PDB-4un3: Crystal structure of Cas9 bound to PAM-containing DNA target

ID or keywords:

Entry

Database: PDB / ID: 4un3

Title

Crystal structure of Cas9 bound to PAM-containing DNA target

Components

CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1
NON-TARGET DNA STRAND
SGRNASubgenomic mRNA
TARGET DNA STRAND

Keywords

HYDROLASE/DNA/RNA / HYDROLASE-DNA-RNA COMPLEX /

CRISPR /

GENOME EDITING / RNP / 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

Biological species

STREPTOCOCCUS PYOGENES (bacteria)
SYNTHETIC CONSTRUCT (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

SAD / Resolution: 2.593 Å

Authors

Anders, C. / Niewoehner, O. / Duerst, A. / Jinek, M.

Citation

Journal: Nature / Year: 2014
Title: Structural Basis of Pam-Dependent Target DNA Recognition by the Cas9 Endonuclease
Authors: Anders, C. / Niewoehner, O. / Duerst, A. / Jinek, M.

History

Deposition	May 25, 2014	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Jul 23, 2014	Provider: repository / Type: Initial release
Revision 1.1	Aug 13, 2014	Group: Database references
Revision 1.2	Oct 1, 2014	Group: Database references

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	4un3.cif.gz	666.1 KB	Display	PDBx/mmCIF format
PDB format	pdb4un3.ent.gz	559.5 KB	Display	PDB format
PDBx/mmJSON format	4un3.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/un/4un3 ftp://data.pdbj.org/pub/pdb/validation_reports/un/4un3	HTTPS FTP

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Related structure data

Related structure data	4un4C 4un5C C: citing same article (ref.)
Similar structure data	5b2t-d 6k57-d 5fw2-d 6k4q-d 5fw3-d 6aeg-d 5b2r-d 4un4-d 6k4p-d 5fq5-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

PDBj /

wwPDB /

NCBI

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Deposited unit

A: SGRNA

B: CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1

C: TARGET DNA STRAND

D: NON-TARGET DNA STRAND

hetero molecules

198 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)	177.723, 68.142, 188.230
Angle α, β, γ (deg.)	90.00, 111.17, 90.00
Int Tables number	5
Space group name H-M	C121

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain	TARGET DNA STRAND Mass: 8552.563 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#4: DNA chain	NON-TARGET DNA STRAND Mass: 3725.465 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain	SGRNA / Subgenomic mRNA Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#2: Protein	CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1 / CAS9 Mass: 158919.047 Da / Num. of mol.: 1 / Mutation: YES Source method: isolated from a genetically manipulated source Source: (gene. exp.) STREPTOCOCCUS PYOGENES (bacteria) / Strain: M1 / Plasmid: PEC-K-MBP / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA 2 References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

#1: RNA chain

SGRNA /

Subgenomic mRNA

Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

#2: Protein

CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1 / CAS9

Mass: 158919.047 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

STREPTOCOCCUS PYOGENES (bacteria) / Strain: M1 / Plasmid: PEC-K-MBP / Production host:

ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): ROSETTA 2
References: UniProt: Q99ZW2,

Hydrolases; Acting on ester bonds

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Non-polymers , 2 types, 413 molecules

#5: Chemical	ChemComp-MG / MAGNESIUM ION Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#6: Water	ChemComp-HOH / water / Water Mass: 18.015 Da / Num. of mol.: 405 / Source method: isolated from a natural source / Formula: H₂O

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Details

Sequence details	N-TERMINAL GAAS SEQUENCE IS DERIVED FROM THE EXPRESSION VECTOR. H840A MUTATION WAS INTRODUCED TO ...N-TERMINAL GAAS SEQUENCE IS DERIVED FROM THE EXPRESSION VECTOR. H840A MUTATION WAS INTRODUCED TO INACTIVATE THE HNH DOMAIN.

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.68 Å³/Da / Density % sol: 54.1 % / Description: NONE
Crystal grow	pH: 8.5 Details: 0.1 M TRIS-ACETATE PH 8.5, 0.3 M KSCN, 15% (W/V) PEG 3350

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Data collection

Diffraction	Mean temperature: 100 K
Diffraction source	Source: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1
Detector	Type: DECTRIS PILATUS / Detector: PIXEL / Date: Nov 28, 2013
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.59→48.15 Å / Num. obs: 126432 / % possible obs: 99.3 % / Observed criterion σ(I): 2 / Redundancy: 6.8 % / R_merge(I) obs: 0.11 / Net I/σ(I): 16.3
Reflection shell	Resolution: 2.59→2.69 Å / Redundancy: 6.3 % / R_merge(I) obs: 0.75 / Mean I/σ(I) obs: 2.4 / % possible all: 93.5

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Processing

Software

Name	Version	Classification
PHENIX	(PHENIX.REFINE: 1.8.2_1309)	refinement
XDS		data reduction
SCALA		data scaling
autoSHARP		phasing

