PDB-8usl: Crystal Structure of Kemp Eliminase HG185 with bound transition s...

ID or keywords:

Entry

Database: PDB / ID: 8usl

Title

Crystal Structure of Kemp Eliminase HG185 with bound transition state analogue, 280 K

Components

Kemp eliminase

Keywords

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

Function / homology

6-NITROBENZOTRIAZOLE

Function and homology information

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
Natural Sciences and Engineering Research Council (NSERC, Canada)		Canada
Canada Foundation for Innovation		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	8usl.cif.gz	370.9 KB	Display	PDBx/mmCIF format
PDB format	pdb8usl.ent.gz	310.5 KB	Display	PDB format
PDBx/mmJSON format	8usl.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	8usl_validation.pdf.gz	1017.8 KB	Display	wwPDB validaton report
Full document	8usl_full_validation.pdf.gz	1018.3 KB	Display
Data in XML	8usl_validation.xml.gz	26.1 KB	Display
Data in CIF	8usl_validation.cif.gz	39.4 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/us/8usl ftp://data.pdbj.org/pub/pdb/validation_reports/us/8usl	HTTPS FTP

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Related structure data

Related structure data	8useC 8usfC 8usgC 8ushC 8usiC 8usjC 8uskC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Kemp eliminase

B: Kemp eliminase

hetero molecules

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

1

A: Kemp eliminase

hetero molecules

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

2

B: Kemp eliminase

hetero molecules

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

Unit cell

Length a, b, c (Å)	51.106, 77.492, 93.846
Angle α, β, γ (deg.)	90.00, 105.51, 90.00
Int Tables number	4
Space group name H-M	P12₁1

#1: Protein	Kemp eliminase Mass: 32860.980 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: Chemical	ChemComp-6NT / 6-NITROBENZOTRIAZOLE Mass: 164.122 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₆H₄N₄O₂ / Feature type: SUBJECT OF INVESTIGATION
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 360 / 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: 2.72 Å³/Da / Density % sol: 54.85 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6 Details: 12.8 mg/mL protein, 100 mM sodium acetate, pH 4.6, 1.6 M ammonium sulfate

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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: Dec 2, 2021
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→90.43 Å / Num. obs: 101929 / % possible obs: 99.7 % / Redundancy: 3.3 % / B_iso Wilson estimate: 14.19 Å² / CC_1/2: 0.994 / R_merge(I) obs: 0.101 / R_pim(I) all: 0.065 / R_rim(I) all: 0.12 / Net I/σ(I): 11.1
Reflection shell	Resolution: 1.55→1.58 Å / Redundancy: 3.3 % / R_merge(I) obs: 0.73 / Mean I/σ(I) obs: 1.5 / Num. unique obs: 5042 / CC_1/2: 0.531 / R_pim(I) all: 0.47 / % possible all: 99.4

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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→90.43 Å / SU ML: 0.17 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 19.5 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1831	4948	4.86 %
R_work	0.161	-	-
obs	0.1621	101787	99.59 %

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→90.43 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4586	0	24	360	4970

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	5311
X-RAY DIFFRACTION	f_angle_d	0.848	7336
X-RAY DIFFRACTION	f_dihedral_angle_d	11.681	1912
X-RAY DIFFRACTION	f_chiral_restr	0.052	802
X-RAY DIFFRACTION	f_plane_restr	0.006	998

