PDB-8usk: Crystal Structure of Kemp Eliminase HG185 in unbound state, 280 K

ID or keywords:

Entry

Database: PDB / ID: 8usk

Title

Crystal Structure of Kemp Eliminase HG185 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: 1.6 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
Revision 1.2	Oct 16, 2024	Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8usk.cif.gz	453.1 KB	Display	PDBx/mmCIF format
PDB format	pdb8usk.ent.gz	309.6 KB	Display	PDB format
PDBx/mmJSON format	8usk.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8usk_validation.pdf.gz	424.7 KB	Display	wwPDB validaton report
Full document	8usk_full_validation.pdf.gz	427 KB	Display
Data in XML	8usk_validation.xml.gz	28.2 KB	Display
Data in CIF	8usk_validation.cif.gz	43.4 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/us/8usk ftp://data.pdbj.org/pub/pdb/validation_reports/us/8usk	HTTPS FTP

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Related structure data

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

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Kemp eliminase

B: Kemp eliminase

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

1

A: Kemp eliminase

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

2

B: Kemp eliminase

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

Unit cell

Length a, b, c (Å)	51.119, 77.444, 93.745
Angle α, β, γ (deg.)	90.000, 105.546, 90.000
Int Tables number	4
Space group name H-M	P12₁1
Space group name Hall	P2yb
Symmetry operation	#1: x,y,z #2: -x,y+1/2,-z

#1: Protein	Kemp eliminase Mass: 34347.547 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.: 538 / Source method: isolated from a natural source / Formula: H₂O
Has protein modification	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: 2.6 Å³/Da / Density % sol: 52.74 %
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: 1.6→90.32 Å / Num. obs: 89399 / % possible obs: 96.4 % / Redundancy: 3.4 % / B_iso Wilson estimate: 14.88 Å² / CC_1/2: 0.992 / R_merge(I) obs: 0.113 / R_pim(I) all: 0.071 / Net I/σ(I): 10.3
Reflection shell	Resolution: 1.6→1.63 Å / Redundancy: 3.5 % / R_merge(I) obs: 0.863 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 4304 / CC_1/2: 0.474 / R_pim(I) all: 0.542 / % possible all: 94.1

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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: 1.6→90.32 Å / SU ML: 0.1795 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 18.8409
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.1833	4342	4.87 %
R_work	0.1605	84890	-
obs	0.1616	89232	96.19 %

Solvent computation

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

Displacement parameters

B_iso mean: 21.24 Å²

Refinement step

Cycle: LAST / Resolution: 1.6→90.32 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4586	0	0	538	5124

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0048	5223
X-RAY DIFFRACTION	f_angle_d	0.8341	7203
X-RAY DIFFRACTION	f_chiral_restr	0.0482	805
X-RAY DIFFRACTION	f_plane_restr	0.0055	974
X-RAY DIFFRACTION	f_dihedral_angle_d	12.1988	1906

