PDB-8use: Crystal Structure of Kemp Eliminase HG649 in unbound state, 280 K

ID or keywords:

Entry

Database: PDB / ID: 8use

Title

Crystal Structure of Kemp Eliminase HG649 in unbound state, 280 K

Components

Kemp eliminase

Keywords

HYDROLASE / De novo enzyme design / computational protein design / Kemp eliminases / biocatalysis / directed evolution / X-ray crystallography

Biological species

Thermoascus aurantiacus (fungus)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.55 Å

Authors

Seifinoferest, B.

Funding support

Canada,

France,

United States, 4items

Organization	Grant number	Country
Canada Foundation for Innovation		Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)		Canada
Human Frontier Science Program (HFSP)		France
Department of Energy (DOE, United States)		United States

Citation

Journal: J.Am.Chem.Soc. / Year: 2024
Title: Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.
Authors: Rakotoharisoa, R.V. / Seifinoferest, B. / Zarifi, N. / Miller, J.D.M. / Rodriguez, J.M. / Thompson, M.C. / Chica, R.A.

History

Deposition	Oct 27, 2023	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Dec 6, 2023	Provider: repository / Type: Initial release
Revision 1.1	Jun 19, 2024	Group: Database references / Category: citation / citation_author Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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 / _citation_author.identifier_ORCID

Structure viewer	Molecule: MolmilJmol/JSmol

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PDBx/mmCIF format	8use.cif.gz	387.4 KB	Display	PDBx/mmCIF format
PDB format	pdb8use.ent.gz	323.5 KB	Display	PDB format
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Validation report

Summary document	8use_validation.pdf.gz	419.5 KB	Display	wwPDB validaton report
Full document	8use_full_validation.pdf.gz	421.7 KB	Display
Data in XML	8use_validation.xml.gz	30.5 KB	Display
Data in CIF	8use_validation.cif.gz	48.5 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/us/8use ftp://data.pdbj.org/pub/pdb/validation_reports/us/8use	HTTPS FTP

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Related structure data

Related structure data	8usfC 8usgC 8ushC 8usiC 8usjC 8uskC 8uslC C: citing same article (ref.)

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Kemp eliminase

B: Kemp eliminase

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

1

A: Kemp eliminase

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

2

B: Kemp eliminase

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

Unit cell

Length a, b, c (Å)	51.130, 77.466, 93.785
Angle α, β, γ (deg.)	90.00, 105.39, 90.00
Int Tables number	4
Space group name H-M	P12₁1

#1: Protein	Kemp eliminase Mass: 34402.652 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermoascus aurantiacus (fungus) / Production host: Escherichia coli (E. coli)
#2: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 690 / 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: 2.6 Å³/Da / Density % sol: 52.74 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion Details: 12.5 mg/mL protein, 0.3 M ammonium sulfate, 30% PEG4000

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Data collection

Diffraction	Mean temperature: 280 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.1 Å
Detector	Type: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jun 15, 2023
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.1 Å / Relative weight: 1
Reflection	Resolution: 1.55→90.42 Å / Num. obs: 101581 / % possible obs: 99.4 % / Redundancy: 3.3 % / B_iso Wilson estimate: 14.58 Å² / CC_1/2: 0.991 / R_merge(I) obs: 0.11 / R_pim(I) all: 0.07 / R_rim(I) all: 0.131 / Net I/σ(I): 7.9
Reflection shell	Resolution: 1.55→1.58 Å / Redundancy: 3.1 % / R_merge(I) obs: 0.77 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 4971 / CC_1/2: 0.505 / R_pim(I) all: 0.513 / R_rim(I) all: 0.928 / % possible all: 98.6

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Processing

Software

Name	Version	Classification
xia2	0.5.492	data reduction
DIALS	Xia2 0.5.492	data reduction
Aimless	Xia2 0.5.492	data scaling
Coot	0.8.9.236	model building
PHASER	v1.13.2998	phasing
PHENIX	v1.13.2998	refinement