Refinement

Method to determine structure:

SAD
Starting model: NONE

Resolution: 2.593→48.154 Å / SU ML: 0.39 / σ(F): 1.01 / Phase error: 28.73 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.252	6332	5 %
R_work	0.2171	-	-
obs	0.2189	65224	98.97 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL

Refinement step

Cycle: LAST / Resolution: 2.593→48.154 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10690	2526	8	405	13629

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.01	13718
X-RAY DIFFRACTION	f_angle_d	0.779	19008
X-RAY DIFFRACTION	f_dihedral_angle_d	19.134	5552
X-RAY DIFFRACTION	f_chiral_restr	0.052	2171
X-RAY DIFFRACTION	f_plane_restr	0.002	1990

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 (%)
2.5927-2.6222	0.3791	160	0.3387	3040	X-RAY DIFFRACTION	74
2.6222-2.653	0.366	216	0.3132	4030	X-RAY DIFFRACTION	100
2.653-2.6854	0.3459	203	0.3036	3909	X-RAY DIFFRACTION	100
2.6854-2.7193	0.393	218	0.304	4197	X-RAY DIFFRACTION	100
2.7193-2.7551	0.3829	208	0.2958	3964	X-RAY DIFFRACTION	100
2.7551-2.7929	0.3347	214	0.2903	4067	X-RAY DIFFRACTION	100
2.7929-2.8328	0.3445	217	0.2949	4084	X-RAY DIFFRACTION	100
2.8328-2.875	0.3149	213	0.281	4018	X-RAY DIFFRACTION	100
2.875-2.92	0.3335	211	0.2827	3985	X-RAY DIFFRACTION	100
2.92-2.9678	0.371	220	0.3067	4093	X-RAY DIFFRACTION	100
2.9678-3.019	0.3431	210	0.2993	4001	X-RAY DIFFRACTION	100
3.019-3.0739	0.3294	217	0.282	4087	X-RAY DIFFRACTION	100
3.0739-3.133	0.3144	210	0.2558	3989	X-RAY DIFFRACTION	100
3.133-3.1969	0.2836	209	0.2502	4026	X-RAY DIFFRACTION	100
3.1969-3.2664	0.2577	218	0.229	4073	X-RAY DIFFRACTION	100
3.2664-3.3424	0.259	214	0.2285	4005	X-RAY DIFFRACTION	100
3.3424-3.426	0.2966	211	0.2314	4059	X-RAY DIFFRACTION	100
3.426-3.5186	0.2418	214	0.229	4047	X-RAY DIFFRACTION	100
3.5186-3.6221	0.2568	217	0.2177	3998	X-RAY DIFFRACTION	100
3.6221-3.7389	0.2663	210	0.2076	4021	X-RAY DIFFRACTION	100
3.7389-3.8725	0.1916	213	0.1918	4061	X-RAY DIFFRACTION	100
3.8725-4.0275	0.2054	211	0.1822	4066	X-RAY DIFFRACTION	100
4.0275-4.2107	0.2408	214	0.186	4016	X-RAY DIFFRACTION	100
4.2107-4.4325	0.1918	213	0.1815	4016	X-RAY DIFFRACTION	100
4.4325-4.71	0.2197	211	0.1691	4073	X-RAY DIFFRACTION	100
4.71-5.0733	0.202	211	0.1661	4022	X-RAY DIFFRACTION	100
5.0733-5.5832	0.2163	215	0.1824	4081	X-RAY DIFFRACTION	100
5.5832-6.3895	0.2484	213	0.1945	4018	X-RAY DIFFRACTION	100
6.3895-8.044	0.2031	214	0.1941	4033	X-RAY DIFFRACTION	100
8.044-48.1619	0.1962	207	0.1853	4022	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	0.0908	0.0816	0.0037	0.0702	-0.0055	0.1209	0.0165	0.0316	-0.0146	0.0247	-0.0268	-0.0366	0.0024	0.1257	-0.0036	-0.0256	-0.1443	-0.0395	0.1632	-0.025	0.0314	95.2594	63.6119	37.4695
2	0.0105	0.0011	-0.0006	0.0094	-0.0056	0.0051	-0.0024	0.039	-0.0273	0.0232	-0.0311	0.039	-0.035	-0.0038	0	0.5292	-0.0396	0.0182	0.3181	-0.0806	0.5123	114.6766	56.0962	66.2952
3	0.1885	-0.1065	0.0232	0.2769	0.0664	0.3458	-0.0087	-0.0443	-0.0245	0.1216	0.0226	-0.0411	0.0393	0.1886	-0.0699	0.1398	-0.0147	-0.0915	0.092	-0.0037	0.1421	100.0829	64.6393	55.117
4	0.0761	0.0544	0.0471	0.1312	-0.0291	0.1193	-0.0098	0.023	-0.0805	0.1253	0.0618	-0.0553	0.0586	-0.0065	0.0362	0.1805	0.0397	-0.0246	0.0622	-0.0395	0.1432	82.2163	66.4306	66.9158
5	0.0194	0.0008	0.0032	0.0115	-0.0024	0.0129	-0.0209	-0.0934	-0.0802	0.1264	-0.0325	0.0231	0.1004	-0.0511	0	0.3964	0.026	-0.0605	0.3176	-0.0038	0.3785	83.9835	38.6548	72.9726
6	0.0324	0.0012	0.0053	0.0324	0.0096	0.0194	0.0141	-0.0467	-0.0148	0.0503	-0.0344	-0.0554	0.0298	-0.0241	0.0039	0.1156	-0.0329	0.0291	0.0743	0.0009	0.1182	55.5236	50.9213	62.3258
7	0.0185	0.0191	-0.0024	0.0613	0.0568	0.0492	-0.0746	0.0158	-0.0777	0.1344	-0.0229	0.0885	0.2225	-0.1059	-0.1724	0.0121	-0.3033	0.0057	0.0415	-0.072	0.0743	60.5735	44.2021	37.1524
8	0.0643	-0.0417	0.0463	0.099	-0.0757	0.0549	0.0219	0.0311	0.0415	0.029	0.0504	-0.0039	0.0808	0.0397	0.0241	0.1525	-0.0029	0.0188	0.1895	-0.0582	0.1305	92.5613	60.0417	42.5298
9	0.0012	-0.0011	-0.0002	0.0019	0.0008	0.0011	0.0075	0.0167	-0.0039	-0.0165	0.0038	0.0485	-0.0008	-0.0043	0.0001	0.6172	0.0727	-0.0683	0.5789	-0.1161	0.4901	109.7609	38.6731	2.1638
10	0.0766	-0.0094	0.066	0.0452	0.0236	0.109	0.0156	0.0469	0.0135	-0.0218	0.0223	0.0098	0.0225	0.0644	0.0706	0.0869	-0.0523	-0.0176	0.1324	-0.0219	0.1457	85.7157	66.9021	29.9779
11	0.004	0.0039	-0.0001	0.0027	-0.0004	0.0001	-0.009	-0.0137	0.0316	0.0053	0.0018	-0.0377	-0.0245	0.0367	-0.0001	0.7267	-0.035	0.032	0.6894	0.0275	0.7331	50.9252	72.2987	36.7413
12	0.1729	0.0103	0.1076	0.0739	-0.0134	0.0729	0.0153	0.0845	-0.1901	-0.1091	0.0086	0.1936	0.1289	-0.0969	0.0131	0.4597	-0.0813	-0.0378	0.4863	-0.143	0.3685	79.5108	42.4677	25.1308
13	0.029	-0.0146	0.0031	0.0386	0.0259	0.0291	-0.0095	-0.0734	0.0058	0.0608	0.0722	0.0433	0.0066	-0.0156	0.0119	0.2348	-0.0379	0.0182	0.2249	0.0322	0.2063	81.5588	62.553	65.6599
14	0.02	-0.002	-0.0026	0.0248	-0.0131	0.0065	0.0106	-0.028	-0.2096	0.0238	0.0007	-0.0786	0.112	0.0553	-0.0001	0.5127	-0.041	0.0433	0.479	-0.1029	0.3869	77.6309	43.1683	25.8268

