PDB-6joo: Crystal structure of Corynebacterium diphtheriae Cas9 in complex ...

ID or keywords:

Entry

Database: PDB / ID: 6joo

Title

Crystal structure of Corynebacterium diphtheriae Cas9 in complex with sgRNA and target DNA

Components

CRISPR-associated protein,CRISPR-associated endonuclease Cas9
Guide RNA
Non-Target DNA
Target DNA

Keywords

HYDROLASE/DNA/RNA / CRISPR-Cas / Cas9 / PAM / DNA endonuclease / Ribonucleoprotein / HYDROLASE-DNA-RNA complex

Function / homology

Function and homology information

endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function

Biological species

Corynebacterium diphtheriae (bacteria)

Corynebacterium diphtheriae NCTC 13129 (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

SAD / Resolution: 2.9 Å

Authors

Hirano, S. / Ishitani, R. / Nishimasu, H. / Nureki, O.

Citation

Journal: Nat Commun / Year: 2019
Title: Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.
Authors: Hirano, S. / Abudayyeh, O.O. / Gootenberg, J.S. / Horii, T. / Ishitani, R. / Hatada, I. / Zhang, F. / Nishimasu, H. / Nureki, O.

History

Deposition	Mar 22, 2019	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Apr 17, 2019	Provider: repository / Type: Initial release
Revision 1.1	May 15, 2019	Group: Data collection / Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

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Data in CIF	6joo_validation.cif.gz	46.1 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/jo/6joo ftp://data.pdbj.org/pub/pdb/validation_reports/jo/6joo	HTTPS FTP

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Related structure data

Similar structure data	5x2g-d 10634 6wbr-d 3zrq-1 3zrr-1 1kv3-3 6a2p-d 2z9u-1 2gv9-2 4our-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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wwPDB /

NCBI

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Deposited unit

A: CRISPR-associated protein,CRISPR-associated endonuclease Cas9

B: Guide RNA

D: Non-Target DNA

C: Target DNA

hetero molecules

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

1

Idetical with deposited unit
defined by author&software
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	139.015, 118.999, 116.272
Angle α, β, γ (deg.)	90.000, 113.590, 90.000
Int Tables number	5
Space group name H-M	C121

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DNA chain , 2 types, 2 molecules DC

#3: DNA chain	Non-Target DNA Mass: 2506.665 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: chemical synthesis / Source: (synth.) Corynebacterium diphtheriae (bacteria)
#4: DNA chain	Target DNA Mass: 8430.446 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: chemical synthesis / Source: (synth.) Corynebacterium diphtheriae (bacteria)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein	CRISPR-associated protein,CRISPR-associated endonuclease Cas9 / SaCas9 Mass: 103966.180 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Corynebacterium diphtheriae (bacteria), (gene. exp.) Corynebacterium diphtheriae NCTC 13129 (bacteria) Gene: Cas9 / Plasmid: pE-SUMO / Strain: NCTC 13129 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): B834 References: UniProt: A0A2T1BQ76, UniProt: Q6NKI3, Hydrolases; Acting on ester bonds
#2: RNA chain	Guide RNA Mass: 36321.402 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription / Source: (synth.) Corynebacterium diphtheriae (bacteria)

#1: Protein

CRISPR-associated protein,CRISPR-associated endonuclease Cas9 / SaCas9

Mass: 103966.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Corynebacterium diphtheriae (bacteria), (gene. exp.)

Corynebacterium diphtheriae NCTC 13129 (bacteria)
Gene: Cas9 / Plasmid: pE-SUMO / Strain: NCTC 13129 / Production host:

Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): B834
References: UniProt: A0A2T1BQ76, UniProt: Q6NKI3, Hydrolases; Acting on ester bonds

#2: RNA chain

Guide RNA

Mass: 36321.402 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription / Source: (synth.)

Corynebacterium diphtheriae (bacteria)

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Non-polymers , 3 types, 6 molecules

#5: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₂H₆O₂
#6: Chemical	ChemComp-ZN / ZINC ION Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 3 / 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: 2.92 Å³/Da / Density % sol: 57.89 % / Mosaicity: 0 °
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 9 Details: 24% PEG 3350, 0.3 M potassium fluoride, 0.2 M lithium sulfate, 0.1 M TRIS hydrochloride

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

SPring-8

/ Beamline: BL41XU / Wavelength: 0.979 Å

Detector

Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Oct 31, 2015

Radiation

Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.979 Å / Relative weight: 1

Reflection

Resolution: 2.9→106.56 Å / Num. obs: 38524 / % possible obs: 100 % / Redundancy: 13.4 % / CC_1/2: 0.999 / R_merge(I) obs: 0.168 / R_pim(I) all: 0.047 / R_rim(I) all: 0.175 / Net I/σ(I): 10.2 / Num. measured all: 515857 / Scaling rejects: 322

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Num. unique obs	CC_1/2	R_pim(I) all	R_rim(I) all	% possible all
2.9-3.03	13.5	3.024	4704	0.802	0.844	3.142	100
10.05-106.56	12.7	0.071	962	0.999	0.021	0.074	99.6

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Processing

Software

Name	Version	Classification
Aimless	0.5.32	data scaling
PHENIX	1.14_3260	refinement
PDB_EXTRACT	3.24	data extraction
DIALS		data reduction
AutoSol		phasing

Refinement

Method to determine structure:

SAD / Resolution: 2.9→67.945 Å / SU ML: 0.51 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 31.64
Details: SF FILE CONTAINS FRIEDEL PAIRS UNDER I/F_MINUS AND I/F_PLUS COLUMNS.

