PDB-6u2m: Crystal structure of a HaloTag-based calcium indicator, HaloCaMP ...

ID or keywords:

Entry

Database: PDB / ID: 6u2m

Title

Crystal structure of a HaloTag-based calcium indicator, HaloCaMP V2, bound to JF635

Components

HaloCaMP V2

Keywords

HYDROLASE / HaloTag / calcium / sensor / fluorescent

Function / homology

Chem-PUJ

Function and homology information

Biological species

Rhodococcus sp. (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT /

molecular replacement / Resolution: 2 Å

Authors

Deo, C. / Schreiter, E.R.

Citation

Journal: Nat.Chem.Biol. / Year: 2021
Title: The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.
Authors: Deo, C. / Abdelfattah, A.S. / Bhargava, H.K. / Berro, A.J. / Falco, N. / Farrants, H. / Moeyaert, B. / Chupanova, M. / Lavis, L.D. / Schreiter, E.R.

History

Deposition	Aug 20, 2019	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Sep 30, 2020	Provider: repository / Type: Initial release
Revision 1.1	Apr 28, 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2	Jun 9, 2021	Group: Database references / Category: citation / citation_author Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3	Oct 11, 2023	Group: Data collection / Database references / Refinement description Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	6u2m.cif.gz	393.4 KB	Display	PDBx/mmCIF format
PDB format	pdb6u2m.ent.gz	320.9 KB	Display	PDB format
PDBx/mmJSON format	6u2m.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/u2/6u2m ftp://data.pdbj.org/pub/pdb/validation_reports/u2/6u2m	HTTPS FTP

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Related structure data

Related structure data	6u32C 5uy1S S: Starting model for refinement C: citing same article (ref.)
Similar structure data	1vc4-3 6wv3-d 2ymu-2 6xt8-1 3huh-d 4j84-d 4exn-1 4mst-d 2bdh-5 4x2e-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 - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: HaloCaMP V2

C: HaloCaMP V2

hetero molecules

113 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: HaloCaMP V2

hetero molecules

defined by author
Evidence: gel filtration
56.7 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

C: HaloCaMP V2

hetero molecules

defined by author
56.7 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	92.560, 60.662, 122.600
Angle α, β, γ (deg.)	90.000, 91.020, 90.000
Int Tables number	3
Space group name H-M	P121

#1: Protein	HaloCaMP V2 Mass: 55849.035 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rhodococcus sp. (bacteria) / Production host: Escherichia coli (E. coli)
#2: Chemical	ChemComp-PUJ / (1E,3S)-1-{10-[2-carboxy-5-({2-[2-(hexyloxy)ethoxy]ethyl}carbamoyl)phenyl]-7-(3-fluoroazetidin-1-yl)-5,5-dimethyldibenz o[b,e]silin-3(5H)-ylidene}-3-fluoroazetidin-1-ium / JF635 Mass: 704.898 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₃₉H₄₈F₂N₃O₅Si / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical	ChemComp-CL / CHLORIDE ION Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#4: Chemical	ChemComp-CA / CALCIUM ION Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#5: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H₂O
Has ligand of interest	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: 3.08 Å³/Da / Density % sol: 60.08 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop Details: 30% (v/v) PEG 200, 100 mM MES/ Sodium hydroxide pH 6.0, and 5% (w/v) PEG 3000

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Data collection

Diffraction

Mean temperature: 100 K / Serial crystal experiment: N

Diffraction source

Source:

SYNCHROTRON / Site:

ALS

/ Beamline: 8.2.1 / Wavelength: 0.9774 Å

Detector

Type: ADSC QUANTUM 315 / Detector: CCD / Date: Mar 22, 2018

Radiation

Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray

Radiation wavelength

Wavelength: 0.9774 Å / Relative weight: 1

Reflection

Resolution: 2→122.58 Å / Num. all: 90798 / Num. obs: 90798 / % possible obs: 98.6 % / Redundancy: 4.6 % / R_pim(I) all: 0.051 / R_rim(I) all: 0.109 / R_sym value: 0.096 / Net I/av σ(I): 3.6 / Net I/σ(I): 6.5 / Num. measured all: 421740

