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

ID or keywords:

Entry

Database: PDB / ID: 5x2h

Title

Crystal 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
sgRNASubgenomic mRNA

Keywords

HYDROLASE/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

Biological species

Campylobacter jejuni subsp. jejuni serotype O:2 (Campylobacter)

Campylobacter jejuni (Campylobacter)
synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

SAD / Resolution: 2.3 Å

Authors

Yamada, M. / Watanabe, Y. / Hirano, H. / Nakane, T. / Ishitani, R. / Nishimasu, H. / Nureki, O.

Citation

Journal: 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

Deposition	Jan 31, 2017	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Mar 29, 2017	Provider: repository / Type: Initial release
Revision 1.1	Feb 26, 2020	Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2	Mar 27, 2024	Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	5x2h.cif.gz	464.8 KB	Display	PDBx/mmCIF format
PDB format	pdb5x2h.ent.gz	369.7 KB	Display	PDB format
PDBx/mmJSON format	5x2h.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/x2/5x2h ftp://data.pdbj.org/pub/pdb/validation_reports/x2/5x2h	HTTPS FTP

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Related structure data

Related structure data	5x2gC C: citing same article (ref.)
Similar structure data	5x2g-d 1lw5-d 1lw4-d 1jg8-d 2a5y-d 5vye-d 3q15-1 2fm1-d 1orr-d 6joo-d Search similar-shape structures by Omokage search (details) Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI
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PDB pages

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Deposited unit

A: CRISPR-associated endonuclease Cas9

B: sgRNA

C: Target DNA strand

D: Non-target DNA strand

hetero molecules

138 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

Idetical with deposited unit
defined by author&software

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 number	19
Space group name H-M	P2₁2₁2₁

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

#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 /

Subgenomic mRNA

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 / Ethylene glycol Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#6: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 275 / Source method: isolated from a natural source / Formula: H₂O

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.59 Å³/Da / Density % sol: 52.46 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / Details: 12.0-14.5% PEG 2000, 0.4M ammonium acetate

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Data collection

Diffraction	Mean temperature: 100 K
Diffraction source	Source: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
Detector	Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: May 19, 2015
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.3→103.91 Å / Num. obs: 64066 / % possible obs: 99.8 % / Redundancy: 10.4 % / CC_1/2: 0.986 / R_pim(I) all: 0.032 / Net I/σ(I): 12.4
Reflection shell	CC_1/2: 0.875 / R_pim(I) all: 0.316

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Processing

Software

Name	Version	Classification
PHENIX	(1.11.1_2575: ???)	refinement
DIALS		data reduction
Aimless		data scaling
AutoSol		phasing

Refinement

Method 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

	R_factor	Num. reflection	% reflection
R_free	0.2311	3144	4.92 %
R_work	0.2	-	-
obs	0.2015	63923	99.62 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å

Refinement step

Cycle: LAST / Resolution: 2.3→72.805 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5925	2715	4	275	8919

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.002	9071
X-RAY DIFFRACTION	f_angle_d	0.515	12829
X-RAY DIFFRACTION	f_dihedral_angle_d	16.032	5123
X-RAY DIFFRACTION	f_chiral_restr	0.034	1515
X-RAY DIFFRACTION	f_plane_restr	0.002	1158

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.3001-2.336	0.3775	140	0.3125	2716	X-RAY DIFFRACTION	99
2.336-2.3743	0.3584	144	0.296	2746	X-RAY DIFFRACTION	100
2.3743-2.4153	0.289	150	0.281	2723	X-RAY DIFFRACTION	100
2.4153-2.4592	0.3471	132	0.2722	2676	X-RAY DIFFRACTION	98
2.4592-2.5065	0.3021	137	0.2692	2672	X-RAY DIFFRACTION	97
2.5065-2.5576	0.2922	144	0.2575	2729	X-RAY DIFFRACTION	100
2.5576-2.6133	0.3226	157	0.2469	2731	X-RAY DIFFRACTION	100
2.6133-2.6741	0.2485	134	0.2447	2750	X-RAY DIFFRACTION	100
2.6741-2.7409	0.2755	156	0.2321	2713	X-RAY DIFFRACTION	99
2.7409-2.815	0.2805	118	0.246	2753	X-RAY DIFFRACTION	100
2.815-2.8979	0.2765	121	0.2429	2756	X-RAY DIFFRACTION	100
2.8979-2.9914	0.2778	139	0.2368	2759	X-RAY DIFFRACTION	100
2.9914-3.0983	0.2606	154	0.2398	2770	X-RAY DIFFRACTION	100
3.0983-3.2224	0.2336	153	0.2136	2727	X-RAY DIFFRACTION	100
3.2224-3.369	0.2398	142	0.2025	2777	X-RAY DIFFRACTION	100
3.369-3.5467	0.2341	153	0.2033	2764	X-RAY DIFFRACTION	100
3.5467-3.7689	0.2461	144	0.1894	2776	X-RAY DIFFRACTION	100
3.7689-4.0598	0.2164	112	0.1756	2820	X-RAY DIFFRACTION	100
4.0598-4.4683	0.1919	173	0.1638	2767	X-RAY DIFFRACTION	100
4.4683-5.1148	0.1687	139	0.152	2830	X-RAY DIFFRACTION	100
5.1148-6.4435	0.2092	129	0.1828	2870	X-RAY DIFFRACTION	100
6.4435-72.8418	0.1785	173	0.1674	2954	X-RAY DIFFRACTION	100

