PDB-6k4p: Crystal structure of xCas9 in complex with sgRNA and DNA (TGG PAM)

ID or keywords:

Entry

Database: PDB / ID: 6k4p

Title

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

Components

CRISPR-associated endonuclease Cas9/Csn1
DNA (28-MER)
non-targeted DNA
sgRNA

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: 2.9 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 25, 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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PDBx/mmCIF format	6k4p.cif.gz	667.1 KB	Display	PDBx/mmCIF format
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Validation report

Summary document	6k4p_validation.pdf.gz	496.6 KB	Display	wwPDB validaton report
Full document	6k4p_full_validation.pdf.gz	533 KB	Display
Data in XML	6k4p_validation.xml.gz	51.2 KB	Display
Data in CIF	6k4p_validation.cif.gz	71.9 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/k4/6k4p ftp://data.pdbj.org/pub/pdb/validation_reports/k4/6k4p	HTTPS FTP

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Related structure data

Related structure data	6k3zC 6k4qC 6k4sC 6k4uC 6k57C 4un3S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	5fw2-d 6k4q-d 6k4u-d 5fw1-d 5fw3-d 6k57-d 4un5-d 6k3z-d 5b2s-d 6k4s-d 5fq5-d 5b2t-d 5b2r-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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Related items in Molecule of the Month

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Deposited unit

A: sgRNA

C: DNA (28-MER)

D: non-targeted DNA

B: CRISPR-associated endonuclease Cas9/Csn1

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
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	177.785, 69.667, 189.016
Angle α, β, γ (deg.)	90.00, 109.52, 90.00
Int Tables number	5
Space group name H-M	C121

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain	DNA (28-MER) Mass: 8552.563 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#3: DNA chain	non-targeted DNA Mass: 3725.465 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain	sgRNA Mass: 27094.166 Da / Num. of mol.: 1 Mutation: D10A, C80L, A262T, R324L, S409T, E480K, E543D, C574E, M694I, H840A, E1219V Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: Protein	CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9 Mass: 158574.844 Da / Num. of mol.: 1 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

#1: RNA chain

sgRNA

Mass: 27094.166 Da / Num. of mol.: 1
Mutation: D10A, C80L, A262T, R324L, S409T, E480K, E543D, C574E, M694I, H840A, E1219V
Source method: obtained synthetically / Source: (synth.)

Streptococcus pyogenes (bacteria)

#4: Protein

CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9

Mass: 158574.844 Da / Num. of mol.: 1
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

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Non-polymers , 2 types, 27 molecules

#5: Chemical	ChemComp-PO4 / PHOSPHATE ION Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO₄
#6: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 22 / 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: 2.88 Å³/Da / Density % sol: 57.23 %
Crystal grow	Temperature: 289 K / Method: vapor diffusion, sitting drop / pH: 8 Details: 0.2M 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: 2.9→50 Å / Num. obs: 48459 / % possible obs: 98.9 % / Redundancy: 3 % / CC_1/2: 0.961 / R_pim(I) all: 0.116 / Net I/σ(I): 6.8
Reflection shell	Resolution: 2.9→3 Å / Redundancy: 2.9 % / Num. unique obs: 4780 / CC_1/2: 0.376 / R_pim(I) all: 0.7 / % possible all: 99.4

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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: 2.9→46.77 Å / Cor.coef. F_o:F_c: 0.929 / Cor.coef. F_o:F_c free: 0.871 / SU ML: 0.411 / Cross valid method: THROUGHOUT / ESU R Free: 0.453 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.26906	2284	5.1 %	RANDOM
R_work	0.20282	-	-	-
obs	0.20625	42600	91.44 %	-

Solvent computation

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

Refinement step

Cycle: 1 / Resolution: 2.9→46.77 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10546	2531	25	22	13124

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.008	0.012	13585
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.018	11363
X-RAY DIFFRACTION	r_angle_refined_deg	1.718	1.553	18890
X-RAY DIFFRACTION	r_angle_other_deg	1.322	1.745	26421
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	8.513	5	1315
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	34.604	22.5	576
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	20.927	15	1981
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	19.627	15	70
X-RAY DIFFRACTION	r_chiral_restr
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	13313
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	2841
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
X-RAY DIFFRACTION	r_mcbond_other
X-RAY DIFFRACTION	r_mcangle_it
X-RAY DIFFRACTION	r_mcangle_other
X-RAY DIFFRACTION	r_scbond_it
X-RAY DIFFRACTION	r_scbond_other
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other
X-RAY DIFFRACTION	r_long_range_B_refined
X-RAY DIFFRACTION	r_long_range_B_other
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 2.895→2.97 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.449	82	-
R_work	0.385	1840	-
obs	-	-	53.81 %

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	0.737	-0.1277	-0.1525	0.4398	-0.1139	0.2599	0.0625	0.1138	0.0641	-0	-0.0162	0.0595	0.135	-0.0423	-0.0463	0.1914	-0.1275	0.0247	0.304	-0.0443	0.0289	-2.1822	27.4971	33.435
2	0.2665	-0.1597	0.0057	0.1036	0.0144	0.0648	-0.0439	-0.0313	-0.1866	0.0677	0.0455	0.1098	0.1752	-0.0458	-0.0015	0.5061	-0.2129	0.0418	0.5147	-0.0094	0.1325	-6.8737	20.1298	51.5779
3	5.4437	1.5492	-1.8359	6.5482	-1.1083	3.7042	0.0271	-0.0008	-1.045	0.0002	0.0764	-0.3587	0.1502	0.48	-0.1035	0.1526	-0.1167	-0.0108	0.2205	-0.0933	0.274	-10.317	8.5056	25.5185
4	0.6393	0.0151	-0.1789	0.2492	-0.0579	0.4929	-0.0549	-0.0896	-0.0223	0.116	0.0567	-0.0196	0.0828	0.0639	-0.0018	0.2313	-0.0823	0.0273	0.2503	-0.0207	0.0133	-5.4535	21.4316	51.8955

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	1 - 82
2	X-RAY DIFFRACTION	2	C	1 - 28
3	X-RAY DIFFRACTION	3	D	2 - 12
4	X-RAY DIFFRACTION	4	B	3 - 1367

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