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- PDB-8kaj: Crystal structure of SpyCas9-crRNA-tracrRNA complex bound to 16nt... -

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Basic information

Entry
Database: PDB / ID: 8kaj
TitleCrystal structure of SpyCas9-crRNA-tracrRNA complex bound to 16nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')
  • DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
  • RNA (34-MER)
  • RNA (65-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: 3.42 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
E: RNA (34-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
I: RNA (65-MER)
J: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)402,25310
Polymers402,25310
Non-polymers00
Water00
1
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
I: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)201,1275
Polymers201,1275
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19590 Å2
ΔGint-161 kcal/mol
Surface area81160 Å2
MethodPISA
2
E: RNA (34-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')
H: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')
J: RNA (65-MER)


Theoretical massNumber of molelcules
Total (without water)201,1275
Polymers201,1275
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18780 Å2
ΔGint-158 kcal/mol
Surface area80970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)144.735, 131.105, 146.631
Angle α, β, γ (deg.)90.000, 103.680, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: RNA chain RNA (34-MER)


Mass: 10912.471 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: crRNA
Source: (synth.) Streptococcus pyogenes serotype M1 (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
#3: DNA chain DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*TP*TP*TP*AP*TP*CP*CP*AP*TP*AP*AP*AP*TP*TP*CP*G)-3')


Mass: 7262.729 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)
#5: RNA chain RNA (65-MER)


Mass: 20968.490 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: tracrRNA
Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria)
Production host: Escherichia coli DH5[alpha] (bacteria)
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: 3.46 Å3/Da / Density % sol: 64.41 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 1.0 M Sodium chloride, 0.1 M Sodium citrate pH 6.0, 12.75% PEG4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL18U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 24, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 3.38→50 Å / Num. obs: 97863 / % possible obs: 99.9 % / Redundancy: 6.1 % / Biso Wilson estimate: 52.3 Å2 / Rpim(I) all: 0.09 / Net I/σ(I): 8.71
Reflection shellResolution: 3.38→3.44 Å / Redundancy: 5.4 % / Num. unique obs: 3645 / Rpim(I) all: 0.493 / % possible all: 99.6

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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_360phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.42→48.03 Å / SU ML: 0.3864 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 24.1207
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2465 3307 3.38 %
Rwork0.237 94556 -
obs0.2373 97863 69.09 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 68.16 Å2
Refinement stepCycle: LAST / Resolution: 3.42→48.03 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21467 5496 0 0 26963
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.012227983
X-RAY DIFFRACTIONf_angle_d1.550938903
X-RAY DIFFRACTIONf_chiral_restr0.1094492
X-RAY DIFFRACTIONf_plane_restr0.00934028
X-RAY DIFFRACTIONf_dihedral_angle_d19.999811332
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.42-3.470.483930.342481X-RAY DIFFRACTION1.42
3.47-3.520.3678110.2818313X-RAY DIFFRACTION5.54
3.52-3.570.2627160.2914501X-RAY DIFFRACTION8.78
3.57-3.630.2494310.2746832X-RAY DIFFRACTION14.71
3.63-3.70.2955540.28971409X-RAY DIFFRACTION24.75
3.7-3.760.2937740.28682215X-RAY DIFFRACTION38.79
3.76-3.840.2388960.27252875X-RAY DIFFRACTION49.98
3.84-3.910.29451010.25653104X-RAY DIFFRACTION54.66
3.91-40.2461260.2593499X-RAY DIFFRACTION61.06
4-4.090.25231380.2453952X-RAY DIFFRACTION69.37
4.09-4.190.26791590.244343X-RAY DIFFRACTION76.06
4.19-4.310.25311540.23834600X-RAY DIFFRACTION81.92
4.31-4.430.28461800.25125081X-RAY DIFFRACTION89.11
4.43-4.580.23721960.24295409X-RAY DIFFRACTION94.49
4.58-4.740.22351920.24295597X-RAY DIFFRACTION97.69
4.74-4.930.23191940.2335560X-RAY DIFFRACTION98.43
4.93-5.150.25552020.22695692X-RAY DIFFRACTION99.28
5.15-5.430.25521950.22575674X-RAY DIFFRACTION99.54
5.43-5.760.21432010.22835697X-RAY DIFFRACTION99.54
5.76-6.210.24731910.23555668X-RAY DIFFRACTION99.68
6.21-6.830.25421930.24685686X-RAY DIFFRACTION99.73
6.83-7.820.22821960.23365690X-RAY DIFFRACTION99.63
7.82-9.830.23622030.20925688X-RAY DIFFRACTION99.68
9.84-48.030.23262010.21595390X-RAY DIFFRACTION94.79

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