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- PDB-5x2h: Crystal structure of Campylobacter jejuni Cas9 in complex with sg... -

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Basic information

Entry
Database: PDB / ID: 5x2h
TitleCrystal structure of Campylobacter jejuni Cas9 in complex with sgRNA and target DNA (AGAAACA PAM)
Components
  • CRISPR-associated endonuclease Cas9
  • Non-target DNA strand
  • Target DNA strand
  • sgRNA
KeywordsHYDROLASE/RNA/DNA / CRISPR-Cas9 / RNA / DNA / Complex / Nuclease / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / defense response to virus / endonuclease activity / DNA binding / RNA binding / metal ion binding
Similarity search - Function
: / : / CRISPR-associated endonuclease CjCas9 wedge domain / CRISPR-associated endonuclease CjCas9 PI-CTD domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / Cas9-type HNH domain / Cas9-type HNH domain profile. ...: / : / CRISPR-associated endonuclease CjCas9 wedge domain / CRISPR-associated endonuclease CjCas9 PI-CTD domain / RuvC endonuclease subdomain 3 / RuvC endonuclease subdomain 3 / CRISPR-associated endonuclease Cas9 / HNH endonuclease / 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
Similarity search - Component
Biological speciesCampylobacter jejuni subsp. jejuni serotype O:2 (Campylobacter)
Campylobacter jejuni (Campylobacter)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.3 Å
AuthorsYamada, M. / Watanabe, Y. / Hirano, H. / Nakane, T. / Ishitani, R. / Nishimasu, H. / Nureki, O.
CitationJournal: Mol. Cell / Year: 2017
Title: Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems
Authors: Yamada, M. / Watanabe, Y. / Gootenberg, J.S. / Hirano, H. / Ran, F.A. / Nakane, T. / Ishitani, R. / Zhang, F. / Nishimasu, H. / Nureki, O.
History
DepositionJan 31, 2017Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 29, 2017Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2020Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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: CRISPR-associated endonuclease Cas9
B: sgRNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,8225
Polymers137,7604
Non-polymers621
Water4,954275
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area18230 Å2
ΔGint-142 kcal/mol
Surface area48920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)102.039, 103.911, 134.458
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain Target DNA strand


Mass: 8467.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target DNA strand


Mass: 2468.670 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 96800.180 Da / Num. of mol.: 1 / Fragment: UNP residues 1-480, 642-984
Source method: isolated from a genetically manipulated source
Details: CjCas9-dHNH
Source: (gene. exp.) Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168) (Campylobacter)
Strain: ATCC 700819 / NCTC 11168 / Gene: cas9, Cj1523c / Production host: Escherichia coli (E. coli) / References: UniProt: Q0P897
#2: RNA chain sgRNA


Mass: 30023.844 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Campylobacter jejuni (Campylobacter)

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Non-polymers , 2 types, 276 molecules

#5: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 275 / 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: 2.59 Å3/Da / Density % sol: 52.46 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: 12.0-14.5% PEG 2000, 0.4M ammonium acetate

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 19, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.3→103.91 Å / Num. obs: 64066 / % possible obs: 99.8 % / Redundancy: 10.4 % / CC1/2: 0.986 / Rpim(I) all: 0.032 / Net I/σ(I): 12.4
Reflection shellCC1/2: 0.875 / Rpim(I) all: 0.316

