PDB-4zt9: Nuclease-inactive Streptococcus pyogenes Cas9 (D10A/H840A, dCas9)...

ID or keywords:

Entry

Database: PDB / ID: 4zt9

Title

Nuclease-inactive Streptococcus pyogenes Cas9 (D10A/H840A, dCas9) in complex with single-guide RNA at 3.1 Angstrom resolution

Components

CRISPR-associated endonuclease Cas9
single-guide RNA

Keywords

HYDROLASE/RNA /

CRISPR-Cas9 / bacteria adaptive immunity / genome editing and regulation / HYDROLASE-RNA 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)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 3.1 Å

Authors

Jiang, F. / Doudna, J.A.

Citation

Journal: Science / Year: 2015
Title: A Cas9-guide RNA complex preorganized for target DNA recognition.
Authors: Jiang, F. / Zhou, K. / Ma, L. / Gressel, S. / Doudna, J.A.

History

Deposition	May 14, 2015	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jul 8, 2015	Provider: repository / Type: Initial release
Revision 1.1	Sep 27, 2023	Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Source and taxonomy Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / entity_src_gen / pdbx_initial_refinement_model / pdbx_struct_assembly / pdbx_struct_assembly_prop / pdbx_struct_oper_list Item: _citation.journal_id_CSD / _database_2.pdbx_DOI ..._citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity_src_gen.pdbx_alt_source_flag / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_prop.type / _pdbx_struct_assembly_prop.value / _pdbx_struct_oper_list.symmetry_operation

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	4zt9.cif.gz	1.1 MB	Display	PDBx/mmCIF format
PDB format	pdb4zt9.ent.gz	937.5 KB	Display	PDB format
PDBx/mmJSON format	4zt9.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/zt/4zt9 ftp://data.pdbj.org/pub/pdb/validation_reports/zt/4zt9	HTTPS FTP

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Related structure data

Related structure data	4zt0SC S: Starting model for refinement C: citing same article (ref.)
Similar structure data	4zt0-2 4zt0-1 5xbl-d 9066 9067 5opk-1 6ifo-2 0064 6ifo-1 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
Related items in Molecule of the Month	#181 - Jan 2015 Cascade and CRISPR similarity (13) #121 - Jan 2010 70S Ribosomes similarity (1) #130 - Oct 2010 Riboswitches similarity (1) #16 - Apr 2001 Aminoacyl-tRNA Synthetases similarity (1) #157 - Jan 2013 Transfer-Messenger RNA similarity (1) #253 - Jan 2021 Expressome similarity (1) #229 - Jan 2019 Fluorescent RNA Aptamers similarity (1) #260 - Aug 2021 Ribonuclease P similarity (1) #15 - Mar 2001 Transfer RNA similarity (1) #249 - Sep 2020 SARS-CoV-2 RNA-dependent RNA Polymerase similarity (1) #146 - Feb 2012 Aminoglycoside Antibiotics similarity (1) #87 - Mar 2007 Zinc Fingers similarity (1) #210 - Jun 2017 Adenine Riboswitch in Action similarity (1) #10 - Oct 2000 Ribosome 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) #226 - Oct 2018 Aminoglycoside Antibiotics and Resistance similarity (1) #105 - Sep 2008 Ribonuclease A similarity (1) #230 - Feb 2019 Initiation Factor eIF4E similarity (1) #161 - May 2013 Ricin similarity (1) #143 - Nov 2011 Toll-like Receptors similarity (1) #106 - Oct 2008 Poly(A) Polymerase similarity (1) #266 - Feb 2022 Oligosaccharyltransferase similarity (1) #81 - Sep 2006 Elongation Factors similarity (1) #104 - Aug 2008 Selenocysteine Synthase similarity (1) #89 - May 2007 Aconitase and Iron Regulatory Protein 1 similarity (1) #227 - Nov 2018 Telomerase similarity (1) #241 - Jan 2020 Twenty Years of Molecules similarity (1)

