PDB-6k4s: Crystal structure of xCas9 in complex with sgRNA and DNA (TGC PAM)

ID or keywords:

Entry

Database: PDB / ID: 6k4s

Title

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

Components

CRISPR-associated endonuclease Cas9/Csn1
non-targeted DNA
sgRNA
targeted DNA

Keywords

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

Streptococcus pyogenes (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 3.01 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 26, 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

Summary document	6k4s_validation.pdf.gz	476.7 KB	Display	wwPDB validaton report
Full document	6k4s_full_validation.pdf.gz	511.4 KB	Display
Data in XML	6k4s_validation.xml.gz	49.9 KB	Display
Data in CIF	6k4s_validation.cif.gz	69.3 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/k4/6k4s ftp://data.pdbj.org/pub/pdb/validation_reports/k4/6k4s	HTTPS FTP

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Related structure data

Related structure data	6k3zC 6k4pC 6k4qC 6k4uC 6k57C 4un3S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	5fw2-d 6k4p-d 5fw3-d 4un5-d 5fw1-d 6k4u-d 6k57-d 6ai6-d 6k3z-d 6k4q-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/Csn1

A: sgRNA

C: targeted DNA

D: non-targeted 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)	177.464, 70.364, 188.899
Angle α, β, γ (deg.)	90.00, 110.42, 90.00
Int Tables number	5
Space group name H-M	C121

#1: Protein	CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9 Mass: 158574.844 Da / Num. of mol.: 1 Mutation: D10A, C80L, A262T, R324L, S409T, E480K, E543D, C574E, 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	targeted DNA Mass: 8592.588 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain	non-targeted DNA Mass: 3685.441 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: 2.92 Å³/Da / Density % sol: 57.88 %
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→50 Å / Num. obs: 43368 / % possible obs: 98.8 % / Redundancy: 6.5 % / CC_1/2: 1 / R_pim(I) all: 0.071 / Net I/σ(I): 12
Reflection shell	Resolution: 3→3.11 Å / Redundancy: 5.5 % / Num. unique obs: 4176 / CC_1/2: 0.543 / R_pim(I) all: 0.578 / % possible all: 96.9

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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: 3.01→49.25 Å / Cor.coef. F_o:F_c: 0.881 / Cor.coef. F_o:F_c free: 0.823 / SU B: 54.535 / SU ML: 0.441 / Cross valid method: THROUGHOUT / ESU R Free: 0.534 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.28531	1905	5 %	RANDOM
R_work	0.23691	-	-	-
obs	0.23934	36346	86.98 %	-

Solvent computation

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

Displacement parameters

B_iso mean: 70.805 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	0.15 Å²	-0 Å²	-1.16 Å²
2-	-	-1.56 Å²	-0 Å²
3-	-	-	1.78 Å²

Refinement step

Cycle: 1 / Resolution: 3.01→49.25 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	9924	2488	0	0	12412

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.009	0.012	12881
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.018	10473
X-RAY DIFFRACTION	r_angle_refined_deg	1.659	1.549	17975
X-RAY DIFFRACTION	r_angle_other_deg	1.359	1.756	24259
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.257	5	1300
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.986	22.635	482
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	18.942	15	1699
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	18.491	15	55
X-RAY DIFFRACTION	r_chiral_restr	0.076	0.2	1814
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	12786
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	2675
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	2.384	4.262	5224
X-RAY DIFFRACTION	r_mcbond_other	2.384	4.263	5223
X-RAY DIFFRACTION	r_mcangle_it	4.124	6.383	6516
X-RAY DIFFRACTION	r_mcangle_other	4.124	6.383	6517
X-RAY DIFFRACTION	r_scbond_it	1.63	3.354	7657
X-RAY DIFFRACTION	r_scbond_other	1.63	3.353	7656
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	2.92	5.069	11460
X-RAY DIFFRACTION	r_long_range_B_refined	5.866	40.623	14981
X-RAY DIFFRACTION	r_long_range_B_other	5.866	40.623	14982
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 3.006→3.084 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.379	84	-
R_work	0.327	1313	-
obs	-	-	43.25 %

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	1.0007	0.2225	-0.1148	0.2408	0.0079	0.9304	0.1399	-0.1853	-0.0258	0.1353	-0.0989	-0.0422	0.1571	0.0637	-0.041	0.3341	-0.3152	0.0587	0.3912	-0.0043	0.2883	106.2744	57.711	226.7738
2	1.8086	0.5439	-0.4927	0.2729	0.0236	0.9543	0.2277	-0.0786	0.0004	0.0654	-0.0806	0.066	-0.0131	0.1313	-0.1471	0.2467	-0.1569	0.0279	0.3682	-0.013	0.338	110.1878	63.6179	209.6784
3	1.6476	-0.288	0.029	0.6311	1.6099	4.5608	0.4199	-0.6213	-0.5562	0.2695	0.0462	-0.0481	0.8316	-0.0357	-0.466	0.562	-0.443	-0.1302	0.5809	0.155	0.2717	105.4183	56.7133	229.4485
4	1.3137	1.4817	-1.2544	1.8142	-0.8271	3.7432	-0.1075	0.0219	-0.26	0.2477	0.1723	-0.3334	1.5724	0.8631	-0.0648	0.9903	0.2275	-0.0817	0.9466	-0.169	0.4087	101.3873	44.5553	201.8785

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	B	3 - 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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