PDB-5x2g: Crystal structure of Campylobacter jejuni Cas9 in complex with sg...

ID or keywords:

Entry

Database: PDB / ID: 5x2g

Title

Crystal structure of Campylobacter jejuni Cas9 in complex with sgRNA and target DNA (AGAAACC 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: 2.4 Å

Authors

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

Funding support

Japan, 2items

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	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	5x2g.cif.gz	466.3 KB	Display	PDBx/mmCIF format
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Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/x2/5x2g ftp://data.pdbj.org/pub/pdb/validation_reports/x2/5x2g	HTTPS FTP

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Related structure data

Related structure data	5x2hC C: citing same article (ref.)
Similar structure data	5x2h-d 6joo-d 5n2e-d 4az5-1 1358 6a2p-d 2fm1-d 5n2g-d 1lw4-d 5n2h-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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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)	104.345, 105.094, 136.521
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: 8492.467 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	Non-target DNA strand Mass: 2444.645 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, 320 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.: 319 / 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: 2.72 Å³/Da / Density % sol: 54.73 %
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: SLS / Beamline: X06SA / Wavelength: 1 Å
Detector	Type: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Aug 28, 2015
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.4→136.52 Å / Num. obs: 59407 / % possible obs: 100 % / Redundancy: 12.9 % / CC_1/2: 0.998 / R_pim(I) all: 0.043 / Net I/σ(I): 13.8
Reflection shell	CC_1/2: 0.757 / R_pim(I) all: 0.529

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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: 2.4→52.547 Å / SU ML: 0.28 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 24.56

	R_factor	Num. reflection	% reflection
R_free	0.2213	2833	4.78 %
R_work	0.1895	-	-
obs	0.191	59303	99.91 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 2.4→52.547 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5929	2715	4	319	8967

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.002	9075
X-RAY DIFFRACTION	f_angle_d	0.498	12834
X-RAY DIFFRACTION	f_dihedral_angle_d	15.884	5123
X-RAY DIFFRACTION	f_chiral_restr	0.035	1515
X-RAY DIFFRACTION	f_plane_restr	0.002	1159

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 (%)
2.4-2.4414	0.348	134	0.3066	2797	X-RAY DIFFRACTION	100
2.4414-2.4858	0.3231	148	0.2747	2788	X-RAY DIFFRACTION	100
2.4858-2.5336	0.2771	141	0.2717	2763	X-RAY DIFFRACTION	100
2.5336-2.5853	0.3076	140	0.2609	2783	X-RAY DIFFRACTION	100
2.5853-2.6416	0.3053	151	0.2459	2785	X-RAY DIFFRACTION	100
2.6416-2.703	0.2897	118	0.2474	2815	X-RAY DIFFRACTION	100
2.703-2.7706	0.3086	152	0.2403	2796	X-RAY DIFFRACTION	100
2.7706-2.8455	0.2876	129	0.2458	2801	X-RAY DIFFRACTION	100
2.8455-2.9292	0.3161	145	0.2489	2791	X-RAY DIFFRACTION	100
2.9292-3.0238	0.3002	156	0.2257	2805	X-RAY DIFFRACTION	100
3.0238-3.1318	0.2555	139	0.2115	2801	X-RAY DIFFRACTION	100
3.1318-3.2572	0.2058	136	0.1982	2819	X-RAY DIFFRACTION	100
3.2572-3.4054	0.2049	143	0.189	2795	X-RAY DIFFRACTION	100
3.4054-3.5849	0.2377	155	0.193	2823	X-RAY DIFFRACTION	100
3.5849-3.8095	0.2167	127	0.1747	2827	X-RAY DIFFRACTION	100
3.8095-4.1035	0.1611	163	0.1586	2825	X-RAY DIFFRACTION	100
4.1035-4.5162	0.1718	147	0.1407	2845	X-RAY DIFFRACTION	100
4.5162-5.1693	0.1909	142	0.137	2859	X-RAY DIFFRACTION	100
5.1693-6.5108	0.1939	100	0.1692	2960	X-RAY DIFFRACTION	100
6.5108-52.5596	0.1721	167	0.1637	2992	X-RAY DIFFRACTION	99

