PDB-6i1l: Crystal structure of FnCas12a in complex with a crRNA guide and s...

ID or keywords:

Entry

Database: PDB / ID: 6i1l

Title

Crystal structure of FnCas12a in complex with a crRNA guide and ssDNA target

Components

CRISPR-associated endonuclease Cas12a
crRNA (40-MER)
ssDNA target strand

Keywords

HYDROLASE / CRISPR-Cas12a / FnCas12a / CRISPR-Cpf1 / FnCpf1

Function / homology

Function and homology information

Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function

Biological species

Francisella tularensis subsp. novicida (bacteria)
synthetic construct (others)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2.98 Å

Authors

Jinek, M. / Swarts, D.C.

Funding support

Germany,

Switzerland, 3items

Organization	Grant number	Country
European Molecular Biology Organization	ALTF 179-2015	Germany
European Molecular Biology Organization	aALTF 509-2017	Germany
Swiss National Science Foundation	SNSF 31003A_149393	Switzerland

Citation

Journal: Mol. Cell / Year: 2019
Title: Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.
Authors: Swarts, D.C. / Jinek, M.

History

Deposition	Oct 29, 2018	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	Jan 23, 2019	Provider: repository / Type: Initial release
Revision 1.1	Feb 20, 2019	Group: Data collection / Database references Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / pdbx_database_proc / pdbx_seq_map_depositor_info Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2	Jan 24, 2024	Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry
Revision 1.3	Oct 23, 2024	Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

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Related structure data

Related structure data	6i1kC 5nfvS S: Starting model for refinement C: citing same article (ref.)
Similar structure data	6gtg-d 5xuz-2 5xuz-1 5b43-d 5xuu-d 6ehm-d 7ccc-d 5xh7-d Search similar-shape structures by Omokage search (details) Search similar-shape structures by Omokage search (details) 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 Cas12a

B: crRNA (40-MER)

C: ssDNA target strand

D: CRISPR-associated endonuclease Cas12a

E: crRNA (40-MER)

F: ssDNA target strand

hetero molecules

345 kDa, 6 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: CRISPR-associated endonuclease Cas12a

B: crRNA (40-MER)

C: ssDNA target strand

hetero molecules

defined by author&software
Evidence: gel filtration
172 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

D: CRISPR-associated endonuclease Cas12a

E: crRNA (40-MER)

F: ssDNA target strand

hetero molecules

defined by author&software
Evidence: gel filtration
173 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	78.800, 188.580, 284.120
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

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Protein / RNA chain / DNA chain , 3 types, 6 molecules ADBECF

#1: Protein	CRISPR-associated endonuclease Cas12a / CRISPR-associated endonuclease Cpf1 / FnCas12a / FnCpf1 Mass: 152194.125 Da / Num. of mol.: 2 / Mutation: E1006Q Source method: isolated from a genetically manipulated source Details: E1006Q Source: (gene. exp.) Francisella tularensis subsp. novicida (strain U112) (bacteria) Strain: U112 / Gene: cas12a, cpf1, FTN_1397 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: A0Q7Q2, deoxyribonuclease I, EC: 3.1.27.2
#2: RNA chain	crRNA (40-MER) Mass: 12754.537 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain	ssDNA target strand Mass: 6081.977 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 127 molecules

#4: Chemical	ChemComp-MG / MAGNESIUM ION Mass: 24.305 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Mg
#5: Chemical	ChemComp-CIT / CITRIC ACID Mass: 192.124 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C₆H₈O₇
#6: Chemical	ChemComp-K / POTASSIUM ION Mass: 39.098 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: K
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 95 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has protein modification	Y

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 3.09 Å³/Da / Density % sol: 60.18 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 1.6 M trisodium citrate

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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: Mar 16, 2018
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2.98→49.14 Å / Num. obs: 87503 / % possible obs: 99.96 % / Redundancy: 26.2 % / CC_1/2: 0.999 / Net I/σ(I): 18.87
Reflection shell	Resolution: 2.98→3.086 Å / Redundancy: 27.6 % / Mean I/σ(I) obs: 2.22 / CC_1/2: 0.675 / % possible all: 100

