PDB-8usf: Crystal Structure of Kemp Eliminase HG649 with bound transition s...

ID or keywords:

Entry

Database: PDB / ID: 8usf

Title

Crystal Structure of Kemp Eliminase HG649 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: 1.45 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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Download

PDBx/mmCIF format	8usf.cif.gz	478.1 KB	Display	PDBx/mmCIF format
PDB format	pdb8usf.ent.gz	328.2 KB	Display	PDB format
PDBx/mmJSON format	8usf.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8usf_validation.pdf.gz	1 MB	Display	wwPDB validaton report
Full document	8usf_full_validation.pdf.gz	1 MB	Display
Data in XML	8usf_validation.xml.gz	31.1 KB	Display
Data in CIF	8usf_validation.cif.gz	50.1 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/us/8usf ftp://data.pdbj.org/pub/pdb/validation_reports/us/8usf	HTTPS FTP

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Related structure data

Related structure data	8useC 8usgC 8ushC 8usiC 8usjC 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

hetero molecules

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

1

A: Kemp eliminase

hetero molecules

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

2

B: Kemp eliminase

hetero molecules

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

Unit cell

Length a, b, c (Å)	51.072, 77.465, 93.811
Angle α, β, γ (deg.)	90.000, 105.276, 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: 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 BL21(DE3) (bacteria)
#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.: 670 / 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: 2.6 Å³/Da / Density % sol: 52.72 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion Details: 12.5 mg/mL protein, 30% PEG4000, 0.3 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: Jun 29, 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: 1.45→90.5 Å / Num. obs: 124369 / % possible obs: 99.7 % / Redundancy: 3.2 % / B_iso Wilson estimate: 12.51 Å² / CC_1/2: 0.996 / R_merge(I) obs: 0.092 / R_pim(I) all: 0.059 / R_rim(I) all: 0.11 / Net I/σ(I): 8.7
Reflection shell	Resolution: 1.45→1.48 Å / Redundancy: 3.1 % / R_merge(I) obs: 0.837 / Mean I/σ(I) obs: 1 / Num. unique obs: 6172 / CC_1/2: 0.452 / R_pim(I) all: 0.552 / % possible all: 99.7

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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: 1.45→90.5 Å / SU ML: 0.1482 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 15.6664
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2

	R_factor	Num. reflection	% reflection
R_free	0.1567	6289	5.06 %
R_work	0.1373	118031	-
obs	0.1383	124320	99.69 %

Solvent computation

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

Displacement parameters

B_iso mean: 19.77 Å²

Refinement step

Cycle: LAST / Resolution: 1.45→90.5 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	4574	0	24	670	5268

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.007	5462
X-RAY DIFFRACTION	f_angle_d	0.8853	7561
X-RAY DIFFRACTION	f_chiral_restr	0.0711	832
X-RAY DIFFRACTION	f_plane_restr	0.0065	1039
X-RAY DIFFRACTION	f_dihedral_angle_d	12.6976	2015

