PDB-8usj: Crystal Structure of Kemp Eliminase HG198 with bound transition s...

ID or keywords:

Entry

Database: PDB / ID: 8usj

Title

Crystal Structure of Kemp Eliminase HG198 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: 1.6 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

Structure viewer	Molecule: MolmilJmol/JSmol

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Validation report

Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/us/8usj ftp://data.pdbj.org/pub/pdb/validation_reports/us/8usj	HTTPS FTP

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Related structure data

Related structure data	8useC 8usfC 8usgC 8ushC 8usiC 8uskC 8uslC 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

C: Kemp eliminase

hetero molecules

104 kDa, 3 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Kemp eliminase

hetero molecules

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

2

B: Kemp eliminase

hetero molecules

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

3

C: Kemp eliminase

hetero molecules

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

Unit cell

Length a, b, c (Å)	46.651, 51.028, 86.296
Angle α, β, γ (deg.)	83.581, 81.118, 87.007
Int Tables number	1
Space group name H-M	P1
Space group name Hall	P1
Symmetry operation	#1: x,y,z

#1: Protein	Kemp eliminase Mass: 34339.488 Da / Num. of mol.: 3 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.: 3 / Source method: obtained synthetically / Formula: C₆H₄N₄O₂ / Feature type: SUBJECT OF INVESTIGATION
#3: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 820 / 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: 1.96 Å³/Da / Density % sol: 37.14 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.6 Details: 6.0 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→50.68 Å / Num. obs: 97130 / % possible obs: 94.3 % / Redundancy: 1.8 % / B_iso Wilson estimate: 10.13 Å² / CC_1/2: 0.981 / R_merge(I) obs: 0.123 / R_pim(I) all: 0.123 / Net I/σ(I): 7.7
Reflection shell	Resolution: 1.6→1.63 Å / R_merge(I) obs: 0.547 / Mean I/σ(I) obs: 1.7 / Num. unique obs: 4660 / CC_1/2: 0.466 / R_pim(I) all: 0.547

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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→50.68 Å / SU ML: 0.2008 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 21.0659
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.19	4795	4.94 %
R_work	0.154	92321	-
obs	0.1558	97116	94.29 %

Solvent computation

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

Displacement parameters

B_iso mean: 16.11 Å²

Refinement step

Cycle: LAST / Resolution: 1.6→50.68 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	6862	0	36	820	7718

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.0039	7761
X-RAY DIFFRACTION	f_angle_d	0.7241	10710
X-RAY DIFFRACTION	f_chiral_restr	0.0464	1189
X-RAY DIFFRACTION	f_plane_restr	0.0052	1443
X-RAY DIFFRACTION	f_dihedral_angle_d	12.1871	2816

