PDB-6k57: Crystal structure of dCas9 in complex with sgRNA and DNA (CGA PAM)

ID or keywords:

Entry

Database: PDB / ID: 6k57

Title

Crystal structure of dCas9 in complex with sgRNA and DNA (CGA PAM)

Components

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

Keywords

HYDROLASE/RNA/DNA / endonuclease / HYDROLASE-RNA-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: 2.98 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 28, 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	6k57_validation.pdf.gz	481.2 KB	Display	wwPDB validaton report
Full document	6k57_full_validation.pdf.gz	529.1 KB	Display
Data in XML	6k57_validation.xml.gz	51.6 KB	Display
Data in CIF	6k57_validation.cif.gz	72 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/k5/6k57 ftp://data.pdbj.org/pub/pdb/validation_reports/k5/6k57	HTTPS FTP

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Related structure data

Related structure data	6k3zC 6k4pC 6k4qC 6k4sC 6k4uC 4un3S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	5fw2-d 5fw3-d 5b2t-d 6k4q-d 5fw1-d 5b2r-d 6k4p-d 5fq5-d 4un3-d 6k3z-d 6k4u-d 5b2s-d 4un5-d 6ai6-d 4un4-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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Related items in Molecule of the Month

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Deposited unit

A: sgRNA

B: CRISPR-associated endonuclease Cas9

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)	177.956, 69.137, 189.243
Angle α, β, γ (deg.)	90.00, 110.34, 90.00
Int Tables number	5
Space group name H-M	C121

#1: RNA chain	sgRNA Mass: 26788.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#2: Protein	CRISPR-associated endonuclease Cas9 / SpCas9 / SpyCas9 Mass: 158588.781 Da / Num. of mol.: 1 / Mutation: D10A, H840A 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
#3: DNA chain	target DNA Mass: 8583.573 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#4: DNA chain	non-target DNA Mass: 3694.455 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Streptococcus pyogenes (bacteria)
#5: 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: 2.86 Å³/Da / Density % sol: 56.99 %
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: Mar 15, 2019
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.9785 Å / Relative weight: 1
Reflection	Resolution: 2.98→50 Å / Num. obs: 44046 / % possible obs: 98.8 % / Redundancy: 6.4 % / CC_1/2: 0.989 / R_pim(I) all: 0.046 / Net I/σ(I): 16.2
Reflection shell	Resolution: 2.98→3.09 Å / Redundancy: 5.7 % / Mean I/σ(I) obs: 2 / Num. unique obs: 4274 / CC_1/2: 0.793 / R_pim(I) all: 0.39 / % possible all: 97.2

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Processing

Software

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

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 4UN3

4un3

Resolution: 2.98→48.89 Å / Cor.coef. F_o:F_c: 0.924 / Cor.coef. F_o:F_c free: 0.859 / SU B: 47.618 / SU ML: 0.384 / Cross valid method: THROUGHOUT / ESU R Free: 0.499 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.27412	2067	5 %	RANDOM
R_work	0.19703	-	-	-
obs	0.20088	38952	91.99 %	-

Solvent computation

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

Displacement parameters

B_iso mean: 57.358 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-0.25 Å²	0 Å²	0.24 Å²
2-	-	-2.78 Å²	-0 Å²
3-	-	-	2.24 Å²

Refinement step

Cycle: 1 / Resolution: 2.98→48.89 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10754	2526	0	22	13302

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.01	0.012	13782
X-RAY DIFFRACTION	r_bond_other_d	0.006	0.018	11725
X-RAY DIFFRACTION	r_angle_refined_deg	1.833	1.555	19106
X-RAY DIFFRACTION	r_angle_other_deg	1.339	1.741	27361
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	8.806	5	1309
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	35.935	22.63	616
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	22.712	15	2144
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	21.01	15	75
X-RAY DIFFRACTION	r_chiral_restr	0.092	0.2	1847
X-RAY DIFFRACTION	r_gen_planes_refined	0.008	0.02	13430
X-RAY DIFFRACTION	r_gen_planes_other	0.004	0.02	2867
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	2.914	5.179	5257
X-RAY DIFFRACTION	r_mcbond_other	2.906	5.179	5256
X-RAY DIFFRACTION	r_mcangle_it	4.846	7.758	6559
X-RAY DIFFRACTION	r_mcangle_other	4.846	7.759	6560
X-RAY DIFFRACTION	r_scbond_it	2.497	5.051	8525
X-RAY DIFFRACTION	r_scbond_other	2.497	5.051	8524
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	4.218	7.464	12548
X-RAY DIFFRACTION	r_long_range_B_refined	7.062	54.648	15999
X-RAY DIFFRACTION	r_long_range_B_other	7.062	54.648	16000
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

LS refinement shell

Resolution: 2.98→3.057 Å / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.335	81	-
R_work	0.316	1544	-
obs	-	-	50.22 %

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	0.052	-0.1053	0.0205	0.2609	-0.0455	0.1074	0.0245	-0.019	0.0127	-0.0395	0.0489	0.0185	0.0356	0.0304	-0.0733	0.0635	-0.0655	0.0204	0.2132	-0.0411	0.0947	86.4095	62.4335	33.5921
2	0.1475	-0.0186	-0.0058	0.1022	-0.065	0.2204	-0.0416	0.046	-0.0142	0.0311	0.0471	0.0138	0.0205	-0.0001	-0.0055	0.0581	-0.0324	0.0447	0.1172	-0.0408	0.046	83.7796	56.3993	52.504
3	0.2354	-0.0964	-0.184	0.0616	0.0906	0.155	-0.1149	0.0417	-0.0669	0.0527	0.0518	0.0401	0.0912	0.0168	0.0631	0.1101	-0.0638	0.0687	0.2436	-0.0107	0.0486	81.5857	55.4415	51.7622
4	2.9883	1.6226	-2.8584	1.8445	-1.9576	2.9115	-0.0746	-0.0001	-0.2441	0.0018	-0.0759	-0.0913	0.0073	0.0348	0.1506	0.0736	-0.1357	0.0794	0.2851	-0.1173	0.1187	78.3076	43.4187	26.5345

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	1 - 81
2	X-RAY DIFFRACTION	2	B	4 - 1364
3	X-RAY DIFFRACTION	3	C	1 - 28
4	X-RAY DIFFRACTION	4	D	2 - 12

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