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 (%)
1.6-1.62	0.3152	136	0.2754	2695	X-RAY DIFFRACTION	93.77
1.62-1.64	0.2797	131	0.2781	2754	X-RAY DIFFRACTION	94.19
1.64-1.66	0.2923	168	0.2581	2755	X-RAY DIFFRACTION	94.05
1.66-1.68	0.2455	141	0.2504	2748	X-RAY DIFFRACTION	94.26
1.68-1.7	0.2815	178	0.2431	2733	X-RAY DIFFRACTION	94.73
1.7-1.72	0.2529	127	0.2312	2795	X-RAY DIFFRACTION	94.66
1.72-1.75	0.2409	158	0.2235	2763	X-RAY DIFFRACTION	94.68
1.75-1.77	0.252	138	0.2144	2786	X-RAY DIFFRACTION	94.9
1.77-1.8	0.2078	168	0.2119	2771	X-RAY DIFFRACTION	95.11
1.8-1.83	0.2137	150	0.2036	2780	X-RAY DIFFRACTION	95.28
1.83-1.86	0.2278	134	0.1962	2791	X-RAY DIFFRACTION	95.28
1.86-1.9	0.2391	141	0.197	2840	X-RAY DIFFRACTION	95.88
1.9-1.93	0.2182	161	0.1916	2793	X-RAY DIFFRACTION	95.82
1.93-1.97	0.212	149	0.1765	2835	X-RAY DIFFRACTION	95.98
1.97-2.02	0.1925	128	0.1784	2795	X-RAY DIFFRACTION	96.21
2.02-2.06	0.2067	139	0.1645	2844	X-RAY DIFFRACTION	96.01
2.06-2.11	0.1723	137	0.1543	2829	X-RAY DIFFRACTION	96.24
2.11-2.17	0.1769	114	0.1538	2841	X-RAY DIFFRACTION	96.04
2.17-2.24	0.1605	143	0.1481	2846	X-RAY DIFFRACTION	96.33
2.24-2.31	0.195	141	0.1522	2849	X-RAY DIFFRACTION	97.17
2.31-2.39	0.178	175	0.1453	2830	X-RAY DIFFRACTION	96.87
2.39-2.49	0.1698	128	0.1475	2889	X-RAY DIFFRACTION	97.23
2.49-2.6	0.1751	145	0.152	2856	X-RAY DIFFRACTION	97.44
2.6-2.74	0.1857	132	0.1564	2900	X-RAY DIFFRACTION	97.68
2.74-2.91	0.1843	161	0.1579	2896	X-RAY DIFFRACTION	98.04
2.91-3.13	0.1701	116	0.1552	2936	X-RAY DIFFRACTION	97.85
3.13-3.45	0.1852	148	0.147	2898	X-RAY DIFFRACTION	98.35
3.45-3.95	0.1409	147	0.1317	2930	X-RAY DIFFRACTION	98.43
3.95-4.97	0.129	167	0.1177	2902	X-RAY DIFFRACTION	98.43
4.97-90.32	0.1634	141	0.1444	3010	X-RAY DIFFRACTION	98.5