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.55→90.42 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 17.5 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1727	4938	4.86 %
R_work	0.1471	-	-
obs	0.1484	101519	99.35 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 1.55→90.42 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4574	0	0	690	5264

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.005	5361
X-RAY DIFFRACTION	f_angle_d	0.811	7421
X-RAY DIFFRACTION	f_dihedral_angle_d	12.58	1975
X-RAY DIFFRACTION	f_chiral_restr	0.048	827
X-RAY DIFFRACTION	f_plane_restr	0.006	1014

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 (%)
1.55-1.57	0.3018	122	0.2633	3166	X-RAY DIFFRACTION	99
1.57-1.59	0.2597	168	0.2468	3181	X-RAY DIFFRACTION	99
1.59-1.61	0.2414	157	0.2419	3201	X-RAY DIFFRACTION	99
1.61-1.63	0.275	136	0.2427	3231	X-RAY DIFFRACTION	99
1.63-1.65	0.258	144	0.2252	3232	X-RAY DIFFRACTION	99
1.65-1.67	0.2564	170	0.2268	3120	X-RAY DIFFRACTION	99
1.67-1.69	0.2445	186	0.2185	3177	X-RAY DIFFRACTION	99
1.69-1.72	0.2343	169	0.2086	3204	X-RAY DIFFRACTION	99
1.72-1.75	0.2186	170	0.2006	3150	X-RAY DIFFRACTION	99
1.75-1.77	0.2544	185	0.2053	3184	X-RAY DIFFRACTION	99
1.77-1.8	0.1967	172	0.194	3198	X-RAY DIFFRACTION	99
1.8-1.84	0.2271	142	0.1772	3218	X-RAY DIFFRACTION	99
1.84-1.87	0.1673	161	0.1649	3210	X-RAY DIFFRACTION	99
1.87-1.91	0.1814	175	0.1577	3191	X-RAY DIFFRACTION	99
1.91-1.95	0.1667	172	0.1489	3210	X-RAY DIFFRACTION	99
1.95-2	0.1586	174	0.1417	3203	X-RAY DIFFRACTION	99
2-2.05	0.1774	192	0.1433	3226	X-RAY DIFFRACTION	100
2.05-2.1	0.1712	161	0.1409	3226	X-RAY DIFFRACTION	100
2.1-2.17	0.1637	170	0.1317	3206	X-RAY DIFFRACTION	100
2.17-2.24	0.1705	165	0.1267	3239	X-RAY DIFFRACTION	100
2.24-2.32	0.1417	146	0.1254	3262	X-RAY DIFFRACTION	100
2.32-2.41	0.1652	152	0.1268	3243	X-RAY DIFFRACTION	100
2.41-2.52	0.1481	145	0.1265	3264	X-RAY DIFFRACTION	100
2.52-2.65	0.1542	144	0.1356	3270	X-RAY DIFFRACTION	100
2.65-2.82	0.1638	169	0.1388	3249	X-RAY DIFFRACTION	100
2.82-3.03	0.166	171	0.1423	3249	X-RAY DIFFRACTION	100
3.03-3.34	0.1528	176	0.1367	3239	X-RAY DIFFRACTION	100
3.34-3.82	0.1532	185	0.1246	3248	X-RAY DIFFRACTION	100
3.82-4.82	0.1377	176	0.114	3245	X-RAY DIFFRACTION	100
4.82-90.42	0.1795	183	0.1444	3339	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	1.3223	0.2411	0.7737	1.5588	0.4857	2.6091	0.029	-0.0048	-0.0753	0.0519	-0.0518	-0.092	0.0637	-0.0005	0.0232	0.1218	-0.0044	-0.0082	0.104	0.0074	0.1366	13.5077	4.8291	36.6507
2	1.0435	-0.0976	-0.0122	0.7938	0.0715	0.2629	0.0359	0.0775	0.0528	-0.0171	-0.0733	0.1157	-0.0263	-0.0617	0.0385	0.1439	0.0056	-0.0184	0.1782	-0.0076	0.1689	-5.0701	11.5823	35.1288
3	1.6182	-0.2449	0.5414	1.2046	-0.3999	0.8091	0.0376	-0.1686	-0.0276	0.0564	-0.0602	0.2074	0.0338	-0.1089	0.0293	0.0977	-0.0136	0.009	0.1552	-0.0313	0.1438	-8.154	13.7822	44.106