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	CHAIN 'B' AND (RESID 4 THROUGH 207 )
2	X-RAY DIFFRACTION	2	CHAIN 'B' AND (RESID 208 THROUGH 299 )
3	X-RAY DIFFRACTION	3	CHAIN 'B' AND (RESID 300 THROUGH 601 )
4	X-RAY DIFFRACTION	4	CHAIN 'B' AND (RESID 602 THROUGH 829 )
5	X-RAY DIFFRACTION	5	CHAIN 'B' AND (RESID 830 THROUGH 909 )
6	X-RAY DIFFRACTION	6	CHAIN 'B' AND (RESID 910 THROUGH 1000 )
7	X-RAY DIFFRACTION	7	CHAIN 'B' AND (RESID 1001 THROUGH 1364 )
8	X-RAY DIFFRACTION	8	CHAIN 'A' AND (RESID 1 THROUGH 32 )
9	X-RAY DIFFRACTION	9	CHAIN 'A' AND (RESID 33 THROUGH 40 )
10	X-RAY DIFFRACTION	10	CHAIN 'A' AND (RESID 41 THROUGH 70 )
11	X-RAY DIFFRACTION	11	CHAIN 'A' AND (RESID 71 THROUGH 81 )
12	X-RAY DIFFRACTION	12	CHAIN 'C' AND (RESID 1 THROUGH 10 )
13	X-RAY DIFFRACTION	13	CHAIN 'C' AND (RESID 11 THROUGH 28 )
14	X-RAY DIFFRACTION	14	CHAIN 'D' AND (RESID 2 THROUGH 12 )

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