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	0.2919	121	0.2755	3185	X-RAY DIFFRACTION	99
1.57-1.59	0.2499	164	0.266	3245	X-RAY DIFFRACTION	100
1.59-1.61	0.3094	164	0.263	3210	X-RAY DIFFRACTION	99
1.61-1.63	0.303	140	0.2554	3223	X-RAY DIFFRACTION	99
1.63-1.65	0.2848	148	0.2482	3283	X-RAY DIFFRACTION	100
1.65-1.67	0.2474	166	0.2334	3147	X-RAY DIFFRACTION	99
1.67-1.69	0.2494	188	0.2273	3216	X-RAY DIFFRACTION	100
1.69-1.72	0.2385	176	0.2225	3202	X-RAY DIFFRACTION	99
1.72-1.75	0.2504	167	0.2094	3196	X-RAY DIFFRACTION	100
1.75-1.77	0.2512	189	0.2049	3199	X-RAY DIFFRACTION	100
1.77-1.8	0.2226	171	0.193	3194	X-RAY DIFFRACTION	100
1.8-1.84	0.2305	141	0.1912	3245	X-RAY DIFFRACTION	100
1.84-1.87	0.215	162	0.1845	3209	X-RAY DIFFRACTION	100
1.87-1.91	0.2089	178	0.1769	3213	X-RAY DIFFRACTION	100
1.91-1.95	0.1734	167	0.168	3218	X-RAY DIFFRACTION	99
1.95-2	0.1811	174	0.1649	3221	X-RAY DIFFRACTION	100
2-2.05	0.198	196	0.1643	3202	X-RAY DIFFRACTION	100
2.05-2.1	0.1833	162	0.1559	3223	X-RAY DIFFRACTION	100
2.1-2.17	0.1814	167	0.1472	3211	X-RAY DIFFRACTION	100
2.17-2.24	0.1619	162	0.1431	3248	X-RAY DIFFRACTION	100
2.24-2.32	0.1674	150	0.1447	3246	X-RAY DIFFRACTION	100
2.32-2.41	0.1528	150	0.1428	3226	X-RAY DIFFRACTION	100
2.41-2.52	0.179	144	0.1431	3275	X-RAY DIFFRACTION	100
2.52-2.65	0.1743	144	0.1507	3256	X-RAY DIFFRACTION	100
2.65-2.82	0.1721	167	0.1582	3240	X-RAY DIFFRACTION	100
2.82-3.03	0.1845	169	0.1598	3251	X-RAY DIFFRACTION	100
3.03-3.34	0.1585	177	0.1637	3219	X-RAY DIFFRACTION	100
3.34-3.82	0.1743	183	0.1391	3249	X-RAY DIFFRACTION	100
3.82-4.82	0.1307	177	0.1201	3249	X-RAY DIFFRACTION	100
4.82-90.43	0.1673	184	0.1378	3338	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	2.4551	0.5297	2.2075	2.2908	1.2015	5.6646	0.0276	0.0082	-0.0481	0.038	-0.0354	-0.1189	0.0013	-0.0319	0.0266	0.0876	-0.0155	0.0063	0.0637	0.0171	0.1046	13.0735	3.6308	36.5909
2	2.24	-0.8262	0.161	1.5711	-0.0383	0.2371	0.0402	0.0398	0.0922	-0.0171	-0.0866	0.1476	-0.0069	-0.0686	0.0481	0.1104	-0.0034	-0.0037	0.1509	-0.0203	0.1403	-6.4738	11.7436	38.0164
3	1.9813	1.0352	-0.189	1.8924	-0.2261	0.5151	0.0696	-0.1253	0.2012	0.052	-0.0999	0.1941	-0.0687	-0.0697	0.0111	0.1212	0.0081	-0.0015	0.1511	-0.052	0.1289	-0.6295	23.8736	44.5064
4	6.6711	3.3633	-5.9586	3.6409	-3.2793	5.9569	0.1494	-0.3791	0.1492	0.2724	-0.2342	0.1352	-0.0449	0.2451	0.0166	0.1655	0.0068	0.0058	0.1756	-0.0688	0.1777	4.7856	29.2618	50.8382
5	0.7423	0.0116	-0.1762	0.8231	0.6263	1.2634	0.0194	-0.0358	0.0039	0.0096	-0.0041	-0.0661	-0.0454	0.0493	-0.0196	0.1081	-0.0007	-0.0102	0.1032	-0.0011	0.1152	17.4355	15.681	38.4702
6	1.7242	0.5147	0.9692	2.5618	1.6416	6.3873	-0.0082	0.0532	0.086	-0.008	0.0063	-0.0921	-0.0309	0.0874	0.0141	0.1142	0.0206	0.0028	0.0747	0.015	0.1268	4.5479	9.5241	6.8241
7	2.5496	1.7836	-1.421	2.8906	-1.4259	1.7289	0.0588	-0.2409	-0.0329	0.1491	-0.1411	0.1893	0.0342	-0.0241	0.0895	0.1702	-0.0017	0.0321	0.1761	-0.0129	0.1441	-11.0145	2.8045	18.4401
8	3.2799	1.7216	-1.9216	3.5557	-1.4524	2.5672	0.0235	0.0335	0.0702	0.0237	0.0141	0.4113	-0.0371	-0.1926	-0.035	0.1453	0.0097	0.0053	0.182	-0.0163	0.1851	-18.2443	0.9781	12.2831
9	2.1482	-1.2334	-0.662	2.2215	0.3132	1.1386	-0.0549	-0.002	-0.1074	0.0544	0.0006	0.2829	0.1224	-0.1195	0.046	0.1422	-0.0227	-0	0.1443	-0.0297	0.151	-12.235	-10.0089	8.1086
10	7.3335	-5.5306	4.4686	6.3905	-3.3395	4.6549	0.1915	0.182	-0.2454	-0.4818	-0.1668	0.193	0.2797	0.1092	-0.0478	0.1941	-0.0264	-0.008	0.1249	-0.0344	0.1645	-11.0845	-16.1799	0.1971
11	2.6623	0.7691	1.3353	1.0208	0.5925	1.345	0.0314	0.0683	-0.1089	-0.096	0.0079	-0.0036	0.0345	0.0192	-0.0395	0.1643	0.0103	0.0109	0.1174	-0.0042	0.1101	2.9502	-5.866	1.2922
12	2.647	0.4558	0.5582	3.1604	-0.4171	6.3722	0.017	0.0909	0.0825	-0.0558	-0.0032	-0.1178	0.0216	0.3343	0.0022	0.0659	0.0096	0.0211	0.0849	-0.0172	0.1153	15.1649	3.2162	7.8785

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 130 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 131 through 181 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 182 through 196 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 197 through 303 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 3 through 45 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 46 through 100 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 101 through 130 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 131 through 181 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 182 through 196 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 197 through 270 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 271 through 303 )

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