PDB pages

PDBj /

wwPDB /

NCBI

Related items in Molecule of the Month

#181 - Jan 2015
Cascade and CRISPR
similarity (13)
#121 - Jan 2010
70S Ribosomes
similarity (1)
#130 - Oct 2010
Riboswitches
similarity (1)
#16 - Apr 2001
Aminoacyl-tRNA Synthetases
similarity (1)
#157 - Jan 2013
Transfer-Messenger RNA
similarity (1)
#253 - Jan 2021
Expressome
similarity (1)
#229 - Jan 2019
Fluorescent RNA Aptamers
similarity (1)
#260 - Aug 2021
Ribonuclease P
similarity (1)
#15 - Mar 2001
Transfer RNA
similarity (1)
#249 - Sep 2020
SARS-CoV-2 RNA-dependent RNA Polymerase
similarity (1)
#146 - Feb 2012
Aminoglycoside Antibiotics
similarity (1)
#87 - Mar 2007
Zinc Fingers
similarity (1)
#210 - Jun 2017
Adenine Riboswitch in Action
similarity (1)
#10 - Oct 2000
Ribosome
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)
#226 - Oct 2018
Aminoglycoside Antibiotics and Resistance
similarity (1)
#105 - Sep 2008
Ribonuclease A
similarity (1)
#230 - Feb 2019
Initiation Factor eIF4E
similarity (1)
#161 - May 2013
Ricin
similarity (1)
#143 - Nov 2011
Toll-like Receptors
similarity (1)
#106 - Oct 2008
Poly(A) Polymerase
similarity (1)
#266 - Feb 2022
Oligosaccharyltransferase
similarity (1)
#81 - Sep 2006
Elongation Factors
similarity (1)
#104 - Aug 2008
Selenocysteine Synthase
similarity (1)
#89 - May 2007
Aconitase and Iron Regulatory Protein 1
similarity (1)
#227 - Nov 2018
Telomerase
similarity (1)
#241 - Jan 2020
Twenty Years of Molecules
similarity (1)

Deposited unit

A: CRISPR-associated endonuclease Cas9

B: single-guide RNA

C: CRISPR-associated endonuclease Cas9

D: single-guide RNA

372 kDa, 4 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: CRISPR-associated endonuclease Cas9

B: single-guide RNA

defined by author&software
186 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

C: CRISPR-associated endonuclease Cas9

D: single-guide RNA

defined by author&software
186 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	102.505, 143.046, 296.145
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

#1: Protein	CRISPR-associated endonuclease Cas9 / Cas9 / CRISPR-Cas9 Mass: 158661.812 Da / Num. of mol.: 2 / Mutation: D10A, H840A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Gene: csn1, cas9, ERS445054_00848, MTB314_0777 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS, Hydrolases; Acting on ester bonds
#2: RNA chain	single-guide RNA Mass: 27501.404 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus pyogenes (bacteria) / Production host: Escherichia coli (E. coli)

#1: Protein

CRISPR-associated endonuclease Cas9 / Cas9 / CRISPR-Cas9

Mass: 158661.812 Da / Num. of mol.: 2 / Mutation: D10A, H840A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Streptococcus pyogenes (bacteria) / Gene: csn1, cas9, ERS445054_00848, MTB314_0777 / Production host:

Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A0C6FZC2, UniProt: Q99ZW2*PLUS,

Hydrolases; Acting on ester bonds

#2: RNA chain

single-guide RNA

Mass: 27501.404 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.)

