PDB-7qr5: SpCas9 bound to FANCF off-target6 DNA substrate

ID or keywords:

Entry

Database: PDB / ID: 7qr5

Title

SpCas9 bound to FANCF off-target6 DNA substrate

Components

(FANCF off-target6 ...) 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: 3.3 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 17, 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	7qr5_validation.pdf.gz	478.4 KB	Display	wwPDB validaton report
Full document	7qr5_full_validation.pdf.gz	494 KB	Display
Data in XML	7qr5_validation.xml.gz	50 KB	Display
Data in CIF	7qr5_validation.cif.gz	69.8 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/qr/7qr5 ftp://data.pdbj.org/pub/pdb/validation_reports/qr/7qr5	HTTPS FTP

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Related structure data

Related structure data	7qqoC 7qqpC 7qqqC 7qqrC 7qqsC 7qqtC 7qquC 7qqvC 7qqwC 7qqxC 7qqzC 7qr0C 7qr1C 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 off-target6 target strand

D: FANCF off-target6 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)	178.307, 66.968, 187.724
Angle α, β, γ (deg.)	90.000, 111.171, 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 off-target6 ... , 2 types, 2 molecules CD

#3: DNA chain	FANCF off-target6 target strand Mass: 8630.567 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	FANCF off-target6 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 , 4 types, 94 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.: 11 / Source method: obtained synthetically / Formula: K
#7: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₂H₆O₂
#8: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 80 / 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: X06SA / Wavelength: 1 Å
Detector	Type: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Feb 26, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 3.3→47.71 Å / Num. obs: 31161 / % possible obs: 98.4 % / Redundancy: 6.5 % / B_iso Wilson estimate: 78.91 Å² / CC_1/2: 0.974 / R_merge(I) obs: 0.51 / Net I/σ(I): 3.67
Reflection shell	Resolution: 3.3→3.42 Å / Redundancy: 5.8 % / R_merge(I) obs: 2.44 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 2977 / CC_1/2: 0.389 / % possible all: 94.3

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Processing

Software

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

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5FQ5

5fq5

Resolution: 3.3→47.71 Å / SU ML: 0.493 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.5614
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2537	1553	5 %
R_work	0.2309	29526	-
obs	0.2321	31079	98.41 %

Solvent computation

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

Displacement parameters

B_iso mean: 84.76 Å²

Refinement step

Cycle: LAST / Resolution: 3.3→47.71 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10897	2535	17	80	13529

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0027	13929
X-RAY DIFFRACTION	f_angle_d	0.5512	19305
X-RAY DIFFRACTION	f_chiral_restr	0.0388	2202
X-RAY DIFFRACTION	f_plane_restr	0.0031	2030
X-RAY DIFFRACTION	f_dihedral_angle_d	18.061	5648

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 (%)
3.3-3.41	0.3829	133	0.3599	2529	X-RAY DIFFRACTION	93.86
3.41-3.53	0.3347	146	0.3139	2721	X-RAY DIFFRACTION	99.2
3.53-3.67	0.3464	133	0.2975	2648	X-RAY DIFFRACTION	99.11
3.67-3.84	0.272	139	0.2661	2692	X-RAY DIFFRACTION	99.26
3.84-4.04	0.2518	149	0.2505	2659	X-RAY DIFFRACTION	99.43
4.04-4.29	0.2562	135	0.2372	2702	X-RAY DIFFRACTION	99.54
4.29-4.62	0.2485	144	0.221	2688	X-RAY DIFFRACTION	99.02
4.62-5.09	0.2613	139	0.2193	2638	X-RAY DIFFRACTION	96.49
5.09-5.82	0.2432	142	0.206	2728	X-RAY DIFFRACTION	99.69
5.82-7.33	0.2305	147	0.215	2756	X-RAY DIFFRACTION	99.52
7.33-47.71	0.1976	146	0.1768	2765	X-RAY DIFFRACTION	97.39

