PDB-7qqx: SpCas9 bound to FANCF off-target5 DNA substrate

ID or keywords:

Entry

Database: PDB / ID: 7qqx

Title

SpCas9 bound to FANCF off-target5 DNA substrate

Components

(FANCF off-target5 ...) 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	7qqx_validation.pdf.gz	467.8 KB	Display	wwPDB validaton report
Full document	7qqx_full_validation.pdf.gz	479.3 KB	Display
Data in XML	7qqx_validation.xml.gz	53.7 KB	Display
Data in CIF	7qqx_validation.cif.gz	78.1 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/qq/7qqx ftp://data.pdbj.org/pub/pdb/validation_reports/qq/7qqx	HTTPS FTP

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Related structure data

Related structure data	7qqoC 7qqpC 7qqqC 7qqrC 7qqsC 7qqtC 7qquC 7qqvC 7qqwC 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
Related items in Molecule of the Month	#181 - Jan 2015 Cascade and CRISPR similarity (16) #253 - Jan 2021 Expressome similarity (2) #121 - Jan 2010 70S Ribosomes similarity (1) #130 - Oct 2010 Riboswitches similarity (1) #16 - Apr 2001 Aminoacyl-tRNA Synthetases similarity (1) #67 - Jul 2005 TATA-Binding Protein similarity (1) #157 - Jan 2013 Transfer-Messenger RNA similarity (1) #168 - Dec 2013 DNA Helicase similarity (1) #255 - Mar 2021 Cisplatin and DNA similarity (1) #87 - Mar 2007 Zinc Fingers similarity (2) #91 - Jul 2007 Thymine Dimers similarity (1) #229 - Jan 2019 Fluorescent RNA Aptamers similarity (1) #260 - Aug 2021 Ribonuclease P similarity (1) #122 - Feb 2010 Enhanceosome similarity (1) #172 - Apr 2014 RecA and Rad51 similarity (1) #227 - Nov 2018 Telomerase similarity (2) #146 - Feb 2012 Aminoglycoside Antibiotics similarity (1) #15 - Mar 2001 Transfer RNA similarity (1) #249 - Sep 2020 SARS-CoV-2 RNA-dependent RNA Polymerase 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) #48 - Dec 2003 Catabolite Activator Protein similarity (1) #84 - Dec 2006 Transposase similarity (1) #10 - Oct 2000 Ribosome similarity (1) #210 - Jun 2017 Adenine Riboswitch in Action 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) #105 - Sep 2008 Ribonuclease A similarity (1) #226 - Oct 2018 Aminoglycoside Antibiotics and Resistance similarity (1) #245 - May 2020 Spliceosomes similarity (1) #262 - Oct 2021 Fifty Years of Open Access to PDB Structures similarity (1) #65 - May 2005 Self-splicing RNA similarity (1) #98 - Feb 2008 Small Interfering RNA 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) #104 - Aug 2008 Selenocysteine Synthase similarity (1) #106 - Oct 2008 Poly(A) Polymerase similarity (1) #143 - Nov 2011 Toll-like Receptors similarity (1) #161 - May 2013 Ricin similarity (1) #230 - Feb 2019 Initiation Factor eIF4E similarity (1) #266 - Feb 2022 Oligosaccharyltransferase similarity (1) #81 - Sep 2006 Elongation Factors similarity (1) #89 - May 2007 Aconitase and Iron Regulatory Protein 1 similarity (1) #241 - Jan 2020 Twenty Years of Molecules similarity (2)

PDB pages

PDBj /

wwPDB /

NCBI

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Deposited unit

A: FANCF sgRNA

B: CRISPR-associated endonuclease Cas9/Csn1

C: FANCF off-target5 target strand

D: FANCF off-target5 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)	178.534, 67.378, 188.387
Angle α, β, γ (deg.)	90.000, 110.903, 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-target5 ... , 2 types, 2 molecules CD

#3: DNA chain	FANCF off-target5 target strand Mass: 8685.603 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	FANCF off-target5 non-target strand Mass: 3662.404 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, 535 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.: 9 / Source method: obtained synthetically / Formula: K
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 524 / 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: Dec 12, 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→45.94 Å / Num. obs: 81607 / % possible obs: 99.1 % / Redundancy: 6.3 % / B_iso Wilson estimate: 53.65 Å² / CC_1/2: 0.998 / R_merge(I) obs: 0.12 / Net I/σ(I): 10.5
Reflection shell	Resolution: 2.4→2.49 Å / Redundancy: 6.2 % / R_merge(I) obs: 1.72 / Mean I/σ(I) obs: 1 / Num. unique obs: 8058 / CC_1/2: 0.438 / % possible all: 98.8

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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→48 Å / SU ML: 0.3483 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.736
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2163	4091	5.02 %
R_work	0.1822	77473	-
obs	0.1839	81564	99.09 %

Solvent computation

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

Displacement parameters

B_iso mean: 69.66 Å²

Refinement step

Cycle: LAST / Resolution: 2.4→48 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10919	2547	11	524	14001