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 (%)
1.45-1.47	0.3171	201	0.288	3942	X-RAY DIFFRACTION	99.71
1.47-1.48	0.315	184	0.2715	3900	X-RAY DIFFRACTION	99.46
1.48-1.5	0.2409	189	0.2612	3950	X-RAY DIFFRACTION	99.3
1.5-1.52	0.2563	232	0.24	3903	X-RAY DIFFRACTION	99.66
1.52-1.54	0.2773	193	0.2409	3896	X-RAY DIFFRACTION	99.78
1.54-1.56	0.2731	226	0.228	3897	X-RAY DIFFRACTION	99.57
1.56-1.58	0.2257	198	0.2057	3939	X-RAY DIFFRACTION	99.61
1.58-1.61	0.2246	229	0.2115	3881	X-RAY DIFFRACTION	99.56
1.61-1.63	0.1918	229	0.1968	3875	X-RAY DIFFRACTION	99.52
1.63-1.66	0.2504	218	0.1962	3936	X-RAY DIFFRACTION	99.5
1.66-1.69	0.2064	212	0.1879	3942	X-RAY DIFFRACTION	99.62
1.69-1.72	0.1949	172	0.1807	3936	X-RAY DIFFRACTION	99.66
1.72-1.75	0.1905	182	0.1738	3947	X-RAY DIFFRACTION	99.3
1.75-1.79	0.1954	209	0.172	3894	X-RAY DIFFRACTION	99.42
1.79-1.83	0.1801	236	0.1577	3923	X-RAY DIFFRACTION	99.71
1.83-1.87	0.1719	218	0.1469	3894	X-RAY DIFFRACTION	99.85
1.87-1.92	0.1723	222	0.1395	3917	X-RAY DIFFRACTION	99.71
1.92-1.97	0.147	195	0.1278	3995	X-RAY DIFFRACTION	99.93
1.97-2.03	0.1531	199	0.1244	3888	X-RAY DIFFRACTION	99.95
2.03-2.09	0.1503	225	0.1217	3957	X-RAY DIFFRACTION	99.95
2.09-2.17	0.1298	215	0.1165	3923	X-RAY DIFFRACTION	99.88
2.17-2.25	0.1243	188	0.11	3969	X-RAY DIFFRACTION	99.9
2.25-2.36	0.132	215	0.1108	3951	X-RAY DIFFRACTION	99.9
2.36-2.48	0.1194	216	0.1089	3939	X-RAY DIFFRACTION	99.86
2.48-2.63	0.1392	218	0.1149	3935	X-RAY DIFFRACTION	99.98
2.63-2.84	0.1416	208	0.1208	3993	X-RAY DIFFRACTION	99.88
2.84-3.12	0.1541	203	0.1218	3952	X-RAY DIFFRACTION	99.74
3.12-3.58	0.1229	186	0.1162	3972	X-RAY DIFFRACTION	99.55
3.58-4.51	0.1167	227	0.1015	3960	X-RAY DIFFRACTION	99.6
4.51-90.5	0.1465	244	0.1297	4025	X-RAY DIFFRACTION	99.74