Refinement TLS params.

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

ID	L11 (°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)
1	1.2819	-0.2738	-0.0507	2.0608	-0.231	1.9696	0.1318	-0.0318	-0.2647	0.0814	0.0087	0.1929	0.4661	0.0088	-0.1331	0.4148	0.0357	-0.0424	0.3832	-0.0049	0.3018	6.4137	-16.8306	65.3145
2	0.9642	-1.3547	-0.931	3.5432	1.8724	0.8705	-0.3551	-0.3627	-0.1626	0.8558	0.2676	0.5341	0.5677	0.111	0.0824	0.8403	0.0862	0.0654	0.564	0.0722	0.4886	-7.0442	-3.2158	88.4874
3	1.534	0.0642	-0.7571	1.56	-0.1118	2.3943	0.1663	0.1395	0.3712	0.0878	0.0867	0.163	-0.4661	-0.2062	-0.1646	0.3613	0.0381	0.0509	0.3252	0.0685	0.3676	8.7235	17.0328	48.0689
4	0.4326	-0.7239	-0.9675	2.094	1.7928	2.0811	0.0267	0.0489	0.0092	-0.0123	-0.1688	0.4132	-0.03	-0.2349	0.1474	0.4424	0.06	0.001	0.4296	-0.0233	0.5257	-8.1376	0.4393	75.0131
5	1.1859	1.6462	1.9494	4.788	2.4163	3.1881	-0.0173	0.388	-0.5421	0.1604	0.3915	-0.8806	0.6943	0.5657	-0.3602	0.6508	0.1781	-0.0235	0.5745	-0.1956	0.5715	14.7196	-32.7386	42.7459
6	0.8939	0.2333	0.2919	0.416	-0.5694	2.1131	0.0771	-0.1173	0.0117	0.2136	-0.0248	-0.1481	-0.1044	0.4441	-0.0537	0.3883	0.0709	-0.0326	0.5013	-0.0357	0.3361	19.4494	-2.5454	71.3379
7	2.9274	1.7169	-2.8053	5.5399	-5.6992	8.3811	0.0176	0.485	0.1439	-0.4752	0.619	0.4906	0.2087	-0.3591	-0.71	0.3783	-0.056	-0.0361	0.4987	0.0659	0.3731	5.8635	-0.057	38.2167
8	0.356	-0.6598	-0.5113	2.3054	1.3437	0.7181	-0.1248	0.2229	-0.0563	0.3732	-0.1898	0.7907	0.1403	-0.2823	0.2945	0.4469	0.0977	0.0464	0.4955	-0.0159	0.7161	-13.435	3.202	77.2474
9	5.2426	-0.2732	0.9009	6.9248	-0.0942	8.8955	0.1562	0.4139	-0.1808	-0.7451	0.1131	0.2962	0.4204	-0.4601	-0.2021	0.3808	-0.0349	-0.0067	0.3758	0.0651	0.3297	9.5924	1.516	36.5386

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 1 through 179 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 180 through 449 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 450 through 984 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 1 through 20 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 21 through 40 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 41 through 93 )
7	X-RAY DIFFRACTION	7	chain 'C' and (resid -7 through 2 )
8	X-RAY DIFFRACTION	8	chain 'C' and (resid 3 through 20 )
9	X-RAY DIFFRACTION	9	chain 'D' and (resid 1 through 8 )

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DNA chain , 2 types, 2 molecules CD

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Protein / RNA chain , 2 types, 2 molecules AB

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Non-polymers , 2 types, 276 molecules

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