PDB-5xh7: Crystal structure of the Acidaminococcus sp. BV3L6 Cpf1 RR varian...

ID or keywords:

Entry

Database: PDB / ID: 5xh7

Title

Crystal structure of the Acidaminococcus sp. BV3L6 Cpf1 RR variant in complex with crRNA and target DNA (TCCA PAM)

Components

CRISPR-associated endonuclease Cpf1
Non-target DNA strand
Target DNA strand
crRNA

Keywords

HYDROLASE/RNA/DNA / nuclease / HYDROLASE-RNA-DNA complex

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

Acidaminococcus sp.

Acidaminococcus sp. BV3L6 (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 2 Å

Authors

Nishimasu, H. / Yamano, T. / Ishitani, R. / Nureki, O.

Citation

Journal: Mol. Cell / Year: 2017
Title: Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1
Authors: Nishimasu, H. / Yamano, T. / Gao, L. / Zhang, F. / Ishitani, R. / Nureki, O.

History

Deposition	Apr 19, 2017	Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0	Jun 14, 2017	Provider: repository / Type: Initial release
Revision 1.1	Jul 26, 2017	Group: Database references / Category: citation Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed
Revision 1.2	Nov 22, 2023	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.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.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

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	5xh7.cif.gz	643.6 KB	Display	PDBx/mmCIF format
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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/xh/5xh7 ftp://data.pdbj.org/pub/pdb/validation_reports/xh/5xh7	HTTPS FTP

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Related structure data

Related structure data	5xh6C 5b43S C: citing same article (ref.) S: Starting model for refinement
Similar structure data	5b43-d 5xh6-d 6ehm-d 4pj3-d 6i1l-1 6dll-d 6lhx-d 6gtf-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
Related items in Molecule of the Month	#181 - Jan 2015 Cascade and CRISPR similarity (2) #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) #18 - Jun 2001 Myosin similarity (1) #19 - Jul 2001 Actin similarity (1)

PDB pages

PDBj /

wwPDB /

NCBI

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Deposited unit

A: CRISPR-associated endonuclease Cpf1

B: crRNA

C: Target DNA strand

D: Non-target DNA strand

hetero molecules

180 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)	80.656, 133.298, 200.020
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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DNA chain , 2 types, 2 molecules CD

#3: DNA chain	Target DNA strand Mass: 10475.724 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: DNA (30-MER) / Source: (synth.) Acidaminococcus sp. BV3L6 (bacteria)
#4: DNA chain	Non-target DNA strand Mass: 2964.957 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: DNA (5'-D(CPAPGPTPCPCPTPCPCPA)-3') / Source: (synth.) Acidaminococcus sp. BV3L6 (bacteria)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein	CRISPR-associated endonuclease Cpf1 / AsCpf1 Mass: 151799.359 Da / Num. of mol.: 1 / Mutation: S542R, K607R Source method: isolated from a genetically manipulated source Source: (gene. exp.) Acidaminococcus sp. (strain BV3L6) (bacteria) Strain: BV3L6 / Gene: cpf1, HMPREF1246_0236 / Production host: Escherichia coli (E. coli) References: UniProt: U2UMQ6, Hydrolases; Acting on ester bonds
#2: RNA chain	crRNA Mass: 13756.139 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: RNA (40-MER) / Source: (synth.) Acidaminococcus sp. BV3L6 (bacteria)

#1: Protein

CRISPR-associated endonuclease Cpf1 / AsCpf1

Mass: 151799.359 Da / Num. of mol.: 1 / Mutation: S542R, K607R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Acidaminococcus sp. (strain BV3L6) (bacteria)
Strain: BV3L6 / Gene: cpf1, HMPREF1246_0236 / Production host:

Escherichia coli (E. coli)
References: UniProt: U2UMQ6, Hydrolases; Acting on ester bonds

#2: RNA chain

crRNA

Mass: 13756.139 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: RNA (40-MER) / Source: (synth.)