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	0.3656	146	0.3001	2979	X-RAY DIFFRACTION	92.05
1.62-1.64	0.3358	155	0.2938	2985	X-RAY DIFFRACTION	91.57
1.64-1.66	0.3323	148	0.2862	3036	X-RAY DIFFRACTION	92.16
1.66-1.68	0.2953	166	0.2739	3001	X-RAY DIFFRACTION	92.77
1.68-1.7	0.3143	174	0.2727	2997	X-RAY DIFFRACTION	92.1
1.7-1.72	0.318	182	0.2628	3073	X-RAY DIFFRACTION	93.97
1.72-1.75	0.278	162	0.2476	2953	X-RAY DIFFRACTION	91.92
1.75-1.77	0.3009	165	0.2337	3056	X-RAY DIFFRACTION	93.39
1.77-1.8	0.2632	175	0.2301	3056	X-RAY DIFFRACTION	93.49
1.8-1.83	0.2502	140	0.2268	3014	X-RAY DIFFRACTION	92.85
1.83-1.86	0.2815	152	0.2148	3094	X-RAY DIFFRACTION	93.73
1.86-1.9	0.2513	134	0.1998	3017	X-RAY DIFFRACTION	92.79
1.9-1.93	0.2344	172	0.1919	3071	X-RAY DIFFRACTION	93.81
1.93-1.97	0.2329	173	0.1841	3014	X-RAY DIFFRACTION	92.46
1.97-2.02	0.1858	147	0.1746	3074	X-RAY DIFFRACTION	94.54
2.02-2.06	0.2221	151	0.1623	3074	X-RAY DIFFRACTION	93.83
2.06-2.11	0.201	164	0.1555	3070	X-RAY DIFFRACTION	93.68
2.11-2.17	0.2057	160	0.152	3070	X-RAY DIFFRACTION	94.22
2.17-2.24	0.1669	166	0.1385	3091	X-RAY DIFFRACTION	94.32
2.24-2.31	0.1757	180	0.1403	3090	X-RAY DIFFRACTION	95.34
2.31-2.39	0.1596	172	0.132	3111	X-RAY DIFFRACTION	95.99
2.39-2.49	0.1819	139	0.1317	3174	X-RAY DIFFRACTION	96.11
2.49-2.6	0.169	172	0.1299	3118	X-RAY DIFFRACTION	95.97
2.6-2.74	0.1587	157	0.1336	3141	X-RAY DIFFRACTION	95.82
2.74-2.91	0.1618	154	0.1348	3156	X-RAY DIFFRACTION	96.53
2.91-3.13	0.1757	163	0.1339	3124	X-RAY DIFFRACTION	96.42
3.13-3.45	0.1393	167	0.1177	3138	X-RAY DIFFRACTION	95.99
3.45-3.95	0.1246	152	0.103	3190	X-RAY DIFFRACTION	96.53
3.95-4.97	0.1162	154	0.0911	3148	X-RAY DIFFRACTION	96.89
4.97-50.68	0.1533	153	0.1252	3206	X-RAY DIFFRACTION	97.42