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	0.176836723592	0.107132740538	-0.19214109674	0.617273982855	-0.0290523379366	0.22098373446	0.013995431359	-0.00676937834839	-0.0748670949836	0.0405175577053	-0.0605002562854	-0.0767620560136	0.0173581917233	-0.0224865236675	-0.00831851196518	0.138347137994	0.00138189869991	-0.0238496851684	0.130313889743	-0.00125444416076	0.140060200801	13.5600240224	5.01218967019	36.734711732
2	0.461274099177	0.325980866544	-0.0238386909628	0.260453732674	-0.00179867195556	0.00891984251099	0.00615102240382	0.057454545269	0.0496676395584	-0.00692552257463	-0.0389060747354	0.124163472806	-0.0340477868123	-0.0595416540354	1.01291606024E-5	0.142583158375	0.011715060854	-0.0215625951766	0.186111747559	-0.0131392769634	0.181217794617	-5.18798339086	12.1653830702	35.0103750909
3	0.318687678058	0.162701502261	0.0541574786437	0.17408015265	0.0188646774876	0.0464110718354	0.0593130083746	-0.0308519381316	0.00556713353921	0.084955181035	-0.113896604916	0.196019163829	0.054547387958	-0.0960352346924	-0.000492477785177	0.128176401245	-0.00394306130274	0.00100554562067	0.199945398398	-0.0315655876452	0.178729746225	-8.1343059479	14.2783950967	43.9671511467
4	0.21168546501	0.161175422217	0.219544618423	0.186953203946	0.159751022831	0.228696900072	0.0120586131845	-0.132276133017	0.209005155074	0.0208796973546	-0.0760139041341	0.237532364417	-0.0623706387095	-0.116392690188	-0.0442159952401	0.122476705056	0.017873000118	0.00682091881761	0.168176097967	-0.054197481398	0.200029120392	-5.10355881085	27.6632251619	44.3355251804
5	0.215112710308	0.0633516017447	0.161631481484	0.627796056872	0.240864956992	0.181591628034	0.052512483235	-0.149508752524	0.113763370304	0.0303958416227	-0.0844859748409	0.0213210937717	-0.0570561356515	-0.0316704468333	-0.0152616638492	0.162266980532	-0.00400655049458	-0.000504007843873	0.182889337261	-0.0288167922448	0.153340519358	6.44156300881	25.0325902144	46.5230116955
6	0.419740262182	0.17159860737	0.150439745627	0.779823418643	0.36487784983	0.367942032011	0.00705806976799	-0.0301458714094	0.00473779019559	0.00570268254855	0.00998497131885	-0.0717947964339	-0.0638533358131	0.0958541821613	-0.0272368479459	0.111692256269	0.00229383272713	-0.011577811141	0.122076539281	-0.00719269562088	0.120187602503	18.678032641	16.6173131733	37.6920172308
7	0.292768543209	-0.00320123101753	0.113326856941	0.425931879415	0.0490769938719	0.181217787831	-0.0299746677505	0.0431368498038	0.116018250388	-0.0135957518912	-0.00504017993895	-0.055718727148	0.00738008423244	0.0086455479676	2.46422273052E-5	0.174074251755	-0.00249539751966	0.000101478687615	0.142865451959	0.00645078120603	0.133543637953	4.51786291809	11.4231180193	6.36060604354
8	0.676500491429	-0.183354186365	0.273094766616	0.387836289548	0.146002641847	0.255927420329	0.0380655221694	-0.195900480219	-0.0277315285991	0.122066979079	-0.0981810856444	0.210292598383	0.0421936245941	-0.0504135456706	-0.0316113997855	0.225376195141	-0.00583524953667	0.0575148019168	0.207518622283	-0.0119971110895	0.19742243303	-11.0971342407	5.03481395117	18.2116479167
9	0.0822651726141	0.0227981075478	-0.0253939094252	0.102068095478	0.142230349657	0.240598594126	0.0261518736492	0.00130946351313	-0.00727249295577	-0.0442211853067	-0.0800580072504	0.30036900546	-0.0120904232586	-0.0732893347071	-0.000302875779759	0.17946515873	0.00290523843106	0.0325308611125	0.206024361178	-0.0138229726332	0.251892224286	-17.9508255165	3.26888484417	12.1202172398
10	0.100622647506	-0.0641486076131	-0.13566793368	0.137170004529	0.106710503693	0.185257291613	-0.0766113619831	0.00472285871765	-0.174519214071	0.161136005447	0.0113322310887	0.362570908886	0.131517650336	-0.124967844327	-2.15310064223E-5	0.202544018766	-0.0193243515953	0.0354035599515	0.191090995362	-0.0152193795023	0.245064353726	-15.9954877668	-10.3780148704	10.7800199947
11	0.240365520326	0.0808511581667	-0.149392501243	0.711954723434	0.313045599163	0.285162119901	0.0209727013727	0.0475827694748	-0.00800179505291	-0.0978002240804	-0.0510973334	0.0582289892351	0.0853489211589	-0.0450923482833	-2.66850436134E-6	0.191462594904	0.00236903371386	-0.00565488569799	0.163663200317	-0.01107544627	0.160763426733	-6.26971372768	-8.4717112849	3.04970205335
12	0.255031765748	0.0120343023136	-0.125235233326	0.372242621793	-0.0460438812848	0.226124540942	-0.0110097838018	0.0847602737137	-0.0341471403724	-0.0910792924604	0.0169227469962	-0.00740453501404	0.0328078449066	0.0443514051201	-1.30231620358E-6	0.175084033138	0.00735166248949	0.00237507708054	0.148009976755	-0.0137728236158	0.118391127829	4.11517662311	-3.25858542484	0.214151945509
13	0.293153423484	0.152360361898	0.0687904264274	0.268387787837	0.230205090103	0.212187412266	0.0303701644923	0.0334352597508	0.05264109691	0.0363216670009	0.0257900885288	-0.0942677015781	0.104748071177	0.135235309074	5.91499811816E-6	0.143188737008	0.0011651099992	0.0155848195112	0.142298527141	-0.0118185789808	0.132644125683	14.7482330823	4.44759637129	8.49411121023

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 163 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 164 through 213 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 214 through 303 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 3 through 45 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 46 through 100 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 101 through 130 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 131 through 163 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 164 through 214 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 215 through 270 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 271 through 303 )

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