACTION99.85
3.37-4.240.23251340.17412607X-RAY DIFFRACTION99.93
4.24-38.760.20451360.16582620X-RAY DIFFRACTION99.89
Refinement TLS params.Method: refined / Origin x: -16.5146904761 Å / Origin y: -0.401937909455 Å / Origin z: -3.73874303122 Å
111213212223313233
T0.366613125273 Å2-0.0108025064893 Å2-0.0204363618839 Å2-0.323349615826 Å2-0.0655766527862 Å2--0.359753063515 Å2
L1.07076401639 °2-0.317069891484 °2-0.427617817391 °2-1.56411664833 °20.789904441375 °2--2.96858620941 °2
S0.0762601972626 Å °0.235658121297 Å °-0.0776866916242 Å °-0.115074641814 Å °-0.161707442719 Å °0.330738332927 Å °-0.405310235732 Å °-0.227824640769 Å °0.0493831869492 Å °
Refinement TLS groupSelection details: all

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