PDB-7ox7: Target-bound SpCas9 complex with TRAC chimeric RNA-DNA guide

ID or keywords:

Entry

Database: PDB / ID: 7ox7

Title

Target-bound SpCas9 complex with TRAC chimeric RNA-DNA guide

Components

CRISPR-associated endonuclease Cas9/Csn1
TRAC non-target DNA strand
TRAC target DNA strand
chimeric RNA-DNA guide

Keywords

HYDROLASE / Cas9 / CRISPR / chRDNA / TRAC / nuclease

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)
synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.6 Å

Authors

Donohoue, P. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.T. / Gibson, J. ...

Funding support

Switzerland, 1items

Citation

Journal: Mol.Cell / Year: 2021
Title: Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.
Authors: Donohoue, P.D. / Pacesa, M. / Lau, E. / Vidal, B. / Irby, M.J. / Nyer, D.B. / Rotstein, T. / Banh, L. / Toh, M.S. / Gibson, J. / Kohrs, B. / Baek, K. / Owen, A.L.G. / Slorach, E.M. / van ...

History

Deposition	Jun 22, 2021	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Sep 15, 2021	Provider: repository / Type: Initial release
Revision 1.1	Jan 31, 2024	Group: Data collection / Refinement description Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	7ox7.cif.gz	837.1 KB	Display	PDBx/mmCIF format
PDB format	pdb7ox7.ent.gz	566.5 KB	Display	PDB format
PDBx/mmJSON format	7ox7.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	7ox7_validation.pdf.gz	333.2 KB	Display	wwPDB validaton report
Full document	7ox7_full_validation.pdf.gz	349.7 KB	Display
Data in XML	7ox7_validation.xml.gz	52.4 KB	Display
Data in CIF	7ox7_validation.cif.gz	74.4 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/ox/7ox7 ftp://data.pdbj.org/pub/pdb/validation_reports/ox/7ox7	HTTPS FTP

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Related structure data

Related structure data	7ox8C 7ox9C 7oxaC 5fq5S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	5b2r-d 5fw2-d 6k4q-d 7ox9-d 5b2s-d 4un3-d 6k4p-d 7ox8-d 6k57-d 5fw3-d 7oxa-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
Related items in Molecule of the Month	#181 - Jan 2015 Cascade and CRISPR similarity (15) #67 - Jul 2005 TATA-Binding Protein similarity (1) #168 - Dec 2013 DNA Helicase similarity (1) #255 - Mar 2021 Cisplatin and DNA similarity (1) #91 - Jul 2007 Thymine Dimers similarity (1) #122 - Feb 2010 Enhanceosome similarity (1) #172 - Apr 2014 RecA and Rad51 similarity (1) #253 - Jan 2021 Expressome similarity (1) #112 - Apr 2009 Oct and Sox Transcription Factors similarity (1) #135 - Mar 2011 Integrase similarity (1) #150 - Jun 2012 Sliding Clamps similarity (1) #180 - Dec 2014 TAL Effectors similarity (1) #227 - Nov 2018 Telomerase similarity (1) #48 - Dec 2003 Catabolite Activator Protein similarity (1) #84 - Dec 2006 Transposase similarity (1) #139 - Jul 2011 DNA Methyltransferase similarity (1) #155 - Nov 2012 Vitamin D Receptor similarity (1) #3 - Mar 2000 DNA Polymerase similarity (1) #40 - Apr 2003 RNA Polymerase similarity (1) #73 - Jan 2006 Topoisomerase similarity (1) #87 - Mar 2007 Zinc Fingers similarity (1) #119 - Nov 2009 Designed DNA Crystal similarity (1) #125 - May 2010 Parvoviruses similarity (1) #131 - Nov 2010 Inteins similarity (1) #160 - Apr 2013 Actinomycin similarity (1) #190 - Oct 2015 Two-component Systems similarity (1) #23 - Nov 2001 DNA similarity (1) #240 - Dec 2019 Hypoxia-Inducible Factors similarity (1) #257 - May 2021 Fetal Hemoglobin similarity (1) #258 - Jun 2021 Glucocorticoid Receptor and Dexamethasone similarity (1) #31 - Jul 2002 p53 Tumor Suppressor similarity (1) #33 - Sep 2002 HIV Reverse Transcriptase similarity (1) #39 - Mar 2003 lac Repressor similarity (1) #45 - Sep 2003 Estrogen Receptor similarity (1) #55 - Jul 2004 DNA Ligase similarity (1) #7 - Jul 2000 Nucleosome similarity (1) #8 - Aug 2000 Restriction Enzymes similarity (1) #92 - Aug 2007 Anabolic Steroids similarity (1) #130 - Oct 2010 Riboswitches similarity (1) #241 - Jan 2020 Twenty Years of Molecules similarity (1)