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	1.0998613802	-0.375631486983	-0.0994778154948	3.37436261322	0.523229972729	2.93275927916	0.039846863684	0.1254800483	0.0035230768359	0.209083828655	0.429333641113	0.621092785602	1.37340251527	-0.608382981464	-0.185634017047	1.31275982755	-0.212669449415	0.0184225323901	1.09378481393	-0.105924336795	1.07098608728	-14.6564547048	6.52078437068	74.347264001
2	0.415558134557	-0.481854186782	0.135529060012	0.141465662928	0.251960662026	0.782648335556	-0.135218234242	-0.333585638292	0.236396928132	0.283495160584	0.18256931781	0.097963377015	0.850233545762	0.429886229675	-0.026796208831	0.738867251186	-0.0493774025454	0.064148464936	0.529884679673	-0.071554681932	0.605469344978	5.78888411595	3.66295618519	36.8816145085
3	0.23480828907	-0.32784899302	-0.0986260617074	1.27731204395	0.185337415219	0.0358924281839	0.449342997953	1.02255732897	-0.416381528343	-0.658291525254	0.225819551377	0.537036599207	0.65771578223	-0.298015218179	-0.0720078163103	1.05574955857	0.143282135696	-0.138084299279	1.6248114151	-0.296664261822	1.02901166024	23.6438509978	-15.424492006	6.12005562193
4	0.938731126574	0.157844546916	-0.154668035614	-0.190382802771	0.518239707433	1.25442761598	-0.0634906340529	0.108947856493	0.0235303184075	0.0191004327779	0.0595692835217	-0.113442112871	-0.153955779904	-0.201463060708	-0.0258557533152	0.482025956956	-0.0431816446294	-0.0543923609882	0.376479762505	0.0103414587994	0.462468136485	-3.61172762483	12.2964611386	31.0338556026
5	0.626672759405	-0.410690111527	0.335385196897	1.37790644433	0.891554134877	1.0368232515	0.503470671431	-0.0175954167473	1.00068654947	-0.102557018495	-0.337418732441	-0.333994752682	-0.812619047274	0.50600244628	-0.0255480470931	1.16747164048	0.0792190279461	0.117371537672	1.04680010043	0.0698732534783	1.11948519764	-39.0315718596	14.9019763867	33.9877873753
6	1.16620393145	0.724136890728	-0.307707282349	0.538210625339	-0.288807191034	0.763028112072	0.200100018637	-0.146385568911	0.105878486985	0.149929724391	-0.156327721697	-0.00424948180992	-0.0390508574231	0.257289437548	-0.0338470745009	0.444581844283	-0.00623473918578	0.017954691984	0.432792298158	-0.0258385843594	0.404244008848	6.05796322784	6.37908302571	37.2894942161
7	1.51982290895	-0.0387837502815	-0.400063362379	0.931956162094	-0.0520190043074	2.61491881595	-0.0494136696463	-0.0552975988413	-0.0889707099753	-0.00504346534489	-0.165085316995	-0.0393663869517	-0.668997230097	-0.715235007599	0.114905962046	0.96705085233	0.0801649612275	-0.00534515548649	0.882332473455	0.0522146254257	0.747597272505	23.8631699456	-3.72388923079	67.0209056145
8	1.92384424307	-0.252012549188	-0.416249729344	1.30070637247	0.654338245305	1.00338548839	-0.058035072206	-0.143253098763	-0.180432746375	0.294153147106	0.122788909701	-0.202857547989	0.157399847118	0.327769975528	-0.0580110959314	0.469408988172	-0.0164933252311	0.0123289878865	0.545159375268	0.0674892758343	0.436076404354	19.8278985544	3.38562114257	38.5906804578
9	1.10983289037	0.794077005934	0.932032388023	0.863210197215	0.292816505338	0.764402646024	-0.115363111976	0.0319795428773	-0.108835028675	0.195531609441	0.108085659097	-0.140730523131	-0.143535893141	-0.0527650738014	0.00665841683626	0.531222036711	0.0603620520325	0.0164147930184	0.518195844724	-0.0608559678767	0.495460369646	-5.94036963061	17.8270630108	76.8316627867
10	1.30689022628	-0.680861803148	0.564354085867	1.15489423995	-0.468189218104	0.533081484072	-0.0647190372073	-0.114784365551	-0.196506650095	0.186194069981	0.0685559596371	-0.0433875029753	0.062922903061	0.04633032	-0.00509103171551	0.699982146807	-0.0400334281351	-0.00260594781856	0.732507554665	0.0298240905905	0.643240147366	-10.4064024886	-12.0144934188	66.4285922949
11	1.50639667475	0.187373186896	-0.330662481966	0.361022174245	-0.133175733084	0.803223904839	-0.0751502858105	0.0376573557088	-0.152692820906	0.00745515420137	0.121789410616	0.00162882884907	0.333652821091	-0.0834863647763	-0.0286279447788	0.564834339992	-0.115987132685	0.017619570316	0.419639005884	-0.0864190245393	0.512928174403	-28.9943013559	-13.7529012158	40.6431165763
12	1.77399454351	-0.906964059009	-0.487248648246	2.59399973143	-0.867686232783	1.69329658306	-0.0779008130524	0.653558231119	-0.363178548797	-0.207156798968	-0.394602737343	0.334980321123	0.764732683326	-0.864583331297	0.309492378026	0.73965812592	-0.120798328258	-0.00758495833826	0.803453423793	-0.0914870904469	0.793383265771	-8.86449951365	-14.7799250217	25.1666804043
13	2.49103020829	0.150960631016	-0.945741915635	0.545351615962	-0.659279626221	0.778479085109	0.12458616127	-0.181937993108	0.133463867222	0.146122988379	0.196843499142	0.39447258716	0.476404145428	-0.0610048802839	-0.197031488364	0.584378944935	-0.0115708468267	-0.0112685982572	0.57975782826	0.0814888197714	0.644902741901	-1.39467266501	6.68991362676	54.8470893162
14	0.558591565917	-0.467731827104	-1.35587926457	0.331377758155	1.1654354106	3.20967023861	-0.298956273867	-0.0133621563626	0.143908427395	0.194567812904	0.425028711136	0.30949103885	0.610982475757	0.0943177164891	0.109147639383	0.928958059572	-0.0321104001918	0.162520058695	1.36583843295	0.0675011674793	0.861160641494	-14.8395110314	2.73964480942	78.0205210278
15	3.7308107138	-0.395302223329	-1.4926708029	3.59916626868	0.330975854779	2.08813682901	0.0486521173117	-0.318173435202	-1.2447618804	-0.102347838366	-0.230533157259	-0.110468773604	1.07200884305	0.202283310959	0.0332836335932	0.733661653377	-0.0112933957914	0.0756369886652	0.647565627069	0.0975956871559	0.745579435232	-11.4381496939	-14.2537058992	23.8352337256

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 43 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 44 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 286 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 287 through 493 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 494 through 751 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 752 through 953 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 954 through 1366 )
12	X-RAY DIFFRACTION	12	chain 'C' and (resid -7 through 2 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 3 through 12 )
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 13 through 20 )
15	X-RAY DIFFRACTION	15	chain 'D' and (resid -2 through 7 )

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