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	R_merge(I) obs	Mean I/σ(I) obs	Num. measured all	Num. unique obs	R_pim(I) all	R_rim(I) all	R_sym value	Net I/σ(I) obs	% possible all
2-2.11	4.4	1.347	0.5	57814	13074	0.696	1.523	1.347	1.1	97.6
2.11-2.24	4.5	0.727	1	56183	12373	0.373	0.82	0.727	2	97.9
2.24-2.39	4.6	0.415	1.8	54142	11674	0.213	0.468	0.415	3.1	98.3
2.39-2.58	4.8	0.256	2.8	52712	10934	0.129	0.288	0.256	4.7	98.6
2.58-2.83	4.7	0.163	4.2	47017	10099	0.085	0.184	0.163	6.6	98.7
2.83-3.16	4.8	0.119	5.2	43922	9151	0.062	0.135	0.119	9.3	99
3.16-3.65	4.7	0.089	6.4	37863	8121	0.048	0.102	0.089	12	99.2
3.65-4.47	4.8	0.073	7.4	33145	6917	0.038	0.083	0.073	14	99.4
4.47-6.32	4.6	0.065	8.1	24768	5400	0.035	0.074	0.065	14	99.6
6.32-74.5	4.6	0.052	6.3	14174	3055	0.029	0.06	0.052	14.4	99.4

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Phasing

Phasing	Method: molecular replacement

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Processing

Software

Name	Version	Classification
REFMAC	5.8.0189	refinement
SCALA	3.3.22	data scaling
PHASER		phasing
PDB_EXTRACT	3.25	data extraction
iMOSFLM		data reduction

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT
Starting model: 5UY1

5uy1

Resolution: 2→122.58 Å / Cor.coef. F_o:F_c: 0.968 / Cor.coef. F_o:F_c free: 0.954 / WR_factor R_free: 0.2433 / WR_factor R_work: 0.2005 / FOM work R set: 0.7524 / SU B: 10.639 / SU ML: 0.135 / SU R Cruickshank DPI: 0.1419 / SU R_free: 0.1381 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.142 / ESU R Free: 0.138 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED

	R_factor	Num. reflection	% reflection	Selection details
R_free	0.2255	4438	4.9 %	RANDOM
R_work	0.1857	-	-	-
obs	0.1877	86312	98.25 %	-

Solvent computation

Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso max: 165.37 Å² / B_iso mean: 52.533 Å² / B_iso min: 28.94 Å²

	B_aniso -1	B_aniso -2	B_aniso -3
1-	-1.16 Å²	0 Å²	0.09 Å²
2-	-	-0.75 Å²	0 Å²
3-	-	-	1.9 Å²

Refinement step

Cycle: final / Resolution: 2→122.58 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	7420	0	110	203	7733
B_iso mean	-	-	84.58	47.8	-
Num. residues	-	-	-	-	928

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.026	0.019	7724
X-RAY DIFFRACTION	r_bond_other_d	0.002	0.02	7086
X-RAY DIFFRACTION	r_angle_refined_deg	2.44	1.956	10492
X-RAY DIFFRACTION	r_angle_other_deg	1.285	2.983	16456
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	7.406	5	924
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	33.783	24.136	382
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	16.937	15	1268
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	21.358	15	54
X-RAY DIFFRACTION	r_chiral_restr	0.163	0.2	1108
X-RAY DIFFRACTION	r_gen_planes_refined	0.014	0.021	8568
X-RAY DIFFRACTION	r_gen_planes_other	0.002	0.02	1582

LS refinement shell

Resolution: 2→2.052 Å / R_factor R_free error: 0 / Total num. of bins used: 20

	R_factor	Num. reflection	% reflection
R_free	0.362	323	-
R_work	0.362	6270	-
all	-	6593	-
obs	-	-	96.91 %

