PDB-6k4u: Crystal structure of xCas9 in complex with sgRNA and DNA (TGA PAM)

ID or keywords:

Entry

Database: PDB / ID: 6k4u

Title

Crystal structure of xCas9 in complex with sgRNA and DNA (TGA PAM)

Components

CRISPR-associated endonuclease Cas9
non-target DNA
sgRNA
target DNA

Keywords

HYDROLASE/DNA / endonuclease / HYDROLASE-DNA 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 serotype M1 (bacteria)

Streptococcus pyogenes (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 3.2 Å

Authors

Chen, W. / Zhang, H. / Zhang, Y. / Wang, Y. / Gan, J. / Ji, Q.

Funding support

China, 1items

Citation

Journal: Plos Biol. / Year: 2019
Title: Molecular basis for the PAM expansion and fidelity enhancement of an evolved Cas9 nuclease.
Authors: Chen, W. / Zhang, H. / Zhang, Y. / Wang, Y. / Gan, J. / Ji, Q.

History

Deposition	May 27, 2019	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Sep 25, 2019	Provider: repository / Type: Initial release
Revision 1.1	Nov 13, 2019	Group: Database references / Category: citation / citation_author Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2	Nov 22, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/k4/6k4u ftp://data.pdbj.org/pub/pdb/validation_reports/k4/6k4u	HTTPS FTP

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Related structure data

Related structure data	6k3zC 6k4pC 6k4qC 6k4sC 6k57C 4un3S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	6k4p-d 6k4q-d 5fw1-d 5fw3-d 5fw2-d 6k57-d 5b2t-d 6k3z-d 4un5-d 6ai6-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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PDB pages	PDBj / wwPDB / NCBI
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Related items in Molecule of the Month

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Deposited unit

B: CRISPR-associated endonuclease Cas9

A: sgRNA

C: target DNA

D: non-target DNA

198 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)	178.005, 70.314, 190.077
Angle α, β, γ (deg.)	90.00, 110.34, 90.00
Int Tables number	5
Space group name H-M	C121

#1: Protein	CRISPR-associated endonuclease Cas9 / SpCas9 / SpyCas9 Mass: 158538.844 Da / Num. of mol.: 1 Mutation: D10A, A262T, R324L, S409T, E480K, E543D, M694I, H840A, E1219V Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes serotype M1 (bacteria) Gene: cas9 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds
#2: RNA chain	sgRNA Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain	target DNA Mass: 8567.574 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain	non-target DNA Mass: 3709.466 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.94 Å³/Da / Density % sol: 58.16 %
Crystal grow	Temperature: 289 K / Method: vapor diffusion, sitting drop / pH: 8 Details: 0.2 M Ammonium phosphate dibasic, 20% w/v Polyethylene glycol 3,350

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Data collection

Diffraction	Mean temperature: 85 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.9785 Å
Detector	Type: AGILENT EOS CCD / Detector: CCD / Date: Sep 21, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.9785 Å / Relative weight: 1
Reflection	Resolution: 3.2→50 Å / Num. obs: 36568 / % possible obs: 99 % / Redundancy: 4.8 % / CC_1/2: 0.995 / R_pim(I) all: 0.08 / Net I/σ(I): 10.2
Reflection shell	Resolution: 3.2→3.31 Å / Redundancy: 4.3 % / Num. unique obs: 3602 / CC_1/2: 0.548 / R_pim(I) all: 0.613 / % possible all: 98.9

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0238	refinement
HKL-3000		data reduction
HKL-3000		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 4UN3

4un3

Resolution: 3.2→49.38 Å / Cor.coef. F_o:F_c: 0.891 / Cor.coef. F_o:F_c free: 0.824 / SU B: 66.918 / SU ML: 0.511 / Cross valid method: THROUGHOUT / ESU R Free: 0.657 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.28201	1564	5.2 %	RANDOM
R_work	0.21982	-	-	-
obs	0.22308	28597	81.8 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å

Displacement parameters

B_iso mean: 76.627 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-1.86 Å²	-0 Å²	0.65 Å²
2-	-	-3.39 Å²	0 Å²
3-	-	-	3.74 Å²

Refinement step

Cycle: 1 / Resolution: 3.2→49.38 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10326	2488	0	0	12814

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.008	0.012	13296
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.018	11049
X-RAY DIFFRACTION	r_angle_refined_deg	1.649	1.553	18499
X-RAY DIFFRACTION	r_angle_other_deg	1.265	1.748	25690
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	8.144	5	1304
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	37.818	22.821	546
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	20.4	15	1902
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	18.474	15	62
X-RAY DIFFRACTION	r_chiral_restr	0.076	0.2	1832
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	13088
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	2748
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	2.796	5.81	5240
X-RAY DIFFRACTION	r_mcbond_other	2.793	5.81	5239
X-RAY DIFFRACTION	r_mcangle_it	4.857	8.699	6536
X-RAY DIFFRACTION	r_mcangle_other	4.857	8.699	6537
X-RAY DIFFRACTION	r_scbond_it	1.893	4.939	8056
X-RAY DIFFRACTION	r_scbond_other	1.893	4.939	8055
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	3.407	7.363	11964
X-RAY DIFFRACTION	r_long_range_B_refined	6.863	57.654	15645
X-RAY DIFFRACTION	r_long_range_B_other	6.863	57.654	15646
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 3.199→3.282 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.312	24	-
R_work	0.316	435	-
obs	-	-	17.1 %

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	1.0042	0.2785	-0.2928	0.1845	0.0542	0.7307	0.0571	-0.1895	-0.0564	0.1193	-0.0783	-0.0162	0.1461	0.1456	0.0212	0.3295	-0.2213	0.018	0.2939	0.0102	0.0105	106.8391	58.5453	229.4409
2	1.805	0.3951	-0.3979	0.3088	-0.1366	0.7261	0.1599	0.0731	0.0651	0.0128	-0.1105	0.1122	-0.0276	0.0235	-0.0494	0.2325	-0.1751	-0.0004	0.2669	-0.0526	0.0467	110.2943	64.6639	211.2676
3	0.7369	-0.1438	-0.1198	0.2988	0.7626	2.1831	0.1323	-0.3315	-0.4917	0.2634	-0.0717	0.0617	0.682	-0.0403	-0.0606	0.5842	-0.4716	-0.0435	0.7412	0.1296	0.3631	105.5255	57.7543	231.2122
4	1.1021	1.2365	-0.2675	1.3913	-0.371	3.0387	0.0752	-0.137	-0.9126	0.0917	-0.1984	-1.0096	0.673	0.848	0.1232	0.4085	-0.0787	0.0394	0.5162	-0.0465	0.8231	101.9591	45.5905	203.6387

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	B	1 - 1365
2	X-RAY DIFFRACTION	2	A	2 - 81
3	X-RAY DIFFRACTION	3	C	2 - 28
4	X-RAY DIFFRACTION	4	D	2 - 12

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