4	2.9061	1.0297	0.0555	0.5273	0.1193	0.186	-0.0727	-0.0342	0.3186	-0.1466	-0.0025	0.3495	-0.0578	-0.101	0.0658	0.1554	0.0172	-0.0275	0.16	-0.0291	0.231	-3.3376	31.962	39.6945
5	1.6728	-0.4009	0.3897	4.7643	-1.8614	2.2352	0.0307	-0.4053	0.3006	0.3205	-0.0527	0.1781	-0.1762	-0.1648	0.0176	0.1235	-0.0147	0.028	0.2201	-0.0805	0.1905	-7.0479	23.163	49.7815
6	0.9366	0.4262	-0.0456	1.2448	0.0484	0.704	0.0485	-0.1484	0.1162	0.0867	-0.0736	0.0444	-0.0561	-0.0492	0.023	0.1419	0.0023	-0.0085	0.1644	-0.0285	0.1474	6.606	24.5397	46.317
7	0.624	0.0096	-0.0013	1.0625	0.5759	1.1432	0.0235	-0.0102	-0.0151	0.0003	0.0102	-0.0912	-0.0422	0.0839	-0.0334	0.1005	-0.0022	-0.012	0.1135	-0.0061	0.1324	18.5661	16.4769	37.4396
8	1.2159	0.1203	0.5329	1.5808	0.6197	2.8186	-0.0378	0.0502	0.1116	-0.0249	-0.0007	-0.0768	-0.0315	0.1093	0.0502	0.1649	0.0048	0.007	0.119	0.0113	0.1416	4.8251	11.5213	6.2029
9	1.3478	0.5364	-0.282	1.2303	-0.2407	0.7641	0.0832	-0.2288	0.0244	0.2675	-0.1126	0.3109	0.0238	-0.097	0.0363	0.2663	-0.0113	0.0698	0.2179	-0.0104	0.2077	-10.9725	4.9105	18.7249
10	0.82	-0.087	-0.2786	0.6425	0.5336	0.9461	-0.025	-0.0245	-0.004	0.1357	-0.0293	0.4049	0.0673	-0.1683	0.044	0.1917	-0.0061	0.0612	0.1953	-0.0155	0.2508	-16.1218	-3.0345	13.0047
11	1.8679	-0.7272	-0.4545	1.3781	0.3044	1.1493	-0.0318	0.1356	-0.0431	0.0288	-0.0238	0.2933	0.046	-0.1663	0.0434	0.1691	-0.0179	-0.0014	0.1827	-0.0192	0.2092	-12.2256	-4.7899	5.0665
12	5.8404	-2.383	1.5829	2.2433	-0.8646	1.8462	-0.0322	0.22	-0.2056	-0.3045	-0.082	0.3524	0.1189	-0.0827	0.1323	0.2179	-0.0258	-0.0198	0.162	-0.0313	0.2114	-10.7077	-14.4043	0.3086
13	1.2783	0.5273	0.5312	1.2284	0.4925	0.9875	0.0244	0.0982	-0.0219	-0.0862	0.0268	-0.0416	0.0365	0.0624	-0.0552	0.1635	0.012	0.0152	0.1358	-0.0066	0.1186	6.7289	-1.3331	3.4937

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 3 through 45 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 46 through 100 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 101 through 130 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 131 through 149 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 150 through 163 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 164 through 213 )
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 214 through 303 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 3 through 45 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 46 through 100 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 101 through 149 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 150 through 181 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 182 through 196 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 197 through 303 )

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