Streptococcus pyogenes (bacteria) / Production host:

Escherichia coli (E. coli)

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Experiment

Experiment	Method: X-RAY DIFFRACTION

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Sample preparation

Crystal	Density Matthews: 2.92 Å³/Da / Density % sol: 57.82 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6 Details: 12% w/v PEG8000, 100 mM sodium cacodylate, pH 6.0, 250 mM sodium chloride

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Data collection

Diffraction	Mean temperature: 100 K
Diffraction source	Source: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 0.98 Å
Detector	Type: ADSC QUANTUM 315r / Detector: CCD / Date: Nov 17, 2014
Radiation	Monochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 0.98 Å / Relative weight: 1
Reflection	Resolution: 3.1→102.5 Å / Num. all: 79819 / Num. obs: 76812 / % possible obs: 96.2 % / Redundancy: 40 % / B_iso Wilson estimate: 68.52 Å² / R_merge(I) obs: 0.068 / Net I/σ(I): 14.5
Reflection shell	Resolution: 3.1→3.27 Å / R_merge(I) obs: 0.499 / % possible all: 89.8

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Processing

Software

Name	Version	Classification
PHENIX	1.9_1692	refinement
MOSFLM		data reduction
SCALA		data scaling
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: PDB entry 4ZT0

4zt0

Resolution: 3.1→49.357 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.17 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.2841	3871	5.04 %
R_work	0.2429	-	-
obs	0.245	76747	96.19 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 3.1→49.357 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	19188	3090	0	0	22278

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.008	22994
X-RAY DIFFRACTION	f_angle_d	0.911	31866
X-RAY DIFFRACTION	f_dihedral_angle_d	13.871	8731
X-RAY DIFFRACTION	f_chiral_restr	0.042	3772
X-RAY DIFFRACTION	f_plane_restr	0.004	3567

