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

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Basic information

Entry
Database: PDB / ID: 6aeb
TitleCrystal structure of xCas9 in complex with sgRNA and target DNA (AAG PAM)
Components
  • DNA (25-MER)
  • DNA (5'-D(*AP*AP*AP*AP*AP*GP*TP*AP*TP*TP*G)-3')
  • DNA Nuclease
  • RNA (95-MER)
KeywordsDNA BINDING PROTEIN/RNA / enzyme / DNA BINDING PROTEIN-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: 3.004 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)405,5448
Polymers405,5448
Non-polymers00
Water181
1
A: RNA (95-MER)
C: DNA (25-MER)
D: DNA (5'-D(*AP*AP*AP*AP*AP*GP*TP*AP*TP*TP*G)-3')
B: DNA Nuclease


Theoretical massNumber of molelcules
Total (without water)202,7724
Polymers202,7724
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18630 Å2
ΔGint-164 kcal/mol
Surface area76500 Å2
MethodPISA
2
E: RNA (95-MER)
G: DNA (25-MER)
H: DNA (5'-D(*AP*AP*AP*AP*AP*GP*TP*AP*TP*TP*G)-3')
F: DNA Nuclease


Theoretical massNumber of molelcules
Total (without water)202,7724
Polymers202,7724
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18650 Å2
ΔGint-164 kcal/mol
Surface area76460 Å2
MethodPISA
Unit cell
Length a, b, c (Å)361.853, 71.130, 198.320
Angle α, β, γ (deg.)90.00, 101.75, 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.: 2 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: DNA chain DNA (25-MER)


Mass: 8558.561 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain DNA (5'-D(*AP*AP*AP*AP*AP*GP*TP*AP*TP*TP*G)-3')


Mass: 3405.273 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: Protein DNA Nuclease


Mass: 158538.844 Da / Num. of mol.: 2
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.: 1 / 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: 3.08 Å3/Da / Density % sol: 60.07 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1M Hepes, PH 7.5, 0.2M Ammonium sulfate, 10% ispropanol, and 16% (w/v) PEG4000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9789 Å
DetectorType: DECTRIS PILATUS3 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: 3→50 Å / Num. obs: 98977 / % possible obs: 99.3 % / Redundancy: 4.2 % / Rmerge(I) obs: 0.119 / Net I/σ(I): 10.1
Reflection shellResolution: 3→3.18 Å

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4OO8

4oo8


Resolution: 3.004→49.452 Å / SU ML: 0.41 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 34.12
Details: The data completeness is 99.3%, calculated by software XDS.
RfactorNum. reflection% reflection
Rfree0.2828 3793 2.01 %
Rwork0.231 --
obs0.2321 98522 97.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.004→49.452 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20820 5530 0 1 26351
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00427416
X-RAY DIFFRACTIONf_angle_d0.69838278
X-RAY DIFFRACTIONf_dihedral_angle_d13.51415866
X-RAY DIFFRACTIONf_chiral_restr0.0424494
X-RAY DIFFRACTIONf_plane_restr0.0033930
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.0035-3.04150.47471300.43046022X-RAY DIFFRACTION86
3.0415-3.08160.44751400.40326845X-RAY DIFFRACTION97
3.0816-3.12380.39481390.38336781X-RAY DIFFRACTION97
3.1238-3.16840.38761350.36776776X-RAY DIFFRACTION97
3.1684-3.21570.44411420.34696873X-RAY DIFFRACTION98
3.2157-3.26590.3751400.32776878X-RAY DIFFRACTION98
3.2659-3.31940.38581420.31876845X-RAY DIFFRACTION98
3.3194-3.37670.45581420.30836939X-RAY DIFFRACTION98
3.3767-3.43810.31541280.29236855X-RAY DIFFRACTION98
3.4381-3.50420.32181360.27946894X-RAY DIFFRACTION98
3.5042-3.57570.32591440.26066893X-RAY DIFFRACTION98
3.5757-3.65340.28031420.25386830X-RAY DIFFRACTION99
3.6534-3.73840.32671490.25117050X-RAY DIFFRACTION99
3.7384-3.83180.33571420.24166833X-RAY DIFFRACTION99
3.8318-3.93540.32291440.23846955X-RAY DIFFRACTION99
3.9354-4.05110.29181410.23546779X-RAY DIFFRACTION98
4.0511-4.18180.28771430.2266916X-RAY DIFFRACTION97
4.1818-4.33120.27331400.2076847X-RAY DIFFRACTION99
4.3312-4.50450.27431420.19926977X-RAY DIFFRACTION99
4.5045-4.70940.24981410.20056937X-RAY DIFFRACTION99
4.7094-4.95740.27061440.19376939X-RAY DIFFRACTION99
4.9574-5.26770.22291440.19836967X-RAY DIFFRACTION99
5.2677-5.67390.27181370.20236946X-RAY DIFFRACTION99
5.6739-6.24390.24621370.22146755X-RAY DIFFRACTION96
6.2439-7.1450.25291450.20676965X-RAY DIFFRACTION99
7.145-8.99320.21881420.17336944X-RAY DIFFRACTION99
8.9932-49.45910.17521420.1656690X-RAY DIFFRACTION96

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