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Processing

Software

Name	Version	Classification
PHENIX	(1.14_3211: ???)	refinement
XDS		data reduction
Coot		model building
PHASER		phasing
XSCALE		data scaling

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5NFV

5nfv

Resolution: 2.98→49.14 Å / SU ML: 0.39 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 22.87

	R_factor	Num. reflection	% reflection
R_free	0.2312	8752	10 %
R_work	0.2038	-	-
obs	0.2066	87497	99.99 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å

Refinement step

Cycle: LAST / Resolution: 2.98→49.14 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	20902	2476	207	52	23637

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.005	24253
X-RAY DIFFRACTION	f_angle_d	0.667	33167
X-RAY DIFFRACTION	f_dihedral_angle_d	16.203	14382
X-RAY DIFFRACTION	f_chiral_restr	0.042	3611
X-RAY DIFFRACTION	f_plane_restr	0.004	3814

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 (%)
2.98-3.0139	0.3774	298	0.3504	2603	X-RAY DIFFRACTION	100
3.0139-3.0493	0.377	287	0.3331	2616	X-RAY DIFFRACTION	100
3.0493-3.0865	0.347	283	0.315	2553	X-RAY DIFFRACTION	100
3.0865-3.1256	0.3511	286	0.2932	2596	X-RAY DIFFRACTION	100
3.1256-3.1667	0.2942	293	0.2759	2598	X-RAY DIFFRACTION	100
3.1667-3.2101	0.2788	278	0.2723	2562	X-RAY DIFFRACTION	100
3.2101-3.2559	0.2962	296	0.2626	2612	X-RAY DIFFRACTION	100
3.2559-3.3045	0.2825	290	0.2586	2605	X-RAY DIFFRACTION	100
3.3045-3.3561	0.2772	282	0.257	2568	X-RAY DIFFRACTION	100
3.3561-3.4111	0.2792	292	0.2416	2631	X-RAY DIFFRACTION	100
3.4111-3.4699	0.2612	289	0.2458	2590	X-RAY DIFFRACTION	100
3.4699-3.533	0.2552	285	0.2419	2591	X-RAY DIFFRACTION	100
3.533-3.6009	0.2502	295	0.2331	2622	X-RAY DIFFRACTION	100
3.6009-3.6744	0.2694	282	0.2315	2555	X-RAY DIFFRACTION	100
3.6744-3.7543	0.2532	296	0.2331	2645	X-RAY DIFFRACTION	100
3.7543-3.8416	0.2654	286	0.2186	2588	X-RAY DIFFRACTION	100
3.8416-3.9376	0.2224	292	0.2009	2638	X-RAY DIFFRACTION	100
3.9376-4.044	0.1989	288	0.1865	2576	X-RAY DIFFRACTION	100
4.044-4.163	0.2101	298	0.183	2648	X-RAY DIFFRACTION	100
4.163-4.2973	0.1995	281	0.1829	2585	X-RAY DIFFRACTION	100
4.2973-4.4508	0.1975	292	0.1735	2640	X-RAY DIFFRACTION	100
4.4508-4.6288	0.1986	299	0.1724	2651	X-RAY DIFFRACTION	100
4.6288-4.8393	0.1906	288	0.1699	2609	X-RAY DIFFRACTION	100
4.8393-5.0942	0.2113	293	0.1673	2621	X-RAY DIFFRACTION	100
5.0942-5.413	0.1977	293	0.1708	2647	X-RAY DIFFRACTION	100
5.413-5.8304	0.2055	296	0.1865	2660	X-RAY DIFFRACTION	100
5.8304-6.4159	0.2404	296	0.196	2659	X-RAY DIFFRACTION	100
6.4159-7.3417	0.2165	298	0.1895	2694	X-RAY DIFFRACTION	100
7.3417-9.2397	0.2016	302	0.1648	2723	X-RAY DIFFRACTION	100
9.2397-49.1468	0.2176	318	0.1939	2859	X-RAY DIFFRACTION	100