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 (%)
3.1-3.1378	0.3939	120	0.3377	2361	X-RAY DIFFRACTION	89
3.1378-3.1776	0.3664	121	0.333	2396	X-RAY DIFFRACTION	90
3.1776-3.2194	0.3434	135	0.3313	2416	X-RAY DIFFRACTION	90
3.2194-3.2635	0.3544	143	0.3134	2410	X-RAY DIFFRACTION	90
3.2635-3.3101	0.3862	140	0.3015	2408	X-RAY DIFFRACTION	91
3.3101-3.3595	0.3236	126	0.302	2406	X-RAY DIFFRACTION	91
3.3595-3.4119	0.3044	118	0.2957	2487	X-RAY DIFFRACTION	91
3.4119-3.4679	0.3483	127	0.2922	2453	X-RAY DIFFRACTION	93
3.4679-3.5276	0.3099	134	0.2809	2525	X-RAY DIFFRACTION	93
3.5276-3.5918	0.3268	157	0.2717	2491	X-RAY DIFFRACTION	94
3.5918-3.6608	0.3538	138	0.2655	2558	X-RAY DIFFRACTION	95
3.6608-3.7355	0.2984	134	0.2462	2538	X-RAY DIFFRACTION	96
3.7355-3.8167	0.2629	142	0.2491	2609	X-RAY DIFFRACTION	97
3.8167-3.9055	0.2715	132	0.249	2632	X-RAY DIFFRACTION	97
3.9055-4.0031	0.2922	142	0.2434	2646	X-RAY DIFFRACTION	98
4.0031-4.1113	0.2764	146	0.2275	2647	X-RAY DIFFRACTION	99
4.1113-4.2322	0.2822	145	0.2382	2660	X-RAY DIFFRACTION	99
4.2322-4.3687	0.2643	136	0.2205	2681	X-RAY DIFFRACTION	100
4.3687-4.5248	0.2669	147	0.2146	2693	X-RAY DIFFRACTION	100
4.5248-4.7058	0.2461	148	0.2073	2707	X-RAY DIFFRACTION	100
4.7058-4.9198	0.2547	127	0.1958	2742	X-RAY DIFFRACTION	100
4.9198-5.1789	0.2357	145	0.2054	2708	X-RAY DIFFRACTION	100
5.1789-5.503	0.2891	143	0.2168	2730	X-RAY DIFFRACTION	100
5.503-5.9272	0.2829	149	0.2393	2727	X-RAY DIFFRACTION	100
5.9272-6.5225	0.2912	129	0.2429	2775	X-RAY DIFFRACTION	100
6.5225-7.4635	0.325	157	0.2265	2740	X-RAY DIFFRACTION	100
7.4635-9.3927	0.2186	129	0.2129	2827	X-RAY DIFFRACTION	100
9.3927-49.3637	0.252	161	0.2465	2903	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	1.4585	0.0856	-0.2587	1.7222	-0.661	1.3475	-0.0593	0.2637	-0.0195	-0.4957	-0.4645	-0.6962	0.4668	0.7119	-0.5086	0.5192	0.2777	0.3222	0.8594	0.4171	0.6002	40.6547	-8.2608	-36.9619
2	2.2998	-0.2486	0.9698	1.9899	-0.6906	3.1383	-0.1906	0.3331	0.5171	-0.8675	-0.5847	-0.283	0.2162	0.1568	0.3806	0.9311	0.2123	0.2519	0.9329	0.2159	0.8833	39.5201	1.4252	-55.477
3	1.1615	-0.2734	-0.5563	4.8954	-2.6451	2.2646	0.1135	0.4932	0.3759	-0.6653	-0.124	-0.1084	-0.1579	0.0906	-0.036	0.6839	-0.0909	-0.0641	0.9939	0.1213	0.7508	3.2472	20.6054	-50.0476
4	2.8057	-0.2474	0.8006	1.9876	-0.8157	2.2389	-0.1764	0.2013	0.5311	0.0641	0.0719	0.1937	-0.3289	0.119	0.0677	0.374	-0.0449	0.0499	0.4211	0.1147	0.4976	4.9917	5.1281	-25.4832
5	3.4027	1.0312	2.8167	1.6395	0.8053	3.9066	-0.3718	-0.2622	0.9224	-0.7551	-0.67	-0.6051	0.4385	0.1504	-0.0529	0.5357	0.0683	0.1457	1.1515	0.3164	0.7389	41.3037	-6.6219	-36.9759
6	1.8285	-1.8907	0.3624	2.3824	-1.5353	3.1908	-0.1451	-0.3959	1.7029	0.362	-0.8624	-0.6301	-0.6433	1.9718	0.5529	0.8196	-0.2441	-0.1052	2.3109	0.5589	1.6171	72.1616	-1.138	-16.4986
7	3.7283	1.3414	-3.8567	2.1905	-0.4813	4.4803	-0.3337	0.8253	0.8522	0.0312	-0.6886	-0.7991	-0.2939	1.8787	0.8159	1.772	-0.7043	-0.7923	3.3655	0.9715	2.779	73.1138	5.6509	-0.957
8	5.0776	-0.4233	0.2561	2.4587	2.2633	2.1927	0.6933	-1.4621	0.7919	1.2595	-0.5438	-1.0618	0.1551	-0.0198	0.2028	1.3488	-0.8789	-0.6876	2.1204	0.9285	1.9407	69.0535	8.4505	-18.6992
9	0.8111	0.0722	-0.4431	0.7203	-0.4385	1.184	-0.2569	-0.0264	-0.5911	-0.5042	-0.571	-0.7042	0.8372	0.4291	-1.6823	0.884	0.1975	0.6351	0.5982	0.6912	0.5005	35.8436	-17.0314	-30.6066