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	2.8178022345	0.311701011873	1.15436181016	1.96670811048	0.5478134679	2.5199751098	0.0349105894312	0.0662170057938	0.0348936354229	-0.0273373761104	-0.040695049548	-0.177132037111	0.0795715976358	0.172085810442	-0.00804547168124	0.110308678882	0.00795093182685	0.0174459389311	0.122420301874	0.0282785953971	0.113848426463	14.5592284355	16.5225191287	35.215611189
2	2.81409447625	-1.29666958463	-0.560355348768	3.4281712347	1.06396862882	2.02125124344	0.113664871242	-0.0587427869676	0.335496651376	-0.0516069695018	0.084207287481	-0.373817311973	-0.336610432651	0.114056768361	-0.0785105823242	0.177265062184	-0.0349888463052	0.0185512769771	0.140515290176	-0.0244114721079	0.179396776744	7.57638541873	38.6638802216	41.7406117825
3	1.20126052204	-0.605426473654	-0.345698915115	1.43983784336	0.279796414634	1.22753370228	0.0873914933423	0.0430045481478	0.158554536005	-0.0332115073687	-0.0505482731433	-0.128683353443	-0.168495961235	-0.0232544832027	-0.0623138012054	0.143882399791	-0.0199736140373	0.0107098625943	0.0998075571934	-0.00100301816634	0.121581743389	4.19351874756	35.5262263665	35.9651722719
4	0.668991631973	0.182655586273	-0.132344078165	1.56537091955	-0.206397777262	0.874997987186	0.0353562761401	-0.00193507905097	0.0187721599885	-0.0302960552087	-0.0185279912206	0.0854610531363	-0.0490292757443	-0.0981999428983	-0.0130140601602	0.102826392878	0.00774592601788	-0.00209007833472	0.130986739648	-0.00317628228363	0.0947238385238	-8.00784696738	28.9087270953	37.1227174957
5	0.580452140406	-1.07046521048	1.78258553284	2.21935242842	-3.69722421228	8.82680365208	0.0077934296391	-0.0659198203674	-0.0818137725947	-0.116463191215	0.107566246141	0.202909830306	0.0986913006522	-0.500292080491	-0.176033621134	0.101054003146	-0.00163945082781	-0.0211751036373	0.196222426463	-0.00354248876387	0.138959224987	-15.6231153421	22.7643217209	34.282960562
6	1.72359660971	-0.709337454464	0.900285740007	0.770550947234	-0.443731039145	1.08283955033	0.0310074417492	-0.0783920415152	-0.0636489176054	-0.0413093321918	-0.00602101929891	0.00423722669814	0.104511121399	-0.0221676230386	-0.0195625102635	0.116024664671	-0.014250706166	0.00131902629626	0.113827764719	0.00697342953038	0.0973938589128	3.35035107245	11.9678606823	40.0964066866
7	3.01883526575	-0.579828829763	-1.46670492057	2.03040593835	0.703745431628	2.63502539615	0.0310930158005	-0.00165143630632	0.0817610705953	0.0117879255046	-0.00895622597496	-0.200487282441	-0.0497745928512	0.0647194498561	-0.0182245197235	0.0887431030989	-0.00113033695306	-0.0224191881287	0.0863830101231	0.0204704598686	0.11724699396	37.8381393322	32.2449256814	11.6010244425
8	1.08275838745	0.715118439397	0.175657231388	1.22417443044	0.441197915688	0.567171059108	-0.00772065130839	0.0520896359151	-0.0677652728603	-0.0447110948945	0.0432123112966	-0.0857301693277	0.0167262583137	0.0227057951032	-0.0357274072213	0.120082450199	0.00691179560754	-0.0054980961968	0.112963296019	0.000845165152191	0.126199219804	30.5670216172	13.3388696214	6.24823229289
9	0.667074736116	0.367192563092	0.372889296657	1.3343391967	0.60999572802	0.806775240403	0.0248483517234	-0.0540992338418	-0.126167233483	0.101959481018	0.0158081297263	-0.0842219485543	0.0525870844727	-0.0251570954828	-0.0159998544815	0.110583108347	0.000819544074735	-0.00973342290121	0.10319634597	0.00588506906012	0.112045295626	24.5598225006	10.6242525601	13.4295094525
10	0.536628578421	0.0982093343533	0.190443360192	1.63818216264	-0.232669914744	0.809524256707	0.0014695563863	0.00578830390123	-0.0171494928959	-0.023761566144	0.00328127257239	0.0950138077847	0.0235870382481	-0.0381618663136	0.00170183170032	0.0862750962132	0.00488364666769	0.00786287387442	0.109064173015	-0.000535241642784	0.0954591559508	14.7041834036	19.0466922988	8.82032869551
11	0.73199660749	0.620944631121	-1.33850563567	3.1460819585	-3.97776561609	7.53548941871	-0.0253538190697	0.132454451544	-0.0311952031276	0.0884251435656	0.109511715392	0.15752382082	-0.136197597096	-0.144183746351	-0.0992409443046	0.0951449163316	0.00539871295299	0.00877701782309	0.128174295146	0.00335449742289	0.117663244861	7.6987599547	25.2932474177	12.4433599651