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	0.4215	-0.0176	0.0146	0.7215	-0.2028	0.4256	0.0397	-0.071	-0.0593	0.0562	-0.0536	-0.0307	0.0112	0.1145	0	0.3743	-0.0102	-0.0436	0.4191	-0.0077	0.4205	-4.8224	-39.6009	81.865
2	0.3965	-0.1953	-0.1512	0.5023	0.0139	0.3411	-0.0988	-0.0209	-0.0722	-0.2535	0.0063	0.1425	0.0938	0.0152	0	0.4788	0.0382	0.0108	0.3263	-0.0017	0.4038	-20.8891	-69.6705	63.4026
3	0.5128	0.3012	-0.2293	0.8737	-0.467	0.558	0.0265	0.007	-0.0388	0.0423	0.1323	0.1376	0.0265	-0.0792	0.075	0.3612	-0.0087	-0.015	0.4073	0.0621	0.4584	-43.2715	-39.7834	88.1155
4	0.0959	0.0494	-0.1282	0.0679	-0.1589	0.4143	0.0247	-0.0678	-0.1099	0.019	0.1092	0.1239	0.0197	-0.1379	-0.0002	0.4385	-0.0249	-0.0655	0.4665	-0.0071	0.6002	-31.0177	-31.8151	67.8827
5	0.2013	0.101	0.0035	0.061	-0.0128	0.1349	-0.1266	-0.117	0.1199	0.0213	-0.4138	-0.0194	0.3767	-0.1258	-0.1421	0.6843	-0.1447	0.0999	0.6352	0.0239	0.8844	-24.3219	-72.8398	82.1502
6	0.0134	0.005	0.014	0.004	0.0045	0.0146	0.0428	-0.2194	-0.0945	-0.1076	0.0136	0.0916	0.0813	0.2111	0.0001	0.4928	-0.0848	-0.1986	0.5936	-0.0157	1.0461	-26.4738	-65.023	79.3118
7	0.1233	0.0566	0.1893	0.0222	0.0847	0.2755	0.1952	-0.2203	-0.1692	0.1999	-0.395	0.0057	0.1011	-0.1383	0.0003	0.4984	0.0124	0.0008	0.5058	-0.0329	0.6222	-17.6033	-39.2272	75.8337
8	0.8416	0.196	-0.232	0.5928	0.0715	0.4502	0.2093	0.2933	0.2045	-0.0612	-0.1542	-0.0345	-0.0242	0.1716	-0.0006	0.4722	0.0083	0.0729	0.605	0.0022	0.4278	-2.2717	-0.5958	21.8112
9	0.4949	0.023	0.1336	-0.0057	-0.0212	0.6667	0.1473	0.155	0.0383	-0.0532	-0.0344	0.0485	-0.0115	-0.1106	0.0003	0.4907	-0.0221	-0.0131	0.5703	0.0374	0.5948	-40.4647	4.7329	25.5625
10	0.7272	0.1003	1.3322	4.3114	-1.6936	3.2809	-0.259	0.5911	-0.1371	-0.4968	-0.4816	-1.5284	-0.171	1.3996	-2.2402	0.3833	0.04	-0.0597	0.795	-0.1095	0.661	15.8377	-37.1832	26.1827
11	0.2317	0.0847	0.1206	0.5043	-0.4365	0.5213	-0.0109	0.0383	-0.0681	-0.1117	0.1308	0.0618	0.1326	0.0487	0	0.5043	-0.0465	0.0028	0.7307	-0.1379	0.4048	-19.6534	-34.064	14.5019
12	0.8402	0.4531	-0.0299	1.1817	0.2644	1.1753	-0.194	0.5029	0.2716	-0.4247	0.2478	0.241	-0.6508	0.0794	0.6716	0.8663	-0.0786	0.0168	0.6256	0.0162	0.36	-26.7763	-23.1886	-14.4932
13	0.0028	0.0116	-0.0074	0.0476	-0.0283	0.0174	-0.1639	0.2376	-0.1575	-0.0349	0.5827	0.5222	-0.117	-0.2327	0.003	0.5834	-0.0173	-0.1247	0.89	-0.1103	0.6461	-22.7659	-38.5231	24.8172
14	0.0345	-0.0227	0.0085	0.0183	0.0012	0.0117	-0.1365	0.3669	-0.2059	-0.2637	-0.1615	0.0385	0.0273	0.127	0.0001	0.8722	0.0011	-0.073	0.7964	-0.1261	0.747	-23.3065	-49.0513	25.746
15	0.0615	-0.0654	-0.0899	0.0682	0.0932	0.1244	0.0664	0.2107	-0.0622	-0.0501	0.0182	0.0134	0.1827	-0.163	-0.0002	0.7342	-0.0468	0.0059	0.7687	-0.1247	0.6062	-8.7529	-28.4246	21.3906
16	0.0107	0.0025	-0.025	0.0061	0.01	0.0781	0.1536	0.987	0.0182	-0.0666	-0.1921	0.3931	-0.3017	0.2952	-0	0.7429	0.1349	-0.1013	1.0518	-0.0422	0.7547	-26.4723	1.4369	15.0903
17	0.0393	0.0173	0.0019	0.0211	-0.0112	0.0096	-0.0552	0.0432	-0.0297	0.1333	0.103	0.3628	-0.2388	-0.0936	-0.0003	0.6991	0.2329	-0.0252	1.28	-0.0071	1.1458	-32.4792	3.4784	8.4372
18	0.0209	0.0189	-0.0394	0.0176	-0.0369	0.076	0.0795	1.1643	-0.3427	-0.005	-0.0469	-0.0067	-0.1764	0.4208	-0.0004	0.7379	0.0444	-0.0428	0.8588	-0.168	0.9146	-19.5793	-6.3295	20.5073
19	0.0633	0.0177	-0.046	0.0237	-0.0058	0.0352	-0.2591	0.4145	-0.2308	-0.0874	-0.1889	0.1451	-0.2356	-0.3465	0.0016	0.6293	0.1568	0.0651	1.0848	-0.165	0.6196	2.0302	-19.0667	19.507
20	0.3698	-0.0843	-0.361	0.465	0.232	0.3968	0.0277	-0.1507	0.3552	0.1148	0.0632	0.096	-0.2387	-0.0013	0.0033	0.5736	-0.0263	0.0634	0.3993	-0.0686	0.5714	-16.1188	0.6009	78.1474