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	0.8435	0.4973	-0.0733	1.8583	0.2651	1.9893	0.1439	-0.0447	-0.2296	0.3495	0.024	0.153	0.4814	0.0236	-0.1415	0.2958	0.0731	-0.0306	0.3173	0.0051	0.2869	6.0573	-16.5327	65.302
2	0.8727	-0.4659	-0.6807	1.6861	0.8988	1.2929	-0.0196	-0.2399	0.0032	0.4325	0.1082	0.493	0.1404	-0.0809	-0.1075	0.4939	0.0706	0.0542	0.4737	0.016	0.4915	-7.5639	4.7532	82.151
3	1.7886	-0.0987	0.178	2.2705	-0.2357	2.2575	0.0076	0.1693	0.2257	-0.1315	0.1329	-0.1212	-0.2454	0.1846	-0.1216	0.2467	-0.0526	0.0332	0.3105	0.0116	0.2399	15.9019	13.6742	43.4627
4	0.7916	-0.3781	-0.2133	2.4645	1.2615	1.9431	-0.0412	0.1341	-0.1119	0.1989	-0.0432	0.3905	0.2551	-0.0961	0.0875	0.2831	0.0903	-0.0049	0.2985	0.0082	0.3551	-0.8567	-8.7539	67.2151
5	0.9023	0.3996	-0.1466	0.2486	-0.3041	1.5636	0.0678	-0.0566	-0.1665	0.2023	0.0574	-0.1616	0.191	0.5104	-0.1933	0.3028	0.1508	-0.1079	0.4511	-0.0392	0.3022	18.0931	-10.0948	61.9978
6	1.6307	-0.3039	0.26	2.9392	-0.6099	1.2248	0.2314	-0.4638	-0.1551	0.4015	0.0323	0.037	-0.0849	0.5543	-0.1299	0.5372	0.0876	-0.1482	0.6769	-0.0395	0.2733	20.9999	-1.7451	81.9603
7	3.7858	0.3002	-2.2986	5.1337	-2.3311	5.4758	0.1563	-0.1571	0.734	-0.575	0.6754	1.0345	0.4251	-0.5689	-0.7812	0.2999	-0.0625	-0.0113	0.5697	0.1171	0.348	8.9191	7.2528	29.9125
8	0.561	0.342	-0.7757	3.5886	-3.5952	4.4365	-0.3021	0.2376	-0.2931	-0.3218	0.2419	0.4028	0.2204	0.0022	0.0426	0.3548	-0.0549	-0.0109	0.3191	0.0554	0.3389	4.8517	-6.0648	46.7432
9	0.1178	-0.06	-0.3666	0.1137	0.3284	0.9879	-0.1188	0.2332	0.1386	0.131	-0.1645	0.7384	0.06	-0.3355	0.2939	0.3499	0.1177	0.0665	0.4574	-0.0631	0.6549	-11.423	0.5174	74.8123
10	0.018	-0.2476	-0.0191	3.553	0.5115	1.097	-0.2702	-0.0606	-0.3935	0.1612	-0.1933	0.5351	0.1086	-0.2384	0.1475	0.4127	0.2275	0.1115	0.478	-0.0313	1.3289	-23.9646	19.2735	89.5516
11	3.3698	-0.084	-0.6003	3.722	-0.2441	6.6726	-0.0003	0.3644	-0.0171	-0.4148	0.2634	0.4662	0.755	-0.4373	-0.19	0.3136	-0.0617	0.0308	0.3821	0.052	0.2625	10.4936	1.8895	37.032

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 715 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 716 through 984 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 1 through 30 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 31 through 80 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 81 through 93 )
7	X-RAY DIFFRACTION	7	chain 'C' and (resid -7 through -3 )
8	X-RAY DIFFRACTION	8	chain 'C' and (resid -2 through 2 )
9	X-RAY DIFFRACTION	9	chain 'C' and (resid 3 through 17 )
10	X-RAY DIFFRACTION	10	chain 'C' and (resid 18 through 20 )
11	X-RAY DIFFRACTION	11	chain 'D' and (resid 1 through 8 )

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DNA chain , 2 types, 2 molecules CD

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Non-polymers , 2 types, 320 molecules

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Assembly

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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, 320 molecules

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Experimental details

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Experiment

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Sample preparation

-
Data collection

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Processing

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About Yorodumi

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Feb 9, 2022. New format data for meta-information of EMDB entries

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Aug 12, 2020. Covid-19 info

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Mar 5, 2020. Novel coronavirus structure data

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Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

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Jul 12, 2017. Major update of PDB

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Yorodumi