PDB-7qqs: SpCas9 bound to FANCF on-target DNA substrate

ID or keywords:

Entry

Database: PDB / ID: 7qqs

Title

SpCas9 bound to FANCF on-target DNA substrate

Components

(FANCF on-target ...) x 2
CRISPR-associated endonuclease Cas9/Csn1
FANCF sgRNA

Keywords

HYDROLASE / crispr / cas9 / off-target / ternary 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 (bacteria)
synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.4 Å

Authors

Pacesa, M. / Jinek, M.

Funding support

Switzerland, 1items

Citation

Journal: Cell / Year: 2022
Title: Structural basis for Cas9 off-target activity.
Authors: Pacesa, M. / Lin, C.H. / Clery, A. / Saha, A. / Arantes, P.R. / Bargsten, K. / Irby, M.J. / Allain, F.H. / Palermo, G. / Cameron, P. / Donohoue, P.D. / Jinek, M.

History

Deposition	Jan 10, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Oct 26, 2022	Provider: repository / Type: Initial release
Revision 1.1	Nov 23, 2022	Group: Database references / Category: citation / citation_author Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._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
Revision 1.2	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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Validation report

Summary document	7qqs_validation.pdf.gz	470.4 KB	Display	wwPDB validaton report
Full document	7qqs_full_validation.pdf.gz	486.5 KB	Display
Data in XML	7qqs_validation.xml.gz	54.9 KB	Display
Data in CIF	7qqs_validation.cif.gz	79.6 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/qq/7qqs ftp://data.pdbj.org/pub/pdb/validation_reports/qq/7qqs	HTTPS FTP

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Related structure data

Related structure data	7qqoC 7qqpC 7qqqC 7qqrC 7qqtC 7qquC 7qqvC 7qqwC 7qqxC 7qqzC 7qr0C 7qr1C 7qr5C 7qr7C 7qr8C 7zo1C 5fq5S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	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

PDBj /

wwPDB /

NCBI

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Deposited unit

A: FANCF sgRNA

B: CRISPR-associated endonuclease Cas9/Csn1

C: FANCF on-target target strand

D: FANCF on-target non-target 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

Unit cell

Length a, b, c (Å)	177.906, 67.055, 187.397
Angle α, β, γ (deg.)	90.000, 111.107, 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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FANCF on-target ... , 2 types, 2 molecules CD

#3: DNA chain	FANCF on-target target strand Mass: 8639.582 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	FANCF on-target non-target strand Mass: 3653.390 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain / Protein , 2 types, 2 molecules AB

#1: RNA chain	FANCF sgRNA Mass: 27068.125 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein	CRISPR-associated endonuclease Cas9/Csn1 / 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 (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

#1: RNA chain

FANCF sgRNA

Mass: 27068.125 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

#2: Protein

CRISPR-associated endonuclease Cas9/Csn1 / 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 (bacteria) / Gene: cas9, csn1, SPy_1046 / Production host:

Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q99ZW2, Hydrolases; Acting on ester bonds

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Non-polymers , 3 types, 566 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.: 10 / Source method: obtained synthetically / Formula: K
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 554 / 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 %
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 7, 2017
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.4→47.72 Å / Num. obs: 81059 / % possible obs: 99.9 % / Redundancy: 6.8 % / B_iso Wilson estimate: 49.91 Å² / CC_1/2: 0.998 / R_merge(I) obs: 0.12 / Net I/σ(I): 13.9
Reflection shell	Resolution: 2.4→2.49 Å / Redundancy: 6.8 % / R_merge(I) obs: 1.59 / Mean I/σ(I) obs: 1.18 / Num. unique obs: 8039 / CC_1/2: 0.487 / % possible all: 99.9

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Processing

Software

Name	Version	Classification
Coot		model building
PHENIX	1.17.1_3660	refinement
XDS		data reduction
XSCALE		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5FQ5

5fq5

Resolution: 2.4→47.72 Å / SU ML: 0.3369 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.1281
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2288	4052	5 %
R_work	0.1971	76985	-
obs	0.1987	81037	99.93 %

Solvent computation

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

Displacement parameters

B_iso mean: 66.13 Å²

Refinement step

Cycle: LAST / Resolution: 2.4→47.72 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10892	2535	12	554	13993

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.003	13922
X-RAY DIFFRACTION	f_angle_d	0.6088	19298
X-RAY DIFFRACTION	f_chiral_restr	0.0395	2201
X-RAY DIFFRACTION	f_plane_restr	0.0036	2029
X-RAY DIFFRACTION	f_dihedral_angle_d	18.116	5646