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	3.059	1.996	-2.0886	2.1199	-2.072	2.0479	0.0166	0.0491	-0.1307	-0.0714	-0.0356	0.0289	0.0485	0.0386	0.019	0.1483	-0.0197	0.0145	0.0477	-0.0179	0.1964	11.079	38.154	75.996
2	1.5921	-3.7662	2.3335	15.1139	7.2582	29.7974	-0.1884	-0.0829	-0.0712	0.7658	-0.0454	0.3268	0.4566	-0.681	0.2338	0.1277	-0.0802	0.0324	0.1812	-0.0709	0.04	14.5466	31.2902	60.7087
3	0.6256	-0.3899	-0.6199	0.92	-0.045	0.8898	0.0778	-0.3586	0.2045	0.1119	0.2441	0.053	-0.1804	0.3401	-0.3219	0.0794	-0.0382	0.0146	0.208	-0.1168	0.2147	16.025	24.7376	45.0532
4	2.2762	0.9382	-0.761	2.4985	1.0911	1.8114	-0.4554	-0.577	-0.3126	0.1745	0.2263	0.2043	0.3982	0.5174	0.2291	0.2027	0.1836	0.045	0.2521	0.1668	0.2158	10.2776	4.5467	50.3778
5	3.7212	4.2196	2.5054	5.694	4.8665	6.5898	-0.2058	-0.1232	-0.3212	0.1566	0.0188	-0.2538	0.6398	0.2021	0.1871	0.2007	0.0869	0.0155	0.0549	0.043	0.2502	17.6347	-3.0227	41.1947
6	1.1972	-0.0306	-0.759	0.5396	-0.0716	0.9352	-0.0554	-0.1031	-0.0953	-0.0845	0.0738	-0.0296	0.068	0.0867	-0.0184	0.0962	0.0107	-0.0332	0.0242	-0.0171	0.2196	13.5045	11.3174	33.7627
7	1.2299	1.1828	3.4568	1.1493	3.3153	9.7994	0.2497	0.1742	-0.0869	0.2582	0.1132	-0.0562	0.8129	0.5238	-0.363	0.2085	0.1002	-0.0359	0.2449	-0.0524	0.2043	13.9949	21.3339	65.9808
8	1.8315	0.8363	-0.2146	1.0288	-0.3619	0.1433	0.2609	-0.1805	0.0384	-0.0098	-0.1861	0.0956	0.0059	0.0793	-0.0748	0.1124	-0.0328	0.0141	0.0587	-0.0697	0.2042	7.2102	30.7691	86.2387
9	1.8527	0.8616	2.7303	1.7402	-0.0243	5.3473	0.0997	0.0339	-0.0514	0.1235	0.1314	0.0368	0.148	-0.0867	-0.2312	0.1473	0.0063	-0.0561	0.0388	-0.0032	0.2035	2.5987	42.2963	73.7193
10	2.7092	-0.2941	0.133	0.7762	-0.4767	1.1315	-0.0927	0.2878	0.2805	0.1172	-0.1162	-0.0418	-0.2183	0.1819	0.209	0.1564	-0.0427	-0.0546	0.0562	0.0534	0.1642	12.8191	49.9831	73.806
11	2.144	2.0869	0.9548	2.2756	1.2734	0.9251	-0.0361	0.041	0.0816	-0.0371	-0.0368	0.0604	-0.001	-0.083	0.0729	0.1286	0.0383	-0.0132	0.0453	0.0412	0.2663	30.8961	26.4791	136.7338
12	0.0537	-0.386	-0.7754	2.845	5.7437	11.6139	-0.0463	0.0424	-0.0151	0.2451	-0.2652	0.1464	0.4541	-0.5557	0.3115	0.2027	-0.0205	0.0271	0.312	0.0561	0.102	28.8231	33.6916	121.9078
13	0.3616	-0.4114	0.5048	1.0725	-0.5603	0.8622	0.0878	-0.2948	-0.117	0.0922	0.2212	-0.0136	0.119	-0.3627	-0.309	0.0941	-0.115	-0.0517	0.2778	0.0745	0.2252	27.9997	39.9834	106.9309
14	4.3886	2.641	0.1764	2.8013	-0.8362	0.74	-0.4172	-0.808	0.4291	0.0774	-0.0349	-0.294	-0.2726	-0.3809	0.4521	0.1439	0.1951	-0.1802	0.3155	-0.287	0.3042	35.1215	60.9782	112.5217
15	0.8617	0.052	0.7457	0.5142	0.0606	0.9286	-0.0847	-0.1499	0.144	-0.1103	0.0316	0.0279	-0.0554	-0.1218	0.0532	0.0787	0	0.0015	0.0413	-0.0311	0.2206	29.9617	54.8929	97.2107
16	1.9001	2.903	-1.576	6.4042	-6.132	8.3649	-0.0274	0.4019	-0.194	0.2206	0.6411	0.1015	-0.4526	-0.4061	-0.6137	0.1373	0.0627	0.0461	0.2533	0.0316	0.1435	30.9521	46.0877	123.5233
17	1.0675	1.1378	0.6132	2.5727	-1.7039	6.917	0.0533	-0.0474	-0.0298	-0.071	-0.0707	0.1436	-0.1669	-0.264	0.0173	0.1176	0.0434	-0.0378	0.1015	-0.0129	0.1244	23.4311	37.675	137.8942
18	2.0302	0.7821	0.2464	0.7813	0.1942	0.1947	0.2566	-0.1901	-0.0563	0.0107	-0.2092	-0.0653	-0.0009	-0.0634	-0.0473	0.0982	-0.0023	-0.0153	0.0661	0.0616	0.2035	35.7964	33.7107	147.4929
19	4.1515	1.0164	-3.7773	4.7335	-0.8976	3.4427	0.1273	0.19	0.2175	0.2149	0.1029	-0.0759	-0.1057	-0.1638	-0.2302	0.1105	0.0662	0.0024	0.0476	-0.0002	0.2056	39.3417	22.1922	134.6044
20	3.065	-0.4113	-0.2414	0.1536	0.4322	1.694	-0.0102	0.2533	-0.2725	0.0681	-0.0715	0.0531	0.1955	-0.1709	0.0817	0.1653	-0.0119	0.021	0.0388	-0.0527	0.187	29.1069	14.6671	134.5818