Acidaminococcus sp. BV3L6 (bacteria)

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Non-polymers , 4 types, 705 molecules

#5: Chemical	ChemComp-NA / SODIUM ION Mass: 22.990 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Na
#6: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#7: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C₂H₆O₂
#8: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 683 / Source method: isolated from a natural source / Formula: H₂O

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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 Å³/Da / Density % sol: 59.05 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 7-10% PEG 3350, 100mM sodium acetate, pH 4.5, 10% 1,6-hexanediol

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Data collection

Diffraction	Mean temperature: 100 K
Diffraction source	Source: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
Detector	Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 18, 2016
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1 Å / Relative weight: 1
Reflection	Resolution: 2→40 Å / Num. obs: 145886 / % possible obs: 99.9 % / Redundancy: 6.4 % / Net I/σ(I): 10.5
Reflection shell	Resolution: 2→2.0227 Å / CC_1/2: 0.841 / R_pim(I) all: 0.466

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Processing

Software

Name	Version	Classification
PHENIX	(1.11.1_2575: ???)	refinement
DIALS		data reduction
Aimless		data scaling
MOLREP		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5B43

5b43

Resolution: 2→39.999 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.7

	R_factor	Num. reflection	% reflection
R_free	0.2144	7402	5.09 %
R_work	0.1827	-	-
obs	0.1843	145320	99.59 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 2→39.999 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	10367	1639	70	683	12759

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.006	12557
X-RAY DIFFRACTION	f_angle_d	0.834	17335
X-RAY DIFFRACTION	f_dihedral_angle_d	16.754	7335
X-RAY DIFFRACTION	f_chiral_restr	0.051	1951
X-RAY DIFFRACTION	f_plane_restr	0.005	1951