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0035	13963
X-RAY DIFFRACTION	f_angle_d	0.6239	19357
X-RAY DIFFRACTION	f_chiral_restr	0.0395	2209
X-RAY DIFFRACTION	f_plane_restr	0.0031	2034
X-RAY DIFFRACTION	f_dihedral_angle_d	18.4496	5666

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	0.3581	147	0.3248	2604	X-RAY DIFFRACTION	98.78
2.43-2.46	0.3288	143	0.3203	2659	X-RAY DIFFRACTION	98.94
2.46-2.49	0.3442	136	0.3096	2661	X-RAY DIFFRACTION	98.73
2.49-2.52	0.3318	139	0.3018	2602	X-RAY DIFFRACTION	98.31
2.52-2.56	0.3241	131	0.2863	2598	X-RAY DIFFRACTION	97.81
2.56-2.59	0.3109	156	0.2778	2671	X-RAY DIFFRACTION	98.13
2.59-2.63	0.3451	137	0.2736	2644	X-RAY DIFFRACTION	99.39
2.63-2.67	0.3378	138	0.272	2646	X-RAY DIFFRACTION	99.25
2.67-2.72	0.3093	134	0.2635	2681	X-RAY DIFFRACTION	99.29
2.72-2.76	0.363	145	0.2605	2630	X-RAY DIFFRACTION	99.36
2.76-2.81	0.2866	139	0.2586	2721	X-RAY DIFFRACTION	99.41
2.81-2.87	0.3136	144	0.2419	2612	X-RAY DIFFRACTION	99.1
2.87-2.93	0.2924	146	0.2452	2695	X-RAY DIFFRACTION	99.65
2.93-2.99	0.2853	135	0.2462	2668	X-RAY DIFFRACTION	99.57
2.99-3.06	0.2839	146	0.233	2684	X-RAY DIFFRACTION	99.79
3.06-3.14	0.2681	135	0.2239	2664	X-RAY DIFFRACTION	99.61
3.14-3.22	0.2712	140	0.2066	2696	X-RAY DIFFRACTION	99.58
3.22-3.32	0.2191	144	0.1953	2634	X-RAY DIFFRACTION	99.61
3.32-3.42	0.2312	140	0.1865	2692	X-RAY DIFFRACTION	99.33
3.42-3.54	0.2231	146	0.1909	2709	X-RAY DIFFRACTION	98.96
3.54-3.69	0.2018	133	0.1747	2612	X-RAY DIFFRACTION	97.97
3.69-3.85	0.1845	134	0.1601	2668	X-RAY DIFFRACTION	98.38
3.85-4.06	0.1687	150	0.1437	2681	X-RAY DIFFRACTION	99.89
4.06-4.31	0.1593	134	0.1356	2710	X-RAY DIFFRACTION	99.75
4.31-4.64	0.1558	144	0.1316	2693	X-RAY DIFFRACTION	99.58
4.64-5.11	0.1575	144	0.126	2707	X-RAY DIFFRACTION	99.34
5.11-5.85	0.1937	143	0.1461	2712	X-RAY DIFFRACTION	99.24
5.85-7.36	0.1892	142	0.1639	2698	X-RAY DIFFRACTION	97.59
7.37-48	0.1692	146	0.1497	2821	X-RAY DIFFRACTION	99.46

