PDB-6wbr: Crystal structure of AceCas9 bound with guide RNA and DNA with 5'...

ID or keywords:

Entry

Database: PDB / ID: 6wbr

Title

Crystal structure of AceCas9 bound with guide RNA and DNA with 5'-NNNCC-3' PAM

Components

CRISPR-associated endonuclease, Csn1 family,CRISPR-associated endonuclease, Csn1 family
DNA (30-MER)
DNA (5'-D(*AP*TP*AP*CP*CP*TP*GP*GP*CP*G)-3')
RNA (94-MER)

Keywords

RNA BINDING PROTEIN/DNA/RNA / DNA endonuclease / CRISPR-Cas9 / HNH / RuvC / RNA BINDING PROTEIN / RNA BINDING PROTEIN-DNA-RNA complex

Function / homology

Function and homology information

endonuclease activity / defense response to virus / DNA binding / RNA binding / zinc ion binding
Similarity search - Function

Biological species

Acidothermus cellulolyticus (bacteria)

Acidothermus cellulolyticus 11B (bacteria)
synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

SAD / Resolution: 2.91 Å

Authors

Li, H. / Das, A.

Funding support

United States, 1items

Citation

Journal: Nat Commun / Year: 2020
Title: The molecular basis for recognition of 5'-NNNCC-3' PAM and its methylation state by Acidothermus cellulolyticus Cas9.
Authors: Das, A. / Hand, T.H. / Smith, C.L. / Wickline, E. / Zawrotny, M. / Li, H.

History

Deposition	Mar 27, 2020	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Nov 18, 2020	Provider: repository / Type: Initial release
Revision 1.1	Jun 2, 2021	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
Revision 1.2	Mar 6, 2024	Group: Advisory / Data collection / Database references Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_unobs_or_zero_occ_atoms Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Summary document	6wbr_validation.pdf.gz	468.8 KB	Display	wwPDB validaton report
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Data in XML	6wbr_validation.xml.gz	38.9 KB	Display
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Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/wb/6wbr ftp://data.pdbj.org/pub/pdb/validation_reports/wb/6wbr	HTTPS FTP

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Related structure data

Related structure data	6wc0C C: citing same article (ref.)
Similar structure data	6wc0-d 6joo-d 6yid-d 2bg5-d 5c3p-d 2eyq-1 5x2g-d 6z89-d 6gos-d 2e9x-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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PDB pages

PDBj /

wwPDB /

NCBI

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Deposited unit

A: CRISPR-associated endonuclease, Csn1 family,CRISPR-associated endonuclease, Csn1 family

B: RNA (94-MER)

C: DNA (30-MER)

D: DNA (5'-D(*AP*TP*AP*CP*CP*TP*GP*GP*CP*G)-3')

152 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)	82.313, 119.348, 177.183
Angle α, β, γ (deg.)	90.000, 90.000, 90.000
Int Tables number	19
Space group name H-M	P2₁2₁2₁
Space group name Hall	P2ac2ab

#1: Protein	CRISPR-associated endonuclease, Csn1 family,CRISPR-associated endonuclease, Csn1 family Mass: 108862.383 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: The sequence is that of the truncated variant. It lacks the HNH catalytic domain.,The sequence is that of the truncated variant. It lacks the HNH catalytic domain.,The sequence is that of ...Details: The sequence is that of the truncated variant. It lacks the HNH catalytic domain.,The sequence is that of the truncated variant. It lacks the HNH catalytic domain.,The sequence is that of the truncated variant. It lacks the HNH catalytic domain. Source: (gene. exp.) Acidothermus cellulolyticus (strain ATCC 43068 / 11B) (bacteria) Strain: ATCC 43068 / 11B / Gene: Acel_1951 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0LWB3
#2: RNA chain	RNA (94-MER) Mass: 30477.963 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Acidothermus cellulolyticus 11B (bacteria)
#3: DNA chain	DNA (30-MER) Mass: 9143.894 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain	DNA (5'-D(APTPAPCPCPTPGPGPCPG)-3') Mass: 3045.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: 56.96 %
Crystal grow	Temperature: 303.15 K / Method: vapor diffusion, hanging drop Details: 0.04 M Citric acid, 0.06 M Bis-tris propane and 15-20% polyethylene glycol 3350