PDB pages

PDBj /

wwPDB /

NCBI

Related items in Molecule of the Month

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similarity (1)
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Deposited unit

A: chimeric RNA-DNA guide

B: CRISPR-associated endonuclease Cas9/Csn1

C: TRAC target DNA strand

D: TRAC 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
Evidence: native gel electrophoresis

Unit cell

Length a, b, c (Å)	177.542, 67.406, 188.136
Angle α, β, γ (deg.)	90.000, 112.542, 90.000
Int Tables number	5
Space group name H-M	C121
Space group name Hall	C2y
Symmetry operation	#1: x,y,z #2: -x,y,-z #3: x+1/2,y+1/2,z #4: -x+1/2,y+1/2,-z

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain	TRAC target DNA strand Mass: 8574.544 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	TRAC non-target DNA strand Mass: 3677.415 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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DNA/RNA hybrid / Protein , 2 types, 2 molecules AB

#1: DNA/RNA hybrid	chimeric RNA-DNA guide Mass: 25995.623 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein	CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9 Mass: 158875.031 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, csn1, SPy_1046 / Plasmid: pMJ841 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta 2(DE3) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

#1: DNA/RNA hybrid

chimeric RNA-DNA guide

Mass: 25995.623 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

#2: Protein

CRISPR-associated endonuclease Cas9/Csn1 / SpCas9 / SpyCas9

Mass: 158875.031 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, csn1, SPy_1046 / Plasmid: pMJ841 / Production host:

Escherichia coli (E. coli) / Strain (production host): Rosetta 2(DE3)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 3 types, 400 molecules

#5: Chemical	ChemComp-MG / MAGNESIUM ION Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical	ChemComp-K / POTASSIUM ION Mass: 39.098 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: K
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 384 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has ligand of interest	N

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2.64 Å³/Da / Density % sol: 53.33 %
Crystal grow	Temperature: 293.15 K / Method: vapor diffusion, hanging drop / pH: 8.5 Details: 0.1 M Tris-acetate pH 8.5, 0.3-0.5 M KSCN, 17-19% PEG3350

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
Detector	Type: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Oct 8, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.6→42.46 Å / Num. obs: 63691 / % possible obs: 99.86 % / Redundancy: 6.8 % / B_iso Wilson estimate: 56.09 Å² / CC_1/2: 0.995 / R_merge(I) obs: 0.2193 / Net I/σ(I): 8.54
Reflection shell	Resolution: 2.6→2.693 Å / Redundancy: 6.8 % / R_merge(I) obs: 2.461 / Mean I/σ(I) obs: 0.74 / Num. unique obs: 6337 / CC_1/2: 0.284 / % possible all: 99.83

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Processing

Software

Name	Version	Classification
Coot		model building
PHENIX	1.15.2_3472	refinement
XDS		data reduction
XSCALE		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5FQ5

5fq5

Resolution: 2.6→42.46 Å / SU ML: 0.3831 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.0583
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.229	3179	4.99 %
R_work	0.209	60474	-
obs	0.21	63653	99.89 %

Solvent computation

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

Displacement parameters

B_iso mean: 79 Å²

Refinement step

Cycle: LAST / Resolution: 2.6→42.46 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10831	2498	16	384	13729

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0034	13819
X-RAY DIFFRACTION	f_angle_d	0.6219	19146
X-RAY DIFFRACTION	f_chiral_restr	0.0407	2179
X-RAY DIFFRACTION	f_plane_restr	0.0038	2013
X-RAY DIFFRACTION	f_dihedral_angle_d	17.0486	8100