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 0 through 329 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 330 through 613 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 825 through 1299 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid -18 through 16 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 17 through 21 )
6	X-RAY DIFFRACTION	6	chain 'C' and (resid -20 through -16 )
7	X-RAY DIFFRACTION	7	chain 'C' and (resid -15 through -1 )
8	X-RAY DIFFRACTION	8	chain 'D' and (resid 1 through 329 )
9	X-RAY DIFFRACTION	9	chain 'D' and (resid 330 through 613 )
10	X-RAY DIFFRACTION	10	chain 'D' and (resid 614 through 824 )
11	X-RAY DIFFRACTION	11	chain 'D' and (resid 825 through 999 )
12	X-RAY DIFFRACTION	12	chain 'D' and (resid 1000 through 1300 )
13	X-RAY DIFFRACTION	13	chain 'E' and (resid -18 through -9 )
14	X-RAY DIFFRACTION	14	chain 'E' and (resid -8 through -4 )
15	X-RAY DIFFRACTION	15	chain 'E' and (resid -3 through 6 )
16	X-RAY DIFFRACTION	16	chain 'E' and (resid 7 through 21 )
17	X-RAY DIFFRACTION	17	chain 'F' and (resid -20 through -16 )
18	X-RAY DIFFRACTION	18	chain 'F' and (resid -15 through -6 )
19	X-RAY DIFFRACTION	19	chain 'F' and (resid -5 through -1 )
20	X-RAY DIFFRACTION	20	chain 'A' and (resid 614 through 824 )

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