	R_factor	Num. reflection	% reflection
R_free	0.2538	3928	5.21 %
R_work	0.2206	-	-
obs	0.2223	38462	99.56 %

Solvent computation

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

Displacement parameters

B_iso max: 392.74 Å² / B_iso mean: 114.8302 Å² / B_iso min: 28.69 Å²

Refinement step

Cycle: final / Resolution: 2.9→67.945 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6292	2755	9	3	9059
B_iso mean	-	-	89.05	40.08	-
Num. residues	-	-	-	-	966

LS refinement shell

Refine-ID: X-RAY DIFFRACTION / R_factor R_free error: 0 / Total num. of bins used: 28

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Num. reflection all	% reflection obs (%)
2.9-2.9354	0.4669	151	0.4237	2524	2675	98
2.9354-2.9726	0.4718	154	0.4053	2531	2685	99
2.9726-3.0117	0.4429	141	0.3618	2482	2623	99
3.0117-3.0529	0.4344	128	0.3361	2605	2733	99
3.0529-3.0966	0.3805	168	0.3312	2460	2628	99
3.0966-3.1428	0.4053	170	0.3211	2564	2734	99
3.1428-3.1919	0.3372	119	0.309	2521	2640	99
3.1919-3.2442	0.3269	119	0.2954	2574	2693	100
3.2442-3.3002	0.3012	134	0.2747	2567	2701	99
3.3002-3.3602	0.3317	146	0.2621	2569	2715	100
3.3602-3.4248	0.3072	124	0.2714	2559	2683	100
3.4248-3.4947	0.2781	134	0.2616	2556	2690	100
3.4947-3.5707	0.2687	161	0.2549	2491	2652	99
3.5707-3.6537	0.2733	143	0.2352	2618	2761	100
3.6537-3.7451	0.2859	151	0.2312	2505	2656	100
3.7451-3.8464	0.1847	159	0.2166	2534	2693	100
3.8464-3.9595	0.211	132	0.219	2607	2739	100
3.9595-4.0873	0.2557	130	0.204	2517	2647	100
4.0873-4.2334	0.2523	110	0.2018	2574	2684	100
4.2334-4.4028	0.2457	157	0.1928	2572	2729	100
4.4028-4.6032	0.1782	145	0.1798	2568	2713	100
4.6032-4.8458	0.2276	132	0.1829	2583	2715	100
4.8458-5.1493	0.2147	131	0.1776	2540	2671	100
5.1493-5.5467	0.216	141	0.1927	2557	2698	100
5.5467-6.1046	0.2672	141	0.2009	2593	2734	100
6.1046-6.9872	0.2584	121	0.2157	2578	2699	100
6.9872-8.8001	0.1942	121	0.1949	2565	2686	100
8.8001-67.9635	0.2365	165	0.1998	2527	2692	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	0.5088	0.2582	0.2699	0.0752	0.1089	-0.1313	-0.1344	0.2965	-0.4079	-0.4389	0.3646	-0.4971	0.1395	0.0128	0.0069	0.8406	0.0939	0.0161	0.575	-0.1411	0.8098	8.7298	-27.0626	28.1817
2	0.3694	0.3299	0.1139	0.2419	-0.0573	0.2729	0.1607	0.2061	0.4157	-0.2423	-0.0502	-0.1803	-0.0612	0.1611	0.126	0.6595	0.0496	0.1267	0.4828	0.3306	0.8359	3.1135	30.8357	21.5219
3	0.4407	0.0935	-0.2499	0.2551	0.2172	0.2733	0.6325	0.2772	0.0164	-1.0027	-0.1459	-0.9546	-0.8833	0.1671	0.4997	0.8308	0.2551	-0.0156	0.4558	0.9416	0.6847	2.946	33.3433	15.3991
4	-0.1112	-0.0485	-0.0656	0.0461	0.3237	0.1968	-0.0362	0.3363	0.2529	-0.3731	-0.1185	-0.236	-0.0202	0.349	-0.1405	0.8405	0.0818	0.3982	1.0348	0.2618	0.7334	28.3802	5.7007	4.4073
5	1.735	-0.4217	-0.4205	1.3884	0.5475	0.6148	-0.0107	-0.0447	0.0049	-0.0763	0.0739	-0.0843	0.0944	0.0432	0.0002	0.3886	0.0462	-0.064	0.3361	0.0171	0.3038	-0.4208	-15.4857	41.1286
6	0.8234	0.7651	-0.0364	1.0787	-0.8163	0.492	0.0711	0.9422	0.6278	-0.1003	0.0166	0.027	0.1173	-0.6239	-0.0013	0.788	0.0305	-0.0018	1.0599	0.1712	0.7093	3.6516	8.7993	19.1179
7	0.0223	-0.0176	-0.0371	-0.0385	0.0128	0.0117	-0.3731	0.3087	0.6912	0.504	0.3673	-0.2687	0.1854	-0.7588	-0.0004	1.376	0.4996	0.335	1.5844	0.1997	2.1079	-28.0767	32.0484	41.2195