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	0.204040107141	-0.228414249717	-0.317833765517	0.446207734386	0.770221554815	3.2396132186	0.0116888980625	-0.01275920709	0.0645696179317	0.186889375927	-0.00855614462834	0.0106853322756	0.355716149431	-0.22356949972	0.0057662828963	0.44580975659	-0.0784653267821	0.0204502514528	0.430235541275	-0.0126066696038	0.428804553429	-1.09094836426	4.88809582106	49.8004590118
2	1.82792786958	0.0738953640077	0.332724321437	0.607016407544	-0.271647061776	1.16172661578	0.202054605827	0.347733677262	-0.21853876271	-0.217180878875	-0.102603236917	0.0119395573274	0.103827949775	0.202131343422	-0.0972651125436	0.376590237023	-0.00692741869494	-0.0737913609781	0.386614838315	-0.0326754654541	0.319528828751	6.72205135857	1.69817751601	21.7689070059
3	1.30194451101	-1.63879466644	0.302302858138	2.28958845957	0.529977126131	3.43030709958	1.03200095973	-1.01046815396	1.68340286585	0.590245985371	-0.651531715266	0.0902138867208	-2.62000812484	0.863190908204	-0.0806002038163	1.40748597169	-0.343782343908	0.270823773261	1.01751759722	0.0206045545748	1.29694688872	-39.1942017998	14.7400645215	34.2058004294
4	0.737100206099	0.244121162198	-0.115920874307	0.330133356426	-0.175701070116	0.780311061113	0.0700204089875	0.106836314265	0.0196964468871	0.0768089229259	-0.06772492319	-0.0502323932539	-0.0253900233824	0.211567475412	-0.00119611454115	0.347116206936	-0.0598577696422	-0.0143833292702	0.329292708349	-0.015909929806	0.330017633907	6.11708559557	6.3546981508	37.6213691562
5	3.58978984748	-0.7288378201	-0.476038704013	2.99060151336	-0.201570293264	4.43798215996	-0.0283853053658	0.261893243475	-0.0296651093355	0.109800307291	-0.0641113653575	0.487972275481	-0.44911528577	-0.810617328096	0.0820055496248	0.704489762045	0.0796826703381	-0.0301507934725	0.622462032503	-0.0337847760458	0.62155090295	24.0253412132	-3.65193797367	67.3946664752
6	1.28731307544	-0.0565056140276	-0.30703099323	1.17119961034	0.496382876939	1.50172263254	0.0478673618199	-0.151720992325	-0.19883787054	0.254004490322	-0.0273781654807	-0.159416082579	0.16308900391	0.447791430286	-0.0367829641603	0.395405126765	-0.0326703472189	-0.0723576082931	0.497878472711	0.0670474531917	0.374730945866	19.9142774349	3.34617542915	38.837635075
7	1.66595549181	0.223481477049	-0.683127375984	1.23149231389	-0.963442231409	2.87229266951	-0.0790561873637	-0.0472496480218	0.0319016678412	0.140027294559	0.0154138433326	-0.171587302513	-0.19105804217	0.119485571424	0.0560779499948	0.409642893711	0.00343514861684	-0.0574195280904	0.287355875109	-0.0401694250966	0.361576236284	0.742593691248	19.108768599	80.8690606501
8	0.530654974886	0.155448742759	0.728850822378	0.448779222941	-0.23927666351	1.17682314214	-0.0217911000072	0.011436624591	-0.0205895836102	0.00446836411314	-0.0474192339821	0.00477137857021	0.0488339695513	0.0141819221445	0.0778823665481	0.401189361624	-0.0286985451382	0.0665168732061	0.419279747173	0.020696535524	0.421008456094	-16.5964255121	4.7685334107	62.8346223881
9	1.40745340215	-0.649218432053	-0.266528846648	1.91783116637	0.0934789357131	1.76188988427	-0.0328654041835	-0.143511968851	-0.133410646997	0.0477564601163	-0.000938625032511	0.0745304214959	-0.0366644895786	0.0169587906637	0.00841432586432	0.61186113733	0.0147341614199	-0.0729006598072	0.565377400553	-0.0344828115981	0.643729954817	-2.828548411	-19.0980338152	71.7429834921
10	1.11674611046	0.237398127452	-0.546857595688	0.400273375485	-0.00497339767395	1.91517666219	-0.131584199822	0.0452456427934	-0.218306396395	0.0317012281731	0.0433747821358	-0.00328828478103	0.594126567776	-0.230128034143	0.0785551207525	0.440731704353	-0.0857520551633	-0.0113225228726	0.290612824794	-0.0127522058886	0.406246413481	-29.2431609056	-12.1279846974	43.8091513442
11	4.73530831026	1.19727198236	1.47154676754	1.7436072579	-1.40869215883	2.6572856126	0.121683429621	0.945709628703	-0.376498228131	-0.187785597593	0.282901322604	0.91996712296	0.400766819803	-1.44948031096	0.0947209324362	1.44435375536	-0.725034290579	0.132304646876	2.17257563139	-0.539163465541	1.06807824279	-16.1180212383	-20.1031412593	18.7101571037
12	0.958368996073	0.2348538461	0.294195464457	0.71170514264	0.780280303109	1.04771739858	0.289509809924	-0.343499609975	-0.173192846276	0.39799409752	-0.29787843847	0.114800550355	0.646234964073	-0.196329773148	0.0409518878465	0.554239841404	-0.0801600976443	-0.0512328814314	0.567673533155	0.0232593465682	0.441064444223	2.08918805589	-2.81161159738	40.3829209037
13	0.618108428626	0.244263917383	0.176636524692	0.0631122297762	-0.0863466599074	1.80537954097	0.0713882628021	-0.0670685465623	0.235520002078	-0.0184455811104	0.220752054587	0.152377587977	0.075161020364	-0.0624213039183	-0.365039023064	0.567851178631	-0.0981828645383	0.0499278327877	0.568066399022	0.0374979028761	0.505994348608	-11.9722471922	5.85686618898	71.8499285419
14	4.18974733895	-0.260276617411	-1.67137699689	4.73406288913	0.874870168695	5.07343307729	-0.10870983618	0.661177475674	-1.11143934486	-0.0951910752635	0.31492967579	-0.158506709265	1.3127920759	0.241012935643	-0.396098906294	0.663865061524	-0.0698995408133	-0.00476897712273	0.645270269705	-0.21151158249	0.727221002742	-11.4292736531	-14.4330774466	23.9093068619

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid -1 through 28 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 29 through 68 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 69 through 82 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 3 through 207 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 208 through 286 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 287 through 493 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 494 through 678 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 679 through 808 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 809 through 909 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 910 through 1366 )
11	X-RAY DIFFRACTION	11	chain 'C' and (resid -7 through -3 )
12	X-RAY DIFFRACTION	12	chain 'C' and (resid -2 through 7 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 8 through 20 )
14	X-RAY DIFFRACTION	14	chain 'D' and (resid -2 through 7 )

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