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 (%)
2.4-2.43	0.3628	134	0.3317	2616	X-RAY DIFFRACTION	99.93
2.43-2.46	0.3433	138	0.3238	2614	X-RAY DIFFRACTION	99.93
2.46-2.49	0.2831	153	0.3055	2682	X-RAY DIFFRACTION	99.82
2.49-2.52	0.3328	126	0.2919	2562	X-RAY DIFFRACTION	99.93
2.52-2.56	0.3428	141	0.2843	2679	X-RAY DIFFRACTION	99.89
2.56-2.59	0.3297	139	0.2818	2647	X-RAY DIFFRACTION	100
2.59-2.63	0.3519	143	0.2817	2624	X-RAY DIFFRACTION	99.96
2.63-2.67	0.3405	141	0.2858	2641	X-RAY DIFFRACTION	100
2.67-2.72	0.2944	135	0.2734	2655	X-RAY DIFFRACTION	100
2.72-2.76	0.365	129	0.2827	2611	X-RAY DIFFRACTION	99.96
2.76-2.81	0.3174	144	0.274	2634	X-RAY DIFFRACTION	99.96
2.81-2.87	0.311	149	0.2524	2646	X-RAY DIFFRACTION	100
2.87-2.93	0.2985	135	0.2466	2661	X-RAY DIFFRACTION	100
2.93-2.99	0.2809	139	0.2407	2625	X-RAY DIFFRACTION	100
2.99-3.06	0.2531	141	0.2489	2670	X-RAY DIFFRACTION	100
3.06-3.14	0.2883	139	0.2371	2594	X-RAY DIFFRACTION	99.93
3.14-3.22	0.2783	132	0.2307	2677	X-RAY DIFFRACTION	99.96
3.22-3.32	0.2402	145	0.2162	2648	X-RAY DIFFRACTION	100
3.32-3.42	0.2553	135	0.2005	2662	X-RAY DIFFRACTION	99.96
3.42-3.54	0.2332	143	0.2001	2663	X-RAY DIFFRACTION	99.96
3.54-3.69	0.225	139	0.1821	2653	X-RAY DIFFRACTION	99.96
3.69-3.85	0.1667	138	0.1724	2644	X-RAY DIFFRACTION	99.89
3.85-4.06	0.1845	144	0.1602	2656	X-RAY DIFFRACTION	99.86
4.06-4.31	0.1714	136	0.1538	2694	X-RAY DIFFRACTION	99.89
4.31-4.64	0.1705	140	0.1438	2659	X-RAY DIFFRACTION	100
4.64-5.11	0.1854	142	0.1465	2679	X-RAY DIFFRACTION	99.93
5.11-5.85	0.2134	143	0.1623	2684	X-RAY DIFFRACTION	99.82
5.85-7.36	0.2081	142	0.1808	2725	X-RAY DIFFRACTION	99.97
7.37-47.72	0.1816	147	0.1634	2780	X-RAY DIFFRACTION	99.59

