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- PDB-9kj6: Crystal Structure of SpCas9 ternary complex, amino acids (1242-12... -

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Basic information

Entry
Database: PDB / ID: 9kj6
TitleCrystal Structure of SpCas9 ternary complex, amino acids (1242-1263) replaced with Gly-Ser linker
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • Non-target Strand of double-stranded DNA
  • Target Strand of double-stranded DNA
  • single-guide RNA
KeywordsDNA BINDING PROTEIN / CRISPR-Cas9
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 ...CRISPR-associated endonuclease Cas9, bridge helix / Bridge helix of CRISPR-associated endonuclease Cas9 / CRISPR-associated endonuclease Cas9, PAM-interacting domain / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / PAM-interacting domain of CRISPR-associated endonuclease Cas9 / : / Cas9 RuvC domain / HNH endonuclease / CRISPR-associated endonuclease Cas9 / Cas9-type HNH domain / Cas9-type HNH domain profile. / HNH nuclease / Ribonuclease H superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.59 Å
AuthorsKim, S. / Bae, J. / Choi, H.J.
Funding support Korea, Republic Of, 1items
OrganizationGrant numberCountry
Samsung Science and Technology FoundationSRFC-MA1801-09 Korea, Republic Of
CitationJournal: To Be Published
Title: Crystal Structure of SpCas9 ternary complex, amino acids (1242-1263) replaced with Gly-Ser linker
Authors: Kim, S. / Choi, H.J.
History
DepositionNov 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9/Csn1
B: single-guide RNA
C: Target Strand of double-stranded DNA
D: Non-target Strand of double-stranded DNA


Theoretical massNumber of molelcules
Total (without water)211,1704
Polymers211,1704
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19520 Å2
ΔGint-155 kcal/mol
Surface area80800 Å2
MethodPISA
Unit cell
Length a, b, c (Å)88.550, 146.850, 222.360
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 156702.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain single-guide RNA


Mass: 32991.609 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain Target Strand of double-stranded DNA


Mass: 13126.428 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target Strand of double-stranded DNA


Mass: 8349.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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: 3.51 Å3/Da / Density % sol: 64.9 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.07 M HEPES (pH 7.5), 0.18 M potassium acetate, 14% (v/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PAL/PLS / Beamline: 5C (4A) / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: May 29, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.59→29.9 Å / Num. obs: 34326 / % possible obs: 98.9 % / Redundancy: 4.5 % / Biso Wilson estimate: 129.34 Å2 / CC1/2: 1 / CC star: 0.999 / Rmerge(I) obs: 0.07705 / Rpim(I) all: 0.03969 / Rrim(I) all: 0.0871 / Net I/σ(I): 14.18
Reflection shellResolution: 3.59→3.718 Å / Rmerge(I) obs: 0.8626 / Mean I/σ(I) obs: 1.96 / Num. unique obs: 3405 / CC1/2: 0.665 / CC star: 0.894 / Rpim(I) all: 0.4341 / Rrim(I) all: 0.9695 / % possible all: 99.88

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5F9R

5f9r


Resolution: 3.59→29.9 Å / SU ML: 0.4966 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.5794
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2705 1716 5 %
Rwork0.228 32591 -
obs0.2301 34307 99.13 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 137.7 Å2
Refinement stepCycle: LAST / Resolution: 3.59→29.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9700 3421 0 0 13121
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002813707
X-RAY DIFFRACTIONf_angle_d0.502219392
X-RAY DIFFRACTIONf_chiral_restr0.0352306
X-RAY DIFFRACTIONf_plane_restr0.00331936
X-RAY DIFFRACTIONf_dihedral_angle_d16.26875179
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.59-3.70.33971430.30712713X-RAY DIFFRACTION99.86
3.7-3.810.34411410.29472672X-RAY DIFFRACTION99.89
3.81-3.950.31911410.27822686X-RAY DIFFRACTION99.61
3.95-4.110.32951420.26952690X-RAY DIFFRACTION99.44
4.11-4.290.29831400.25472670X-RAY DIFFRACTION98.91
4.29-4.520.30121430.23982715X-RAY DIFFRACTION99.44
4.52-4.80.28281420.22142690X-RAY DIFFRACTION99.44
4.8-5.170.27991430.2122716X-RAY DIFFRACTION99.31
5.17-5.690.2681430.22682719X-RAY DIFFRACTION99.2
5.69-6.50.26751440.24172732X-RAY DIFFRACTION98.97
6.51-8.170.27511440.22832743X-RAY DIFFRACTION98
8.17-29.90.21211500.18752845X-RAY DIFFRACTION97.75

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