10	4.007	-0.9287	-1.3773	4.7056	0.5535	1.9552	0.3467	0.7859	-0.0895	-0.1449	-0.9442	0.0493	0.5252	0.4282	0.7788	0.623	0.1757	0.1679	0.5217	0.045	0.5355	8.4122	-25.9279	-22.031
11	2.7027	-0.7088	-4.1773	5.4823	4.4951	8.9529	1.2369	1.2353	0.1195	-0.4546	-1.0181	-0.5219	0.0007	-1.1134	-0.2559	0.691	0.1822	0.0691	0.6233	0.1258	0.6274	-2.3704	-20.2646	-23.1056
12	1.8415	-0.1209	0.7954	0.7346	-0.0901	1.0542	-0.014	0.2348	-0.0608	0.0269	-0.0243	0.2087	-0.0626	-0.1272	0.0874	0.4693	-0.0128	0.0128	0.4554	0.0005	0.2829	11.587	-57.5812	-35.6071
13	1.1859	-1.454	-1.6308	3.0408	0.4028	3.6921	-0.2245	-0.0093	-0.539	-0.0145	-0.0274	0.1988	0.4529	0.0387	0.3071	0.5168	-0.0593	0.0129	0.6339	-0.1075	0.7589	-0.2665	-79.6665	-41.7403
14	2.5938	0.2418	-1.2458	1.6523	0.8277	2.3451	-0.0639	0.6206	-0.2593	-0.4806	0.186	-0.4673	-0.0182	0.2531	-0.1076	0.6719	0.0251	0.0401	0.804	-0.0131	0.5324	23.2794	-59.5007	-59.5069
15	4.2425	-0.9665	0.0929	1.1168	0.0671	1.9159	-0.0088	0.4666	-0.6917	0.0623	-0.0299	-0.0373	0.1794	0.1707	0.1018	0.4722	-0.011	-0.0887	0.5164	-0.0859	0.4631	45.6264	-71.0189	-21.2401
16	5.1547	1.491	-1.1469	2.1488	-0.6072	3.1533	-0.4713	0.0848	-0.3481	0.0316	0.2718	-0.0383	0.3393	-0.0061	0.224	0.4358	0.0564	-0.0315	0.386	-0.0135	0.2693	7.9445	-59.713	-34.053
17	5.4187	0.0216	-2.4798	2.672	-0.3535	4.2387	0.0908	-2.7956	-0.1574	2.8086	0.5728	1.0458	-0.9361	0.4609	-0.7121	1.669	0.2049	0.5477	2.033	0.0522	1.1628	-21.8611	-68.9402	-4.8371
18	5.1569	2.4542	-1.4901	6.8105	1.3424	6.5985	0.4749	-1.0124	-0.3078	2.0688	0.1717	0.1237	0.8967	-0.5789	-0.6469	1.1103	-0.0199	0.2604	1.1407	0.0879	0.8888	-17.2524	-72.9683	-17.0627
19	1.3803	-0.1192	0.9724	1.3535	0.1597	1.1853	0.0689	-0.0883	0.3681	0.2551	-0.1344	-0.0579	-0.1982	0.1166	0.0871	0.526	0.1064	-0.0477	0.4152	-0.0616	0.2692	16.7742	-49.3718	-29.402
20	6.5883	-5.1257	4.4483	6.9907	-2.3518	8.0893	0.7603	2.0068	0.9507	-0.2847	-0.8291	-0.3617	-0.9274	0.9695	0.075	0.8947	-0.0322	-0.1301	0.7244	0.1604	0.8005	48.61	-42.7091	-22.0325

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 3 through 207 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 208 through 692 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 693 through 909 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 910 through 1368 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 11 through 26 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 27 through 31 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 32 through 37 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 38 through 44 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 45 through 68 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 69 through 76 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 77 through 82 )
12	X-RAY DIFFRACTION	12	chain 'C' and (resid 4 through 207 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 208 through 395 )
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 396 through 730 )
15	X-RAY DIFFRACTION	15	chain 'C' and (resid 731 through 1367 )
16	X-RAY DIFFRACTION	16	chain 'D' and (resid 11 through 28 )
17	X-RAY DIFFRACTION	17	chain 'D' and (resid 29 through 36 )
18	X-RAY DIFFRACTION	18	chain 'D' and (resid 37 through 44 )
19	X-RAY DIFFRACTION	19	chain 'D' and (resid 45 through 68 )
20	X-RAY DIFFRACTION	20	chain 'D' and (resid 69 through 82 )

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Feb 9, 2022. New format data for meta-information of EMDB entries

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