8	0.1159	0.2671	-0.3949	0.3689	0.4376	0.971	0.1517	0.0999	0.3429	0.1079	0.0627	0.1811	-0.237	0.1646	0.0001	0.7415	-0.0262	0.0946	0.7156	-0.0117	1.0763	6.2663	19.6511	43.0014
9	-0.0139	0.1408	-0.0475	0.3268	-0.0441	0.2282	0.2527	-0.0297	0.4472	-0.2206	-0.0582	-0.4181	-0.0462	0.2907	0.0001	0.7875	-0.1402	0.0097	0.8869	-0.01	1.0497	11.6978	12.7386	46.4198
10	0.2624	-0.1374	0.1864	-0.025	0.234	0.4898	-0.1513	-0.2282	0.2457	-0.194	0.219	-0.8463	-0.1418	0.1929	-0.0001	0.6969	0.0592	0.0543	0.8612	-0.0483	1.1097	22.8783	-11.1378	40.1116
11	0.0855	-0.1165	0.0402	0.1537	-0.0419	-0.0452	0.0453	-0.3258	0.4407	-0.4312	0.2014	0.395	0.269	-0.4095	-0.0003	0.7502	-0.0137	-0.0663	0.8382	0.0735	0.8219	-17.661	-5.76	42.9674
12	0.0374	0.0729	0.0366	0.0822	0.041	0.0147	0.0913	-0.1643	-0.0084	-0.6669	-0.2066	0.3704	-0.2297	0.3315	-0.0003	0.9189	-0.0166	-0.175	0.951	0.1036	1.0301	-21.6664	-12.5517	44.1581
13	-0.011	0.0056	-0.0194	-0.0091	0.0348	0.0807	-0.0029	-0.1369	-0.0482	-0.5304	-0.1709	0.2734	-0.0746	-0.207	0	0.9236	0.0458	-0.0301	0.928	0.1616	0.8536	-12.7846	3.3152	32.1897
14	0.0386	-0.0445	0.0453	0.0033	-0.0284	0.0322	0.0853	0.3087	0.0685	0.0288	-0.2098	0.0559	0.8908	-0.1625	-0	1.3281	-0.0047	0.1136	1.3353	0.0979	0.9187	2.8835	10.5901	14.5549
15	-0.0037	0.0012	-0.0045	0.0169	-0.0148	0.0434	0.2787	0.1435	0.3392	-0.2787	0.0597	0.0141	0.1732	-0.195	-0.0002	1.3885	-0.0167	0.267	1.3869	-0.1053	1.1542	15.7069	-2.6867	12.5074
16	0.004	0.0153	0.0056	0.0147	0.0158	0.0028	0.1359	0.091	-0.0418	-0.1508	0.0004	-0.1588	0.1947	0.084	0	2.232	-0.0027	0.0394	1.9347	-0.4824	1.468	14.0981	-13.6335	-2.583
17	0.0022	-0.0051	0.0053	0.0146	-0.011	0.0138	-0.0742	0.224	0.0347	0.0586	-0.0344	-0.1613	0.1381	0.0927	-0	2.8342	0.3513	0.418	2.0318	-0.2756	2.0921	28.2145	-18.3378	-4.5362

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	chain 'A' and (resid -1 through 52 )	A	-1 - 52
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 53 through 181 )	A	53 - 181
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 182 through 231 )	A	182 - 231
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 232 through 466 )	A	232 - 466
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 467 through 1084 )	A	467 - 1084
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 1 through 30 )	B	1 - 30
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 31 through 40 )	B	31 - 40
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 41 through 60 )	B	41 - 60
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 61 through 75 )	B	61 - 75
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 76 through 113 )	B	76 - 113
11	X-RAY DIFFRACTION	11	chain 'D' and (resid 5 through 12 )	D	5 - 12
12	X-RAY DIFFRACTION	12	chain 'C' and (resid 1 through 5 )	C	1 - 5
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 6 through 10 )	C	6 - 10
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 11 through 15 )	C	11 - 15
15	X-RAY DIFFRACTION	15	chain 'C' and (resid 16 through 20 )	C	16 - 20
16	X-RAY DIFFRACTION	16	chain 'C' and (resid 21 through 25 )	C	21 - 25
17	X-RAY DIFFRACTION	17	chain 'C' and (resid 26 through 28 )	C	26 - 28

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