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- PDB-8kak: Crystal structure of SpyCas9 in complex with sgRNA and 18nt target DNA -

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Basic information

Entry
Database: PDB / ID: 8kak
TitleCrystal structure of SpyCas9 in complex with sgRNA and 18nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (26-MER)
  • DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
  • RNA (98-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / Nuclease / RNA BINDING PROTEIN-RNA-DNA complex
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) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes serotype M1 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.6 Å
AuthorsChen, Y. / Chen, J. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32022047 China
CitationJournal: Nat.Biotechnol. / Year: 2025
Title: Trans-nuclease activity of Cas9 activated by DNA or RNA target binding.
Authors: Chen, J. / Chen, Y. / Huang, L. / Lin, X. / Chen, H. / Xiang, W. / Liu, L.
History
DepositionAug 3, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Apr 23, 2025Group: Database references / Structure summary / Category: citation / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (98-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (26-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
E: RNA (98-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (26-MER)
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)403,22810
Polymers403,0368
Non-polymers1922
Water1086
1
A: RNA (98-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (26-MER)
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,6145
Polymers201,5184
Non-polymers961
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18840 Å2
ΔGint-167 kcal/mol
Surface area80390 Å2
MethodPISA
2
E: RNA (98-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (26-MER)
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,6145
Polymers201,5184
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18830 Å2
ΔGint-165 kcal/mol
Surface area80490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.742, 104.970, 184.309
Angle α, β, γ (deg.)75.819, 78.675, 72.631
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

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DNA chain , 2 types, 4 molecules CGDH

#3: DNA chain DNA (26-MER)


Mass: 7924.158 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: TS
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)
#4: DNA chain DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')


Mass: 3394.230 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: NTS
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)

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RNA chain / Protein , 2 types, 4 molecules AEBF

#1: RNA chain RNA (98-MER)


Mass: 31610.740 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Production host: Escherichia coli DH5[alpha] (bacteria)
#2: Protein CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9


Mass: 158588.781 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 2 types, 8 molecules

#5: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: 3.22 Å3/Da / Density % sol: 61.76 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 0.15 M Ammonium sulfate, 0.1 M MES pH 6.0, 14.75% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97852 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 14, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97852 Å / Relative weight: 1
ReflectionResolution: 3.6→50 Å / Num. obs: 52766 / % possible obs: 94.6 % / Redundancy: 2.7 % / Biso Wilson estimate: 30.01 Å2 / Rpim(I) all: 0.176 / Net I/σ(I): 4.11
Reflection shellResolution: 3.6→3.72 Å / Redundancy: 2.6 % / Num. unique obs: 2595 / Rpim(I) all: 0.708 / % possible all: 93.2

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-30007.21data scaling
HKL-30007.21data reduction
Coot0.7.2.1model building
PHENIX1.17.1_3660phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.6→24.97 Å / SU ML: 0.5191 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 28.245
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2971 2061 4.96 %
Rwork0.2649 39469 -
obs0.2665 41530 73.71 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 37.42 Å2
Refinement stepCycle: LAST / Resolution: 3.6→24.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21654 5478 10 6 27148
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.011828178
X-RAY DIFFRACTIONf_angle_d1.59539146
X-RAY DIFFRACTIONf_chiral_restr0.21084480
X-RAY DIFFRACTIONf_plane_restr0.00614062
X-RAY DIFFRACTIONf_dihedral_angle_d23.112111466
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.6-3.680.334970.2775172X-RAY DIFFRACTION4.76
3.68-3.770.3764340.2816583X-RAY DIFFRACTION16.34
3.77-3.870.3484700.29351126X-RAY DIFFRACTION32.23
3.87-3.990.30391120.28781883X-RAY DIFFRACTION52.83
3.99-4.120.33791240.28382526X-RAY DIFFRACTION70.91
4.12-4.260.37361650.28443022X-RAY DIFFRACTION85.12
4.26-4.430.2951830.27153108X-RAY DIFFRACTION87.18
4.43-4.630.27991520.27113304X-RAY DIFFRACTION92.14
4.63-4.870.29761490.26433351X-RAY DIFFRACTION93.36
4.87-5.180.27221650.26093322X-RAY DIFFRACTION93.14
5.18-5.570.31881800.26463380X-RAY DIFFRACTION94.45
5.57-6.130.30241920.27683364X-RAY DIFFRACTION94.95
6.13-70.29651400.28133492X-RAY DIFFRACTION96.44
7-8.750.25082080.24013438X-RAY DIFFRACTION96.46
8.75-24.970.24661800.22343398X-RAY DIFFRACTION95.87

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