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 (%)
2.6-2.64	0.3727	142	0.3571	2597	X-RAY DIFFRACTION	99.71
2.64-2.68	0.3631	122	0.3554	2613	X-RAY DIFFRACTION	99.93
2.68-2.72	0.3708	142	0.3392	2606	X-RAY DIFFRACTION	100
2.72-2.77	0.3397	133	0.3301	2596	X-RAY DIFFRACTION	99.89
2.77-2.82	0.3053	145	0.3131	2634	X-RAY DIFFRACTION	100
2.82-2.88	0.3112	139	0.3086	2587	X-RAY DIFFRACTION	99.82
2.88-2.93	0.3428	141	0.3085	2636	X-RAY DIFFRACTION	99.86
2.93-3	0.3088	122	0.2963	2567	X-RAY DIFFRACTION	99.89
3-3.07	0.3421	151	0.3047	2676	X-RAY DIFFRACTION	99.96
3.07-3.14	0.3205	137	0.2781	2570	X-RAY DIFFRACTION	99.96
3.14-3.23	0.2694	127	0.2562	2644	X-RAY DIFFRACTION	99.89
3.23-3.32	0.2376	148	0.2326	2567	X-RAY DIFFRACTION	99.93
3.32-3.43	0.2311	137	0.2212	2633	X-RAY DIFFRACTION	100
3.43-3.55	0.2797	136	0.2175	2661	X-RAY DIFFRACTION	99.93
3.55-3.7	0.2415	138	0.2053	2602	X-RAY DIFFRACTION	99.89
3.7-3.86	0.2056	135	0.1911	2624	X-RAY DIFFRACTION	99.93
3.86-4.07	0.185	143	0.1779	2636	X-RAY DIFFRACTION	99.89
4.07-4.32	0.2061	140	0.1742	2655	X-RAY DIFFRACTION	100
4.32-4.66	0.207	134	0.1633	2630	X-RAY DIFFRACTION	100
4.66-5.12	0.1901	138	0.1589	2652	X-RAY DIFFRACTION	100
5.12-5.86	0.1923	143	0.1688	2667	X-RAY DIFFRACTION	99.96
5.86-7.38	0.1915	141	0.183	2683	X-RAY DIFFRACTION	99.75
7.38-40.55	0.1562	145	0.1563	2738	X-RAY DIFFRACTION	99.45