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Processing

Software
NameVersionClassification
PHENIX(1.11.1_2575: ???)refinement
DIALSdata reduction
Aimlessdata scaling
AutoSolphasing
RefinementMethod to determine structure: SAD / Resolution: 2.3→72.805 Å / SU ML: 0.31 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.68
RfactorNum. reflection% reflection
Rfree0.2311 3144 4.92 %
Rwork0.2 --
obs0.2015 63923 99.62 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.3→72.805 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5925 2715 4 275 8919
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0029071
X-RAY DIFFRACTIONf_angle_d0.51512829
X-RAY DIFFRACTIONf_dihedral_angle_d16.0325123
X-RAY DIFFRACTIONf_chiral_restr0.0341515
X-RAY DIFFRACTIONf_plane_restr0.0021158
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3001-2.3360.37751400.31252716X-RAY DIFFRACTION99
2.336-2.37430.35841440.2962746X-RAY DIFFRACTION100
2.3743-2.41530.2891500.2812723X-RAY DIFFRACTION100
2.4153-2.45920.34711320.27222676X-RAY DIFFRACTION98
2.4592-2.50650.30211370.26922672X-RAY DIFFRACTION97
2.5065-2.55760.29221440.25752729X-RAY DIFFRACTION100
2.5576-2.61330.32261570.24692731X-RAY DIFFRACTION100
2.6133-2.67410.24851340.24472750X-RAY DIFFRACTION100
2.6741-2.74090.27551560.23212713X-RAY DIFFRACTION99
2.7409-2.8150.28051180.2462753X-RAY DIFFRACTION100
2.815-2.89790.27651210.24292756X-RAY DIFFRACTION100
2.8979-2.99140.27781390.23682759X-RAY DIFFRACTION100
2.9914-3.09830.26061540.23982770X-RAY DIFFRACTION100
3.0983-3.22240.23361530.21362727X-RAY DIFFRACTION100
3.2224-3.3690.23981420.20252777X-RAY DIFFRACTION100
3.369-3.54670.23411530.20332764X-RAY DIFFRACTION100
3.5467-3.76890.24611440.18942776X-RAY DIFFRACTION100
3.7689-4.05980.21641120.17562820X-RAY DIFFRACTION100
4.0598-4.46830.19191730.16382767X-RAY DIFFRACTION100
4.4683-5.11480.16871390.1522830X-RAY DIFFRACTION100
5.1148-6.44350.20921290.18282870X-RAY DIFFRACTION100
6.4435-72.84180.17851730.16742954X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.2819-0.2738-0.05072.0608-0.2311.96960.1318-0.0318-0.26470.08140.00870.19290.46610.0088-0.13310.41480.0357-0.04240.3832-0.00490.30186.4137-16.830665.3145
20.9642-1.3547-0.9313.54321.87240.8705-0.3551-0.3627-0.16260.85580.26760.53410.56770.1110.08240.84030.08620.06540.5640.07220.4886-7.0442-3.215888.4874
31.5340.0642-0.75711.56-0.11182.39430.16630.13950.37120.08780.08670.163-0.4661-0.2062-0.16460.36130.03810.05090.32520.06850.36768.723517.032848.0689
40.4326-0.7239-0.96752.0941.79282.08110.02670.04890.0092-0.0123-0.16880.4132-0.03-0.23490.14740.44240.060.0010.4296-0.02330.5257-8.13760.439375.0131
51.18591.64621.94944.7882.41633.1881-0.01730.388-0.54210.16040.3915-0.88060.69430.5657-0.36020.65080.1781-0.02350.5745-0.19560.571514.7196-32.738642.7459
60.89390.23330.29190.416-0.56942.11310.0771-0.11730.01170.2136-0.0248-0.1481-0.10440.4441-0.05370.38830.0709-0.03260.5013-0.03570.336119.4494-2.545471.3379
72.92741.7169-2.80535.5399-5.69928.38110.01760.4850.1439-0.47520.6190.49060.2087-0.3591-0.710.3783-0.056-0.03610.49870.06590.37315.8635-0.05738.2167
80.356-0.6598-0.51132.30541.34370.7181-0.12480.2229-0.05630.3732-0.18980.79070.1403-0.28230.29450.44690.09770.04640.4955-0.01590.7161-13.4353.20277.2474
95.2426-0.27320.90096.9248-0.09428.89550.15620.4139-0.1808-0.74510.11310.29620.4204-0.4601-0.20210.3808-0.0349-0.00670.37580.06510.32979.59241.51636.5386
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 179 )
2X-RAY DIFFRACTION2chain 'A' and (resid 180 through 449 )
3X-RAY DIFFRACTION3chain 'A' and (resid 450 through 984 )
4X-RAY DIFFRACTION4chain 'B' and (resid 1 through 20 )
5X-RAY DIFFRACTION5chain 'B' and (resid 21 through 40 )
6X-RAY DIFFRACTION6chain 'B' and (resid 41 through 93 )
7X-RAY DIFFRACTION7chain 'C' and (resid -7 through 2 )
8X-RAY DIFFRACTION8chain 'C' and (resid 3 through 20 )
9X-RAY DIFFRACTION9chain 'D' and (resid 1 through 8 )

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