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9792 Å
Detector	Type: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 18, 2019
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.9792 Å / Relative weight: 1
Reflection	Resolution: 2.9→142.6 Å / Num. obs: 55715 / % possible obs: 98.3 % / Observed criterion σ(I): 2 / Redundancy: 7.2 % / B_iso Wilson estimate: 90.9 Å² / CC_1/2: 0.976 / R_merge(I) obs: 0.476 / R_pim(I) all: 0.099 / R_rim(I) all: 0.486 / Net I/σ(I): 6.1
Reflection shell	Resolution: 2.9→3 Å / Redundancy: 7.4 % / R_merge(I) obs: 2 / Mean I/σ(I) obs: 0.9 / Num. unique obs: 4272 / CC_1/2: 0.362 / R_pim(I) all: 0.781 / % possible all: 97.5

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Processing

Software

Name	Version	Classification
PHENIX	1.18.2_3874	refinement
PDB_EXTRACT	3.25	data extraction
XDS		data reduction
AutoProcess		data scaling
PHENIX		phasing

Refinement

Method to determine structure:

SAD / Resolution: 2.91→74.65 Å / SU ML: 0.4832 / Cross valid method: FREE R-VALUE / σ(F): 2 / Phase error: 28.9275
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.2661	1901	5.04 %
R_work	0.2241	35851	-
obs	0.2262	37752	96.24 %

Solvent computation

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

Displacement parameters

B_iso mean: 101.34 Å²

Refinement step

Cycle: LAST / Resolution: 2.91→74.65 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	7400	2760	0	0	10160

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0066	10650
X-RAY DIFFRACTION	f_angle_d	1.1278	15071
X-RAY DIFFRACTION	f_chiral_restr	0.0582	1748
X-RAY DIFFRACTION	f_plane_restr	0.0066	1476
X-RAY DIFFRACTION	f_dihedral_angle_d	20.3527	2538

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 (%)
2.91-2.98	0.3688	144	0.3506	2271	X-RAY DIFFRACTION	87.72
2.98-3.06	0.3488	130	0.31	2465	X-RAY DIFFRACTION	93.45
3.06-3.15	0.3171	142	0.3004	2470	X-RAY DIFFRACTION	94.64
3.15-3.25	0.3084	142	0.3022	2475	X-RAY DIFFRACTION	95.44
3.25-3.37	0.3592	142	0.2859	2531	X-RAY DIFFRACTION	95.94
3.37-3.5	0.3356	131	0.2808	2534	X-RAY DIFFRACTION	96.35
3.5-3.66	0.317	130	0.275	2525	X-RAY DIFFRACTION	96.34
3.66-3.85	0.3458	141	0.2667	2582	X-RAY DIFFRACTION	97.32
3.85-4.1	0.3045	122	0.2509	2600	X-RAY DIFFRACTION	97.91
4.1-4.41	0.2853	124	0.214	2621	X-RAY DIFFRACTION	97.72
4.41-4.86	0.223	137	0.1887	2642	X-RAY DIFFRACTION	98.62
4.86-5.56	0.218	142	0.2006	2616	X-RAY DIFFRACTION	98.22
5.56-7	0.2671	125	0.197	2699	X-RAY DIFFRACTION	98.26
7-74.65	0.1925	149	0.1741	2820	X-RAY DIFFRACTION	99