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	4.15948026116	-0.806990817449	0.773693346762	1.8275321656	-0.0986349463845	1.48317235277	0.676047603515	0.927487684741	-0.754132137343	0.110644259426	0.125990090665	-0.517845797716	1.1832105647	0.432681079708	0.399256454528	-0.000198133138203	0.00827720138767	0.108167471998	0.29529698303	-0.10973006748	0.0296560733891	-12.5255720854	6.03042271043	71.1376318942
2	0.349737165541	0.282780409437	0.544069492927	0.411151939924	-0.305121499905	0.361933773915	0.128552122409	0.172112109784	0.0294599866558	-0.0308007796528	-0.0370430879943	0.0380540530165	0.122563286628	0.0462118935057	0.410351795033	0.351674662313	-0.00621227932772	-0.0660731245247	0.361436662487	-0.0432979231452	0.345186292397	5.12438882951	3.06819862871	25.7128186422
3	0.00526438236892	0.00594200802024	0.0260711634377	0.0417586370516	-0.0178484260525	-0.0234275250996	0.133921844097	-0.113983026489	0.479419998305	-0.396404364749	0.335405976152	0.13587558221	-0.325197869331	0.338613797689	3.90681575445E-6	0.910837216127	-0.231734491435	0.246800112656	1.17831045551	0.132220285082	1.18129453775	-38.8464109068	16.6099850514	34.4116591808
4	0.254449308382	0.0330959143766	-0.00163600020359	-0.0526163029979	-0.182777353011	0.547742967886	0.0241575542853	0.117050497454	0.0128248300109	0.0237343508423	-0.0337620900569	0.03000927596	0.00114812362345	0.113642213419	-0.000911971557524	0.307828633991	-0.0506297019575	0.0102313969864	0.320096090889	-0.0309925696479	0.374915038178	-1.07494685701	7.58485270708	31.2771465133
5	0.26481250057	0.0969695814979	0.416126976919	-0.203280594864	-0.482547589216	0.273711318105	-0.1729358169	0.105881501979	0.0950867217542	0.256645098626	-0.0676471381656	0.130078536264	-0.416228279373	0.298281392973	-0.0234234033954	0.646450807013	0.0316941269769	0.0736879182088	0.475649552102	-0.01293403993	0.384113280581	28.2829609591	0.200415036979	64.0843103821
6	0.846091391207	-0.249202342065	-0.307380067647	0.127782709774	0.117619959359	0.698746694733	0.00116426808089	-0.132993988345	-0.0275677872485	0.0912852143261	-0.0100381394785	0.086856585154	-0.0122378257596	0.290534911069	3.91026761167E-5	0.326463356244	-0.0328552360711	-0.0213552612812	0.370951760383	0.0240749026969	0.326790518359	16.4507695947	5.37406723896	39.6832945226
7	0.398636643578	0.163728827908	0.178352055178	0.689473653675	0.170820970045	0.831788366155	-0.0311834390152	-0.0698902928595	0.0463979300168	0.201627001601	0.1130415401	-0.0822719012785	-0.188940761507	0.121518021238	4.48280668638E-5	0.431739514395	-0.00672737579737	-0.00639745992355	0.267262468461	-0.0326153637739	0.336697481799	-2.48063139188	18.7839982824	82.8013466728
8	0.366654016397	0.304260323982	0.364210260457	0.722217598769	-0.0646325384591	0.31722979639	0.115295069539	-0.147001850129	-0.290038136767	0.0907938550381	0.0159347965681	-0.476990773677	0.283461075169	0.081944645746	-0.000988531737291	0.496053085769	0.057487941893	-0.0696770865788	0.57680791444	-0.0274059616784	0.77404947191	-10.6981815933	-8.62587534976	64.9954478312
9	0.985092123547	0.13525678155	-0.30781933659	0.415397819363	0.0511158953511	0.841035451583	-0.0707958423579	0.0730275275129	-0.0737950593476	0.0508455918744	0.0798435634039	-0.00747717197342	0.293512810914	-0.0248034468097	-0.000441161164408	0.369219139777	-0.0813397883734	-0.024040963485	0.249310492701	-0.0398324346663	0.334686355608	-29.6446045667	-11.4014152218	43.1465011334
10	0.0346324400895	-0.00234342827295	0.00953546986814	0.00167906491687	-0.0253119122201	0.00545686033604	0.0432754880169	0.134150742524	-0.149569871155	0.0903431000629	-0.0423417645854	0.0337626868411	-0.017573034473	-0.210378785912	0.0107685459682	1.28047006455	-0.589218834338	-0.0529484042881	1.56508179571	-0.352699362444	0.933256737815	-15.9150486952	-20.8866835369	18.9873837887
11	0.0251558774664	0.00774216010929	0.00477642031899	0.00220057722024	-0.0311609819233	0.0275630766903	0.0145548285598	-0.122994501184	-0.317842911913	-0.0633661234071	-0.0230837635077	0.00813926497598	0.337080728415	0.0315227859702	-5.92987488976E-5	0.488807224888	-0.0866784715516	0.0046897117366	0.5741022926	-0.0756066705116	0.589015391142	-2.58601173911	-9.13030614196	31.0662570293
12	-0.0198035674481	-0.00761483859797	-0.0405484691413	-0.010691178873	0.059160284986	-0.0861906772415	0.0699865232575	-0.0487902509569	0.296239568423	0.047718422234	0.316544888061	0.0371255215042	0.281826421858	0.33310174937	0.000154483834261	0.516233639085	-0.127698440203	0.047651067152	0.488461941456	-0.0688765599065	0.486896792365	-1.22933959055	7.08772215261	54.9807126707
13	-0.00606329245654	0.00437980760809	0.0178430544593	-0.00618163295325	-0.00295466833998	0.00243721919686	-0.0335966451911	-0.124424254084	0.250504893585	0.405228327612	0.0842446476163	0.17011338265	0.193536342313	-0.0799540020757	0.000248912889656	1.2441921903	-0.11811716744	0.201038131254	0.615503331154	0.00312315257178	0.819308413529	-14.0943960996	-0.932022793403	74.5366545023
14	0.0996403617612	-0.0603642200714	0.0265986818803	0.0483784338259	-0.00669939810208	0.0462143208803	0.0692960639629	-0.00435628036384	-0.0453488261406	0.0292141824196	-0.123620022491	0.00725178115223	0.231192508728	-0.142263883552	-0.00662796373275	0.839653836166	-0.0479872956791	0.0098624086878	0.576961877808	-0.0904228475133	0.701163659964	-22.1188158861	9.81885866236	82.3581883071
15	0.480862402522	0.0215097123615	-0.0610893424731	0.225928823448	-0.0424587963747	-0.0139956065849	-0.00707830565075	0.193729683517	-0.88673188138	0.0952581165355	0.435737479617	0.194832672996	0.766159994755	0.35016886878	0.0214851928081	0.594509527277	-0.0709385767671	-0.0153785479015	0.471581876004	-0.211536672556	0.50695356343	-11.5507142271	-14.0593090428	23.7856529579

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 1 through 10 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 11 through 70 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 71 through 81 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 3 through 163 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 164 through 300 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 301 through 512 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 513 through 692 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 693 through 909 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 910 through 1366 )
10	X-RAY DIFFRACTION	10	chain 'C' and (resid -7 through -3 )
11	X-RAY DIFFRACTION	11	chain 'C' and (resid -2 through 2 )
12	X-RAY DIFFRACTION	12	chain 'C' and (resid 3 through 12 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 13 through 17 )
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 18 through 20 )
15	X-RAY DIFFRACTION	15	chain 'D' and (resid -3 through 7 )

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