12	1.21204253938	0.414747748472	-0.541461989364	0.816300520844	-0.35261972851	0.737031444669	-0.00209748135101	0.0501737478783	0.0377774429969	0.0063859747751	0.0180811466393	-0.0448134870068	-0.011547588547	-0.000693227557649	-0.0129262957261	0.0931982416548	0.00812518254615	-0.00706555391313	0.103588472043	-0.00307835366491	0.0979199070889	26.1784134616	36.5933609454	7.17644352394
13	2.48818425599	-1.11330560376	1.7473862397	2.18374488082	-1.73073625038	4.24508443231	-0.0413791935855	-0.0651374026965	-0.0623609205938	0.0576114171748	0.000587585614744	-0.0184333073296	0.0941214412514	-0.0806118994619	0.0708154061374	0.0821497115379	0.00490881549387	0.00794598029372	0.0731596846885	-0.00529594385802	0.0869000457263	13.4012990196	-8.64225974691	73.702852644
14	3.19869441007	0.312520777553	1.23530761995	2.66411036526	-0.445810834007	1.57454000446	-0.0598624584533	0.0347910565836	0.0636922281992	-0.119906040019	0.0367937583496	0.113844804158	-0.0121488695341	0.0150505324998	0.0100074793957	0.0921686497396	0.00626490289319	0.00249367150691	0.0876006513247	-0.0161720354718	0.0945558859352	4.761235399	0.100413562931	66.0628186838
15	0.885150920991	0.0644065243096	-0.293967745192	1.35633211741	-0.423164963631	0.730658888741	0.0381823557649	0.0148640517693	0.0609277338404	-0.0640604436885	0.00566130091017	0.075587306044	-0.0217591448662	-0.00654665553289	-0.0582851214927	0.0893825780585	0.000283018065985	-0.0144837870664	0.0812306092311	-0.00405337052953	0.0986065329912	14.2642694576	14.5218698328	63.8263569213
16	0.813043540428	-0.131503832499	-0.55017505233	1.74572037761	-0.644213381943	1.35251946717	0.0724574668837	-0.0449773579788	0.100133930198	0.0609381962571	-0.0273458861144	-0.0484160086577	-0.116973206991	-0.00358019751027	-0.0382547994675	0.0769555810318	0.00662907418811	-0.0111193754761	0.0903380262146	-0.0100137383575	0.094079552154	21.7301208395	18.1680777089	69.4406064155
17	0.719825403815	-0.188545187833	-0.236698580543	1.28540763087	0.0494510687827	0.9846301674	0.00453534096911	-0.00183387284737	0.0525212299511	-0.0841903500375	0.00343881621771	-0.107478587868	0.0243014328425	0.0456040202502	-0.0148080202536	0.0749766986112	0.00144176344787	0.00596223557495	0.0983907276653	0.00139671199875	0.101650953105	30.2308156715	9.49188127803	64.1826154072
18	0.908304751772	0.482254120012	0.551044940464	2.54004715405	2.50628924337	4.15653286917	0.00571562101039	0.019333223981	0.00422501537336	-0.102212379966	0.0452338990606	-0.190747089423	0.0422256274677	0.203772500248	-0.0364261241626	0.0769701685775	0.00234895291325	0.0116992435025	0.113685359339	0.00635126176611	0.14444051986	38.0200075339	3.04371905752	65.2497592655
19	1.18720403964	0.626104771326	0.266451484675	0.987430813208	0.225818902305	0.409445555613	-0.0368414023809	0.0106557794546	-0.0735364398972	-0.074262680687	0.0247632156857	-0.087582258484	0.0408135726442	0.0186747198424	0.0092183141735	0.111528418942	0.00395176988655	0.00929741318862	0.0935760098064	0.00457430943464	0.1066099971	23.9895086554	-6.46040615783	65.0603463385
20	3.4120451232	-0.637552470172	0.737109938375	2.4412534106	-0.136714856995	2.97725235323	-0.0223223047496	0.163101101616	-0.00255526670514	-0.150566797128	0.0525675079176	0.155731712218	-0.0530853320104	-0.0566913780772	-0.0486828660473	0.0924086093923	-0.017088733927	-0.00780803617674	0.0637909714915	-0.0184164192039	0.0964711078189	9.54457409698	-11.6961522901	59.9781789688

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 63 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 64 through 130 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 131 through 181 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 182 through 196 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 197 through 302 )
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 181 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 182 through 196 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 197 through 303 )
13	X-RAY DIFFRACTION	13	chain 'C' and (resid 4 through 22 )
14	X-RAY DIFFRACTION	14	chain 'C' and (resid 23 through 45 )
15	X-RAY DIFFRACTION	15	chain 'C' and (resid 46 through 100 )
16	X-RAY DIFFRACTION	16	chain 'C' and (resid 101 through 130 )
17	X-RAY DIFFRACTION	17	chain 'C' and (resid 131 through 181 )
18	X-RAY DIFFRACTION	18	chain 'C' and (resid 182 through 196 )
19	X-RAY DIFFRACTION	19	chain 'C' and (resid 197 through 270 )
20	X-RAY DIFFRACTION	20	chain 'C' and (resid 271 through 303 )

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