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	1.00745869489	-0.126537682727	0.0624579121124	0.346373162003	0.264604234251	0.475984947599	-0.596305330649	0.148055361262	-0.453929392903	0.221305348931	0.216621701622	0.246542265398	0.7818269161	-0.460483034043	-0.0472458664609	0.605962725718	-0.125469643765	0.0706211359485	0.695688202876	-0.158315447584	0.618199943992	-14.6646853248	6.92645698755	74.2976619006
2	0.451763190122	0.0474114845629	-0.0232899827406	0.485279014475	0.0888734091758	0.111897604116	0.0173670945672	0.0454255885071	0.0539836520423	0.0726740046122	0.0753116531804	0.0101194799147	0.251130303009	0.129992290593	0.00828453065328	0.399529774509	-0.0167497218614	-0.0173384436133	0.435709185602	0.0012488764724	0.407425195174	5.6326364995	3.88393467149	36.9608136715
3	0.133429369827	-0.117188050851	-0.0787661236993	0.121946675334	0.0719252129353	0.0640178402015	0.453147626846	0.185279984033	0.496509837448	-0.82212625788	-0.128792875055	0.0453523255	0.121819980183	0.124792740175	0.00372367709441	0.96517894753	0.257330131348	-0.245556533842	1.08241169061	-0.256234022983	0.754602495748	22.2960915785	-15.9828596358	1.55095887335
4	0.555335498215	0.398925411047	0.424821748153	0.455249418049	0.108188682364	0.460807724883	0.0879158244917	0.0363316843444	0.119238526741	-0.0766641145354	0.0467824707327	0.0239048876424	-0.0187187857373	0.0213850680409	0.000113797611082	0.39888314118	-0.0182595736101	-0.0305044002496	0.399920313501	0.0168563148796	0.416744735965	1.28946101763	7.79762233399	28.27042385
5	0.0996429778144	-0.143316757567	-0.0108916606174	0.124623657882	0.0423765509801	0.0340308898509	0.547723780875	-0.19808204661	0.50857610847	-0.573305729726	-0.212101513316	0.451517528172	-0.943046861452	0.0391223856904	0.000634994932026	0.911797049898	-0.110755315178	0.202665964792	0.765176451396	0.0730439187167	0.858013931545	-39.276325745	14.84189433	33.7472976584
6	0.779373569464	0.294962788689	-0.0998087677425	0.209588581193	-0.186388676619	0.510675996391	0.132968423605	0.0140216140897	0.0276666857346	0.0854027265653	-0.110952672242	-0.00319714105467	-0.0521161718201	0.126678600747	0.0044852727372	0.355525705562	-0.0729370676185	-0.00938329022591	0.330567208415	-0.0199221597809	0.360105937735	5.86846175555	6.56988071131	37.3153640271
7	0.184800964436	-0.141259868458	0.533186384937	-0.0333373123797	-0.510521563134	0.604646586035	-0.0594836303646	0.054600021489	-0.0747889091789	0.0772872408426	-0.110334020432	0.0464164276408	-0.178396341422	-0.0485406767597	-5.44310311799E-5	0.620120657661	0.0602251042992	-0.0242904547878	0.57371963893	-0.0306758514896	0.565779152043	26.254328531	-3.33966745646	56.3944690367
8	0.298379697993	-0.309173353615	-0.202900856066	-0.060840696098	0.190993096106	1.04245142353	0.0276454409223	-0.0466656027106	-0.0118378722652	0.126903878819	-0.0178456848987	-0.0156721849574	-0.001812313009	0.117383873978	-2.09688689335E-5	0.407427168227	-0.0660966519083	-0.0294086738931	0.338028822317	0.00493428249335	0.424071324586	9.62114914805	9.14429386448	50.186819568
9	0.195535007374	0.106382583397	0.353691281972	0.422946494731	-0.0282090371847	0.797268918701	-0.110447965366	0.0158537576703	-0.00609364101404	0.0878427403286	0.0615169627742	-0.0393855661688	-0.19026782154	0.00678466016026	-1.06398039984E-5	0.392944395311	0.0042753927646	0.00423532763512	0.35122344794	-0.0247385272523	0.378806473685	-5.31401557442	18.6128020209	74.8502751586
10	0.54598841644	0.112969285387	0.154526386932	0.00523012277883	0.0385064592787	0.827117198985	-0.0195842712832	-0.150489583629	-0.0879341347672	0.110343409997	0.0530263228343	-0.103628266777	0.205884332032	0.111669981521	-3.42069127705E-5	0.487954414403	-0.0384391696966	-0.00573881896733	0.439440520554	0.0081585508674	0.505000262054	-16.3683195087	-11.6352063641	65.2266550176
11	1.09084601836	0.215949075832	-0.330040109466	0.408954922286	-0.158164069483	0.834352168533	-0.0499847163771	0.106240595421	-0.133928684273	0.0157102343343	0.0415846995853	0.0508886188593	0.291348280517	-0.130269911576	-0.000115026599584	0.399939762935	-0.120555465861	-0.00563439183337	0.275748616222	-0.0676664814532	0.372200090357	-28.0971190501	-13.9650095323	36.6918983937
12	0.0497186893223	-0.00581389073087	-0.0113667757483	0.116554933445	-0.00864602775878	0.00893551637633	0.101236607128	0.0755972922328	-0.129010384087	0.233758097429	0.0430651932392	-0.0411817640221	0.0185350008291	-0.31789479276	-0.0377637941988	1.01641277062	-0.557108216337	0.0247270289608	1.61687126424	-0.552923650547	0.768737177728	-15.9410030553	-21.3573532889	19.2555421999
13	0.779180915708	0.602005569938	-0.700377519873	0.438878508411	-0.523671169188	0.646097730543	0.15042262449	-0.201086640627	0.0232135013359	0.525883505425	-0.152846920558	-0.0781420064983	0.356131086958	0.346091015007	0.0129320880083	0.384571760738	-0.00549813026677	-0.0220134399038	0.236903653881	-0.00609513796485	0.409314112264	-2.31304800419	-9.17926078343	31.2704985426
14	-0.0958645242051	0.0661281245729	0.0878283234623	-0.0380451380836	0.154552492244	0.0924952055688	0.00093145933365	-0.0171589811226	0.188546013205	0.0216438101857	0.111832724274	0.0722752208009	0.177637099559	-0.0334899265639	-2.50952099135E-5	0.491478813544	-0.0544585537782	0.0541744578505	0.488325693136	0.0272509926893	0.420263275324	-7.06390921993	5.36405185051	65.1231879046
15	0.202151602879	0.215255750541	-0.092982614354	0.281975240521	-0.0320137030894	0.0528492537494	0.218643168863	0.452564553704	-0.728546644781	-0.0182227430111	0.0782651255467	0.235680875795	0.654286297454	-0.0547249450641	0.00781629941607	0.669299271495	-0.0432021414822	-0.0402551858902	0.511468051904	-0.157400301494	0.659864484599	-11.6299883685	-14.3718554313	23.8209690843

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid -1 through 8 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 9 through 28 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 29 through 38 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 39 through 68 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 69 through 82 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 3 through 207 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 208 through 342 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 343 through 551 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 552 through 750 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 751 through 1031 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 1032 through 1367 )
12	X-RAY DIFFRACTION	12	chain 'C' and (resid -7 through -3 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid -2 through 2 )
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 3 through 20 )
15	X-RAY DIFFRACTION	15	chain 'D' and (resid -2 through 7 )

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