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	5.407823773	1.31547164225	5.41116381628	1.35633154889	1.23074000959	8.01956138847	0.158159477808	-0.0759238395802	-0.0340254893844	0.16644180557	-0.0154757754096	-0.292308615407	0.0517494089558	0.428788417096	-0.138518450903	0.659264898748	-0.0497020940064	-0.0092956410459	0.475384925554	0.00616472197016	0.527651535237	-18.5807707833	9.88234893575	-10.7222173502
2	0.577441976665	0.234065559668	-0.310066278647	1.05638111354	-1.26601319893	2.28843725958	0.0823321487209	0.0220286691722	0.253960187828	0.255305814031	-0.0940377385175	0.032593227328	-0.281720008392	0.0294040736947	-0.00780552157829	0.809842967806	-0.0854936793175	-0.0631737017951	0.689667909262	-0.0203441104285	0.749524135613	-9.11556306921	31.4341610581	-18.5540161157
3	6.01567343122	3.5254827068	0.0811813342003	2.90783974937	0.162801376658	-0.215298103289	-0.139614868603	0.258530400027	-0.162459028727	-0.403884833899	0.195273875971	-0.36670172868	0.121525011255	-0.0481599832414	-0.114211698263	0.764039565865	0.0257760413637	-0.00563210494102	0.648913930316	0.0502313284502	0.597809520374	-17.6328639263	20.0674272772	-38.6558648972
4	1.9462689325	0.251874750346	0.505193812792	1.34718143089	-0.143486400281	1.89309988822	0.032654791547	0.127629054903	-0.167411231202	0.0862095858664	-0.018475809084	0.232253557981	0.0385095155064	-0.0860491825589	-0.0307097112414	0.595824311162	-0.0448175909911	-0.0437727062365	0.405867288	-0.0645904689909	0.584636155509	-45.748382441	-5.88384807732	-21.9385978924
5	0.105020160857	0.38062451892	0.0660980413972	6.64773603598	-6.38642507877	5.70124823698	0.418898161352	0.151278511066	0.104958599683	0.592365985845	0.246976397138	1.2082579061	-0.696377398041	-0.0488157720404	-0.66617828764	0.951347145825	0.0365137165491	-0.00278545129178	0.640346856066	0.083688866324	0.730434586897	-22.6587867365	24.0899140308	-20.7810871959
6	8.03356200056	0.979499667322	2.52477783397	7.31270410269	-1.27638463444	8.08699414592	1.29668566989	-1.40369664261	-0.244253779213	1.57367962969	-0.0250696702558	0.647651334424	1.37622716663	-0.308687973874	-1.27979900174	1.49473002398	-0.255299834526	-0.0465400726056	0.887492774704	0.213020385808	0.787641804599	-20.9702849678	-1.3681556467	15.2104245195
7	2.25915393233	-3.9297476315	3.49879577518	7.67151869273	-5.6324689266	6.4129544622	0.838242181887	-1.80340145464	-1.43484284116	-0.00743802319798	1.92085376763	3.24220411971	2.72058887422	-0.721911496703	-2.66764250859	2.79468982144	-0.649799068435	0.171106717246	1.87602187017	0.376111541987	1.76649027309	-30.012332839	-7.24222240332	25.2551841932
8	6.12220738743	1.66193945293	4.54929947254	0.715921019521	0.235339333707	8.41880987016	0.18171357545	0.0733715138868	-0.241435163128	0.440872490801	-0.0229731545026	0.00443460590383	0.329326111329	0.597977328553	-0.0964578861008	0.786276000586	-0.0277902561049	0.00480406005331	0.466823890453	0.0449304096494	0.704778361651	-17.3690729655	-0.938422138636	-0.186441177806
9	3.24733996962	0.581965483896	-3.78341416235	7.80703619219	-3.90706021897	5.5096173241	0.420905218915	1.21678731406	-1.76916503987	0.676165032749	-1.03587826759	-1.04849645577	-0.357154698877	2.92665761594	0.553933255841	1.06725284374	0.0859448756042	0.134569162798	2.69938579413	-0.233711099039	1.82026129991	2.45376055417	-10.7757271088	-17.0744727226
10	4.03426518653	-2.00334055756	1.08757385741	7.74936799453	3.19204669768	7.35738746108	-0.209898550941	0.635776109826	0.0135540977508	0.435682880794	0.507708798966	-1.33972304395	0.45250753602	1.76891408352	-0.334378578677	0.858020486981	0.123605486871	0.0316970453095	0.828229184738	0.00267385508381	0.739856752442	-17.5084151532	-4.38898401843	-22.5610110001
11	3.53076430537	-3.57702727512	2.39181612601	3.58456170992	-2.43666470831	1.99688588814	0.0411107949982	1.39236529954	2.23723458778	-1.69238306183	-0.330964022582	-1.32860315607	1.65142046515	2.3846213653	0.122022512699	1.39360637688	0.134457284788	0.258788532976	1.51510891754	0.127537075046	1.25952792818	-25.2170778973	-7.29496915586	-46.4988244162
12	8.40911415711	-4.6483205611	-1.65295944344	5.37416497014	0.209554781522	7.49597629082	0.517985191794	0.144778593967	0.469869329007	0.784455614169	-0.0083272540014	-2.38124475215	0.391970757226	1.07546701161	-0.797987096441	1.05287306635	0.0373587544388	-0.243495849805	0.905825062618	0.0333913501471	1.55925674619	-24.9774503386	-1.88765820446	-42.1497679747
13	7.21081136221	-0.908457219359	-2.02858252409	3.80757377827	-2.02603625485	4.47617017646	-0.256765840627	-0.801601233624	0.612546783085	0.266490932683	0.135412622774	1.1361819615	0.427302020683	0.136683784183	0.0924086397242	0.810520889494	0.0174910975097	-0.097086224565	0.524115306473	-0.0537861465231	0.596024784566	-51.3340069596	-7.60286749154	-5.4230622238
14	3.99859517391	5.33312037337	-3.17784228295	7.79298853182	-1.69015871623	2.6212693401	0.819786663805	0.0168664375709	0.166984506776	0.979428325486	-0.201236949771	0.82736893906	-0.574523851013	0.0598928191246	-0.791767846897	0.857436513393	-0.0057961776084	-0.0982201591244	0.54249097283	0.142255071932	0.833025133382	-21.8921837489	17.0950312659	-13.4760869239
15	5.09169660869	2.203980161	-1.18830128803	1.86478659822	1.09715149946	3.96314736181	0.306970759894	0.680856212601	0.694064853604	0.200698636185	0.2286200142	1.45300072457	-0.586828335139	-0.547462142221	-0.448250118686	1.14537729844	0.138107292419	-0.0934330536683	0.794843569261	0.203862490431	0.786407592803	-28.4189956692	38.922038694	-33.9168182269
16	7.61493521061	0.333702140909	-4.33709554351	4.81535991014	-2.64305754841	4.96250674281	-0.301064614593	-0.569886285556	-0.359232204981	-0.161610367916	-0.138743432296	0.490682701129	0.157185093904	-0.392625365769	0.402912426627	0.957428403525	-0.150858038091	-0.0272913939896	0.694790064744	-0.072258364627	0.654511345236	-51.2060919546	-9.38554081793	-4.41583606018

Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

ID	Refine TLS-ID	Selection details	Auth asym-ID	Label asym-ID	Auth seq-ID	Label seq-ID
1	1	chain 'A' and (resid 7 through 158 )	A	A	7 - 158	1 - 152
2	2	chain 'A' and (resid 159 through 392 )	A	A	159 - 392	153 - 380
3	3	chain 'A' and (resid 393 through 702 )	A	A	393 - 702	381 - 522
4	4	chain 'A' and (resid 703 through 1134 )	A	A	703 - 1134	523 - 942
5	5	chain 'B' and (resid 2 through 21 )	B	B	2 - 21
6	6	chain 'B' and (resid 22 through 31 )	B	B	22 - 31
7	7	chain 'B' and (resid 32 through 41 )	B	B	32 - 41
8	8	chain 'B' and (resid 42 through 51 )	B	B	42 - 51
9	9	chain 'B' and (resid 52 through 61 )	B	B	52 - 61
10	10	chain 'B' and (resid 62 through 76 )	B	B	62 - 76
11	11	chain 'B' and (resid 77 through 81 )	B	B	77 - 81
12	12	chain 'B' and (resid 82 through 94 )	B	B	82 - 94
13	13	chain 'C' and (resid -10 through -1 )	C	C	-10 - -1
14	14	chain 'C' and (resid 1 through 10 )	C	C	1 - 10
15	15	chain 'C' and (resid 11 through 20 )	C	C	11 - 20
16	16	chain 'D' and (resid 1 through 10 )	D	D	1 - 10

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