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 (%)
2-2.0227	0.3044	234	0.2558	4553	X-RAY DIFFRACTION	99
2.0227-2.0465	0.2898	275	0.2414	4448	X-RAY DIFFRACTION	99
2.0465-2.0715	0.2716	247	0.2376	4561	X-RAY DIFFRACTION	99
2.0715-2.0977	0.2711	226	0.2263	4511	X-RAY DIFFRACTION	99
2.0977-2.1253	0.2887	229	0.2324	4607	X-RAY DIFFRACTION	99
2.1253-2.1544	0.2596	217	0.2199	4534	X-RAY DIFFRACTION	99
2.1544-2.1852	0.242	257	0.2137	4570	X-RAY DIFFRACTION	99
2.1852-2.2178	0.2403	200	0.2062	4540	X-RAY DIFFRACTION	99
2.2178-2.2525	0.2497	242	0.2112	4553	X-RAY DIFFRACTION	99
2.2525-2.2894	0.262	237	0.2019	4562	X-RAY DIFFRACTION	99
2.2894-2.3289	0.2462	252	0.2	4561	X-RAY DIFFRACTION	100
2.3289-2.3712	0.2299	263	0.1973	4572	X-RAY DIFFRACTION	99
2.3712-2.4168	0.2492	260	0.1913	4505	X-RAY DIFFRACTION	100
2.4168-2.4661	0.2428	256	0.1942	4602	X-RAY DIFFRACTION	100
2.4661-2.5197	0.2507	262	0.1971	4550	X-RAY DIFFRACTION	100
2.5197-2.5784	0.2112	234	0.1913	4561	X-RAY DIFFRACTION	100
2.5784-2.6428	0.2303	243	0.1892	4599	X-RAY DIFFRACTION	100
2.6428-2.7143	0.2357	233	0.1928	4579	X-RAY DIFFRACTION	100
2.7143-2.7941	0.2111	243	0.1944	4632	X-RAY DIFFRACTION	100
2.7941-2.8843	0.247	251	0.2061	4592	X-RAY DIFFRACTION	100
2.8843-2.9873	0.232	256	0.2126	4606	X-RAY DIFFRACTION	100
2.9873-3.1069	0.2308	259	0.2089	4577	X-RAY DIFFRACTION	100
3.1069-3.2482	0.2369	241	0.2034	4645	X-RAY DIFFRACTION	100
3.2482-3.4194	0.2397	285	0.1941	4542	X-RAY DIFFRACTION	100
3.4194-3.6335	0.2183	262	0.19	4645	X-RAY DIFFRACTION	100
3.6335-3.9138	0.2044	208	0.1733	4666	X-RAY DIFFRACTION	100
3.9138-4.3073	0.1807	249	0.1539	4682	X-RAY DIFFRACTION	100
4.3073-4.9296	0.1793	248	0.1414	4703	X-RAY DIFFRACTION	100
4.9296-6.207	0.1841	265	0.1632	4747	X-RAY DIFFRACTION	100
6.207-40.0067	0.1657	268	0.159	4913	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	0.5044	0.1054	-0.4961	0.2969	-0.3023	1.7402	0.1401	-0.0505	0.1424	0.1023	0.0235	0.1173	-0.4085	-0.2536	-0.1701	0.37	0.0348	-0.0091	0.3303	0.0228	0.3712	20.3167	6.5344	-18.9959
2	0.1844	-0.1692	-0.1368	0.2758	-0.3097	1.4887	0.0005	-0.0717	0.0761	0.0694	0.0365	-0.0084	-0.3065	0.0335	-0.0319	0.3513	-0.0349	0.0036	0.2645	-0.014	0.3666	37.0307	4.0822	-26.1486
3	0.4954	0.093	0.1458	0.6242	-0.1741	0.816	0.0358	-0.0451	0.0121	0.0659	-0.0667	-0.0145	0.0007	0.1943	0.0282	0.3265	-0.0111	-0.0183	0.2999	0.039	0.2922	47.2032	-15.8382	-31.3563
4	0.8558	0.3998	-0.4752	1.5216	-2.1265	4.0695	-0.0077	-0.2363	-0.1228	0.0689	0.0073	0.0871	0.1045	-0.0575	-0.0052	0.2914	0.0103	0.0206	0.2256	-0.0092	0.2916	30.4768	-38.5159	-15.7887
5	2.2144	-1.3048	-1.1411	2.4193	0.2759	3.2387	0.0353	-0.112	-0.0537	-0.0675	-0.0859	-0.2964	-0.1765	0.6023	0.0434	0.2445	-0.0423	-0.0265	0.3875	0.0512	0.3533	57.8808	-7.594	-39.1946
6	1.0128	-0.3614	0.0397	0.8722	-0.0942	0.3479	0.0426	-0.1585	0.002	0.0894	0.0142	-0.0157	-0.1007	-0.0345	-0.0443	0.3151	0.0118	-0.0151	0.2801	0.037	0.2531	38.5035	-1.9575	-36.4192
7	0.756	-0.3506	1.7663	0.4367	-0.6534	4.5043	0.2921	-0.1238	0.0315	0.0755	0.0198	-0.0716	-0.0107	-0.0359	-0.3389	0.3939	-0.0555	-0.0283	0.4403	-0.0046	0.318	36.5875	-0.8437	-7.8342
8	3.4697	4.2856	-1.699	9.0964	1.8275	4.9069	0.2913	-0.2732	-0.614	0.9812	-0.1687	-0.5068	0.697	0.9823	-0.1421	0.4938	0.0835	-0.0761	0.5429	0.0652	0.4993	33.1114	-10.0547	10.5782
9	5.479	-2.6016	-3.0437	7.3956	3.552	2.4123	-0.3159	-0.2008	0.0463	0.436	-0.303	-0.9778	0.3352	1.8849	0.6377	0.5082	0.1174	-0.03	0.8905	0.1206	0.3901	36.8461	-10.706	2.5711
10	0.2382	-0.0778	0.5312	0.7185	-1.4241	3.4261	0.1733	0.0745	0.0302	0.0577	0.0683	0.0105	-0.0973	0.091	-0.2494	0.3282	0.0277	0.0014	0.3187	-0.0186	0.2811	32.642	1.628	-20.5028
11	2.1933	2.9792	-0.9213	7.8726	1.4438	2.2941	0.2572	0.2929	1.4463	0.0544	-0.591	-0.0718	-1.3218	0.0176	0.2045	0.7944	0.0259	-0.0598	0.3293	0.1205	0.7919	22.1428	24.3429	-51.2284
12	4.26	-0.6113	0.5387	1.6635	-1.1434	1.901	-0.18	0.8943	1.1362	-0.928	0.1128	0.099	-0.8692	-0.4046	0.0495	0.9813	0.0511	0.0236	0.4331	0.1548	0.59	22.436	25.224	-51.5204

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 1 through 325 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 326 through 804 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 805 through 1024 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 1025 through 1307 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid -19 through -5 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid -4 through 5 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 6 through 15 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 16 through 20 )
9	X-RAY DIFFRACTION	9	chain 'C' and (resid -19 through -15 )
10	X-RAY DIFFRACTION	10	chain 'C' and (resid -14 through 0 )
11	X-RAY DIFFRACTION	11	chain 'C' and (resid 1 through 10 )
12	X-RAY DIFFRACTION	12	chain 'D' and (resid -10 through -1 )

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