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- PDB-8kai: Crystal structure of SpyCas9-crRNA-tracrRNA complex bound to 17nt... -

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Basic information

Entry
Database: PDB / ID: 8kai
TitleCrystal structure of SpyCas9-crRNA-tracrRNA complex bound to 17nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (25-MER)
  • 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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix 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, PAM-interacting domain / CRISPR-associated endonuclease Cas9, bridge helix / CRISPR-associated endonuclease Cas9, REC lobe / REC lobe of CRISPR-associated endonuclease Cas9 / Bridge helix 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: 3.49 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: 2024
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (25-MER)
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 (25-MER)
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)402,86210
Polymers402,86210
Non-polymers00
Water724
1
A: RNA (34-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (25-MER)
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)201,4315
Polymers201,4315
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19540 Å2
ΔGint-157 kcal/mol
Surface area80870 Å2
MethodPISA
2
E: RNA (34-MER)
F: CRISPR-associated endonuclease Cas9/Csn1
G: DNA (25-MER)
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)201,4315
Polymers201,4315
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18760 Å2
ΔGint-157 kcal/mol
Surface area80960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)144.300, 130.186, 146.413
Angle α, β, γ (deg.)90.000, 104.021, 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

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RNA chain , 2 types, 4 molecules AEIJ

#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)
#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)

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DNA chain , 2 types, 4 molecules CGDH

#3: DNA chain DNA (25-MER)


Mass: 7566.922 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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Protein / Non-polymers , 2 types, 6 molecules BF

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


Mass: 158588.781 Da / Num. of mol.: 2 / Mutation: A10D,A840H
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
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: 3.31 Å3/Da / Density % sol: 62.86 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 1.0 M Sodium chloride, 0.1 M Sodium citrate pH 6.0, 13% 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 PILATUS3 6M / Detector: PIXEL / Date: Jun 8, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97852 Å / Relative weight: 1
ReflectionResolution: 3.4→50 Å / Num. obs: 73038 / % possible obs: 99.7 % / Redundancy: 5 % / Biso Wilson estimate: 62.14 Å2 / Rpim(I) all: 0.126 / Net I/σ(I): 5.2
Reflection shellResolution: 3.4→3.46 Å / Redundancy: 4.2 % / Num. unique obs: 3650 / Rpim(I) all: 0.514 / % possible all: 99.4

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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: 3.49→48.57 Å / SU ML: 0.5209 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 27.9603
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2939 2617 4.94 %
Rwork0.2248 50306 -
obs0.2282 52923 79.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 76.88 Å2
Refinement stepCycle: LAST / Resolution: 3.49→48.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21467 5536 0 4 27007
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.014128027
X-RAY DIFFRACTIONf_angle_d1.779438971
X-RAY DIFFRACTIONf_chiral_restr0.1154500
X-RAY DIFFRACTIONf_plane_restr0.01064030
X-RAY DIFFRACTIONf_dihedral_angle_d15.97075870
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.49-3.550.581630.246180X-RAY DIFFRACTION2.4
3.56-3.620.3354250.2774354X-RAY DIFFRACTION10.82
3.62-3.70.3805360.2715717X-RAY DIFFRACTION21.65
3.7-3.780.368730.25991217X-RAY DIFFRACTION36.95
3.78-3.870.3266990.26532099X-RAY DIFFRACTION62.64
3.87-3.960.34751210.27132810X-RAY DIFFRACTION83.1
3.96-4.070.34061560.25673089X-RAY DIFFRACTION92.66
4.07-4.190.31261420.24893238X-RAY DIFFRACTION96.41
4.19-4.320.32171670.24063314X-RAY DIFFRACTION98.81
4.32-4.480.32261810.2373302X-RAY DIFFRACTION99.37
4.48-4.660.30072000.22643294X-RAY DIFFRACTION99.32
4.66-4.870.28961660.21873325X-RAY DIFFRACTION99.54
4.87-5.130.30221630.21553347X-RAY DIFFRACTION99.83
5.13-5.450.31971840.23349X-RAY DIFFRACTION99.86
5.45-5.870.27941730.20563343X-RAY DIFFRACTION99.91
5.87-6.450.28171700.21833356X-RAY DIFFRACTION99.91
6.46-7.390.27982050.21683333X-RAY DIFFRACTION99.92
7.39-9.290.25641880.19483372X-RAY DIFFRACTION99.69
9.3-48.570.22811650.20463367X-RAY DIFFRACTION97.06

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