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- PDB-8kam: Crystal structure of SpyCas9 in complex with sgRNA and 16nt target DNA -

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Basic information

Entry
Database: PDB / ID: 8kam
TitleCrystal structure of SpyCas9 in complex with sgRNA and 16nt target DNA
Components
  • CRISPR-associated endonuclease Cas9/Csn1
  • DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*CP*TP*TP*CP*AP*AP*TP*TP*AP*GP*AP*AP*CP*AP*CP*G)-3')
  • 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: 3.91 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 (98-MER)
B: CRISPR-associated endonuclease Cas9/Csn1
C: DNA (5'-D(*CP*AP*AP*TP*AP*CP*CP*TP*CP*TP*TP*CP*AP*AP*TP*TP*AP*GP*AP*AP*CP*AP*CP*G)-3')
D: DNA (5'-D(*TP*TP*TP*AP*GP*GP*TP*AP*TP*TP*G)-3')


Theoretical massNumber of molelcules
Total (without water)200,8754
Polymers200,8754
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18980 Å2
ΔGint-160 kcal/mol
Surface area80760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)199.034, 70.387, 186.963
Angle α, β, γ (deg.)90.000, 109.616, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

#1: RNA chain RNA (98-MER)


Mass: 31610.740 Da / Num. of mol.: 1
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.: 1
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*CP*TP*TP*CP*AP*AP*TP*TP*AP*GP*AP*AP*CP*AP*CP*G)-3')


Mass: 7281.746 Da / Num. of mol.: 1 / 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.: 1 / Source method: obtained synthetically / Details: NTS
Source: (synth.) Streptococcus pyogenes serotype M1 (bacteria)
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
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: 3.17 Å3/Da / Density % sol: 61.17 %
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: BL18U1 / Wavelength: 0.97915 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 15, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3.91→50 Å / Num. obs: 19972 / % possible obs: 88.3 % / Redundancy: 3.5 % / Biso Wilson estimate: 4.82 Å2 / Rpim(I) all: 0.194 / Net I/σ(I): 2.8
Reflection shellResolution: 3.91→3.98 Å / Num. unique obs: 984 / Rpim(I) all: 0.516

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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: 3.91→38.78 Å / SU ML: 0.6119 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 27.9253
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.29 1554 10.06 %
Rwork0.2601 13892 -
obs0.2631 15446 35.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 32.06 Å2
Refinement stepCycle: LAST / Resolution: 3.91→38.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10816 2717 0 1 13534
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006214049
X-RAY DIFFRACTIONf_angle_d1.346119515
X-RAY DIFFRACTIONf_chiral_restr0.09482236
X-RAY DIFFRACTIONf_plane_restr0.00422028
X-RAY DIFFRACTIONf_dihedral_angle_d21.72035714
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.91-4.040.2799180.2259159X-RAY DIFFRACTION4.63
4.04-4.180.2784410.2336370X-RAY DIFFRACTION10.58
4.19-4.350.3487670.2336611X-RAY DIFFRACTION17.42
4.35-4.550.29861040.2533930X-RAY DIFFRACTION26.47
4.55-4.790.32981220.25051085X-RAY DIFFRACTION30.7
4.79-5.090.27951110.25551033X-RAY DIFFRACTION29.31
5.09-5.480.27521040.2677915X-RAY DIFFRACTION26.11
5.48-6.030.31951150.28721050X-RAY DIFFRACTION29.79
6.03-6.90.342120.33721884X-RAY DIFFRACTION53.18
6.9-8.670.33993220.31642782X-RAY DIFFRACTION79.22
8.68-38.780.22273380.21873073X-RAY DIFFRACTION87.46

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