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	1.13417864106	0.151006662504	0.480666470896	1.47715088044	0.611713438645	2.31559762734	0.0207391299463	0.0188345847974	-0.0710675096667	0.0156712905267	-0.0259256115333	-0.0808307080801	0.0554186558358	0.000567885908095	0.00757564850522	0.108648141925	-0.00820432928551	-0.0061814536508	0.0918664511413	0.00889651406795	0.121644111235	13.6942581385	4.77569979269	36.5639987906
2	0.895790033109	0.057897358435	0.0392694431911	0.804147502566	0.0421434801035	0.360536725028	0.0146781796154	0.0540621886611	0.0437672988643	-0.0207519266213	-0.0439973862667	0.143617355806	-0.0197629654255	-0.0624756591169	0.0266985488198	0.105934518195	0.00194096573735	-0.0162555212763	0.138988141473	-0.0103932270531	0.125364843419	-5.2968902199	11.9281544978	34.9688655419
3	1.60030170719	-0.196920724977	0.617214137593	1.0340882082	-0.283704866795	0.689514761006	0.0150691160321	-0.166699655768	0.0162922381621	0.0674292546233	-0.0183056219719	0.208661911978	0.0120022624731	-0.116013619841	0.0151244236808	0.0904731736422	-0.0123113993613	0.00799361414356	0.144461038303	-0.028205063371	0.126425389567	-8.77560343516	14.5608910435	43.9146235377
4	0.956598886873	0.490889884641	0.00138079360341	0.79156119624	0.0749839042703	0.523138605503	0.0275236259641	-0.12065442498	0.153083338953	0.0339140366574	-0.0700656034058	0.13246805769	-0.0384765489661	-0.0817133830146	0.0329959049971	0.115567437342	0.00533822457047	-0.00272066634476	0.149438764542	-0.0331270349484	0.144855217506	-0.710927563052	25.2125318223	44.7821534674
5	1.94824247163	0.88162469764	-1.69533979873	1.0789656822	-1.06402864996	2.99119840451	0.0488868921549	-0.216563691861	0.170935298173	0.201816726892	-0.111970767471	0.136210501289	-0.0060977910422	-0.00458288911597	0.0771161923212	0.151460699208	0.00187083600564	0.00214362350083	0.15764556913	-0.0589182041313	0.167400610035	5.0104293355	30.8222153554	50.8569107194
6	0.584109010746	-0.0135604889712	-0.119396646854	0.830627876506	0.446982112505	1.08624925439	0.0223991746066	-0.0173820619022	0.0017717757286	-0.00394547796489	-0.00541697975992	-0.0608812129887	-0.0439480107727	0.0370968006028	-0.0245719354837	0.0900767912521	-0.00195023343087	-0.0105421211296	0.0961166761972	-0.00221141847167	0.107230315359	17.5720330163	17.4526752219	38.1398714461
7	1.02291941253	0.225686952238	0.440606866992	1.47854018904	0.583161321836	2.75081439639	-0.0197148488692	0.0312901515723	0.0946833511133	-0.00545090171643	0.00431512311192	-0.0688678566199	-0.110600710315	0.0765464820619	0.0238115612832	0.151554811444	0.0067670776325	5.8677491456E-5	0.105898356783	0.0110553963152	0.124431559354	4.69723315309	11.3485777057	6.62398742209
8	0.974326669796	0.247135941635	-0.214111387042	1.06755578278	-0.11614819286	0.626046297352	0.0809006311317	-0.178609028837	0.070402599412	0.285252604328	-0.0583306351233	0.348953186647	0.0123879314849	-0.0954430876469	-0.0113888908784	0.232592498836	-0.00768966467258	0.0621553297239	0.185011975267	-0.0138691038178	0.175490079419	-10.7143828102	5.17888749811	18.4512887586
9	1.15644837568	0.323582328448	-0.690109935943	1.20697728321	-0.396629111171	1.43894050503	0.0187602176169	-0.0267236784781	0.0924836332718	0.0949141993824	0.00640381115246	0.460891516645	0.00224527576072	-0.184397406551	-0.0284872874528	0.154994090381	0.00297011483708	0.0403691895698	0.171900864291	-0.0192863255718	0.242457471842	-18.0614666679	2.89433504728	12.5068047918
10	1.11304289704	-0.293030082905	-0.295878843203	1.31922725189	0.25534481572	0.771961784134	-0.0391407300006	0.0362323068058	-0.0749070912113	0.110948788935	-0.0154694750479	0.296435456731	0.121776434771	-0.117107563295	0.0401672842182	0.172682028736	-0.0208829818976	0.0167712172481	0.154818322257	-0.0102778704316	0.189233657448	-12.455067112	-8.72246835084	8.48173783522
11	4.05033748314	-1.4914540167	1.33819893986	1.73720804146	-0.510228610463	1.10074447694	-0.0694691773379	0.155447064775	-0.106028294692	-0.227211325251	-0.0231636742609	0.313391375759	0.120051968774	-0.0490561975464	0.0893859224097	0.206197497783	-0.0152387186805	-0.0199356267686	0.151995971727	-0.0260613457999	0.191034320516	-11.293997961	-14.3152457414	0.535599647379
12	1.46497385587	0.209104718986	0.442908398411	0.88899917835	0.280235984289	0.766403261876	0.0382253162076	0.0303923477584	-0.0758511199156	-0.0871173985781	0.0142464552788	0.00477657432329	0.0626830331996	-0.0264623910909	-0.0325539478254	0.155023544319	-0.00147947485566	0.00132506441693	0.11656176075	-0.00551190322739	0.11159207452	0.971693459091	-6.39309141231	2.0773080608
13	1.4603874515	0.138268503347	0.0206095221909	1.6215800639	0.661807398691	1.09856692622	-0.00801060074453	0.171312618258	-0.0097008211924	-0.198308067834	0.0127623198564	0.00923903660344	-0.0432281282784	0.0818482785392	-0.0292072817279	0.175216862199	0.00045486879019	0.0189976035093	0.138788181903	0.00349351194873	0.114647291995	6.45987640338	-0.667356091284	-0.57727929684
14	1.75173782846	0.261605197331	0.819113135956	2.13177174798	-0.0404800824561	1.94160961775	0.0477129922935	0.0732909694312	0.0443469211354	0.000792817922895	0.0172929567756	-0.118425813118	0.041284197641	0.238979592696	-0.0399940119861	0.111097102079	0.00409862803378	0.0123527975971	0.104224878455	-0.013595420567	0.113263219396	14.9700890098	4.0659414189	8.70967416505

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 181 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 182 through 196 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 197 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 181 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 182 through 196 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 197 through 244 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 245 through 270 )
14	X-RAY DIFFRACTION	14	chain 'B' and (resid 271 through 303 )

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