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- PDB-6aeg: Crystal structure of xCas9 in complex with sgRNA and target DNA (... -

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Basic information

Entry
Database: PDB / ID: 6aeg
TitleCrystal structure of xCas9 in complex with sgRNA and target DNA (GAT PAM)
Components
  • DNA (25-MER)
  • DNA (5'-D(*AP*AP*AP*GP*AP*TP*TP*AP*TP*TP*G)-3')
  • DNA nuclease
  • RNA (95-MER)
KeywordsDNA BINDING PROTEIN/DNA/RNA / enzyme / DNA / nuclease / DNA BINDING PROTEIN-DNA-RNA 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) / RNA (> 100) / CRISPR-associated endonuclease Cas9/Csn1
Similarity search - Component
Biological speciesStreptococcus pyogenes (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.701 Å
AuthorsGuo, M. / Ren, K. / Zhu, Y. / Huang, Z.
CitationJournal: Cell Res. / Year: 2019
Title: Structural insights into a high fidelity variant of SpCas9.
Authors: Guo, M. / Ren, K. / Zhu, Y. / Tang, Z. / Wang, Y. / Zhang, B. / Huang, Z.
History
DepositionAug 4, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 27, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (95-MER)
C: DNA (25-MER)
D: DNA (5'-D(*AP*AP*AP*GP*AP*TP*TP*AP*TP*TP*G)-3')
B: DNA nuclease


Theoretical massNumber of molelcules
Total (without water)202,7724
Polymers202,7724
Non-polymers00
Water2,198122
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18830 Å2
ΔGint-155 kcal/mol
Surface area75490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)190.480, 70.460, 183.529
Angle α, β, γ (deg.)90.00, 106.48, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: RNA chain RNA (95-MER)


Mass: 32269.199 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: DNA chain DNA (25-MER)


Mass: 8567.574 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain DNA (5'-D(*AP*AP*AP*GP*AP*TP*TP*AP*TP*TP*G)-3')


Mass: 3396.259 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: Protein DNA nuclease


Mass: 158538.844 Da / Num. of mol.: 1
Mutation: D10A, A263T, R324L, S409I, E480K, E543D, M694I, H840A, E1219V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Production host: Escherichia coli (E. coli)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 122 / 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: 2.91 Å3/Da / Density % sol: 57.76 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1M Hepes, pH 7.5, 0.2M Ammonium sulfate, 10% ispropanol, 16% (w/v) PEG4000, 30%(v/v) (+/-)-2-Methyl-2,4-pentanediol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9789 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Apr 18, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9789 Å / Relative weight: 1
ReflectionResolution: 2.7→50 Å / Num. obs: 60687 / % possible obs: 94.1 % / Redundancy: 2.8 % / Rmerge(I) obs: 0.082 / Net I/σ(I): 9.1
Reflection shellResolution: 2.7→2.75 Å

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
HKL-2000data reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6AEB

6aeb


Resolution: 2.701→44.972 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 29.73
Details: The data completeness is 94.1%, calculated by software HKL2000.
RfactorNum. reflection% reflection
Rfree0.2625 3344 3.56 %
Rwork0.2147 --
obs0.2164 56285 74.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.701→44.972 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10475 2765 0 122 13362
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00513764
X-RAY DIFFRACTIONf_angle_d0.76219205
X-RAY DIFFRACTIONf_dihedral_angle_d13.677980
X-RAY DIFFRACTIONf_chiral_restr0.0432247
X-RAY DIFFRACTIONf_plane_restr0.0041976
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7009-2.73950.3772580.32641693X-RAY DIFFRACTION34
2.7395-2.78040.3668680.30021913X-RAY DIFFRACTION38
2.7804-2.82380.3306870.30912250X-RAY DIFFRACTION44
2.8238-2.87010.3489920.30922475X-RAY DIFFRACTION50
2.8701-2.91960.3391130.3182982X-RAY DIFFRACTION58
2.9196-2.97270.36811230.32363214X-RAY DIFFRACTION64
2.9727-3.02980.42091290.29983488X-RAY DIFFRACTION70
3.0298-3.09170.29831390.28613730X-RAY DIFFRACTION74
3.0917-3.15890.36711410.26693893X-RAY DIFFRACTION77
3.1589-3.23230.32271450.25883944X-RAY DIFFRACTION79
3.2323-3.31310.27791520.24644136X-RAY DIFFRACTION82
3.3131-3.40270.26761500.25064144X-RAY DIFFRACTION82
3.4027-3.50280.30311560.24424244X-RAY DIFFRACTION83
3.5028-3.61580.2691630.21954384X-RAY DIFFRACTION87
3.6158-3.7450.30381650.21734495X-RAY DIFFRACTION89
3.745-3.89480.26411600.20034512X-RAY DIFFRACTION89
3.8948-4.0720.23981680.19954489X-RAY DIFFRACTION89
4.072-4.28650.25421640.18114382X-RAY DIFFRACTION88
4.2865-4.55480.23781630.17364297X-RAY DIFFRACTION85
4.5548-4.90610.23231670.16974421X-RAY DIFFRACTION88
4.9061-5.39910.20571690.17154525X-RAY DIFFRACTION89
5.3991-6.17860.24071580.18494389X-RAY DIFFRACTION88
6.1786-7.77790.22361520.19454363X-RAY DIFFRACTION86
7.7779-44.97850.16071620.17054229X-RAY DIFFRACTION84

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