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Auth asym-ID	Auth seq-ID
1	X-RAY DIFFRACTION	1	A	4 - 26
2	X-RAY DIFFRACTION	2	A	27 - 31
3	X-RAY DIFFRACTION	3	A	32 - 109
4	X-RAY DIFFRACTION	4	A	110 - 159
5	X-RAY DIFFRACTION	5	A	160 - 179
6	X-RAY DIFFRACTION	6	A	202 - 335
7	X-RAY DIFFRACTION	7	A	336 - 351
8	X-RAY DIFFRACTION	8	A	352 - 419
9	X-RAY DIFFRACTION	9	A	420 - 436
10	X-RAY DIFFRACTION	10	A	437 - 489
11	X-RAY DIFFRACTION	11	C	4 - 26
12	X-RAY DIFFRACTION	12	C	27 - 31
13	X-RAY DIFFRACTION	13	C	32 - 109
14	X-RAY DIFFRACTION	14	C	110 - 154
15	X-RAY DIFFRACTION	15	C	155 - 337
16	X-RAY DIFFRACTION	16	C	338 - 345
17	X-RAY DIFFRACTION	17	C	346 - 357
18	X-RAY DIFFRACTION	18	C	358 - 419
19	X-RAY DIFFRACTION	19	C	420 - 436
20	X-RAY DIFFRACTION	20	C	437 - 489

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