PDB-8brq: Crystal structure of a surface entropy reduction variant of penic...

ID or keywords:

Entry

Database: PDB / ID: 8brq

Title

Crystal structure of a surface entropy reduction variant of penicillin G acylase from Bacillaceae i. s. sp. FJAT-27231

Components

(Penicillin G ...) x 2

Keywords

HYDROLASE / Penicillin / Acylase / Surface entropy reduction / Crystal contact engineering / Crystallizability

Function / homology

Function and homology information

hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides / antibiotic biosynthetic process / metal ion binding
Similarity search - Function

Biological species

Bacillus sp. FJAT-27231 (bacteria)

Method

X-RAY DIFFRACTION /

SYNCHROTRON /

MOLECULAR REPLACEMENT / Resolution: 1.63 Å

Authors

Wichmann, J. / Mayer, J. / Mattes, H. / Lukat, P. / Blankenfeldt, W. / Biedendieck, R.

Funding support

Germany, 1items

Citation

Journal: Cryst.Growth Des. / Year: 2023
Title: Multistep Engineering of a Penicillin G Acylase for Systematic Improvement of Crystallization Efficiency
Authors: Wichmann, J. / Mayer, J. / Hintmann, M. / Lukat, P. / Blankenfeldt, W. / Biedendieck, R.

History

Deposition	Nov 23, 2022	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 3, 2023	Provider: repository / Type: Initial release
Revision 1.1	Jun 19, 2024	Group: Data collection / Category: chem_comp_atom / chem_comp_bond

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8brq.cif.gz	455.8 KB	Display	PDBx/mmCIF format
PDB format	pdb8brq.ent.gz	375.5 KB	Display	PDB format
PDBx/mmJSON format	8brq.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8brq_validation.pdf.gz	451.5 KB	Display	wwPDB validaton report
Full document	8brq_full_validation.pdf.gz	451.8 KB	Display
Data in XML	8brq_validation.xml.gz	34.8 KB	Display
Data in CIF	8brq_validation.cif.gz	55.5 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/br/8brq ftp://data.pdbj.org/pub/pdb/validation_reports/br/8brq	HTTPS FTP

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Related structure data

Related structure data	8brrC 8brsC 8brtC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - FunctionF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI
Related items in Molecule of the Month	#166 - Oct 2013 Proteasome similarity (1) #80 - Aug 2006 AAA+ Proteases similarity (1) #176 - Aug 2014 Dynein similarity (1) #60 - Dec 2004 Ubiquitin similarity (1)

Deposited unit

A: Penicillin G acylase

B: Penicillin G acylase

hetero molecules

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

1

Idetical with deposited unit
defined by author&software
Evidence: gel filtration

Unit cell

Length a, b, c (Å)	61.252, 102.105, 140.079
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P2₁2₁2₁

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Penicillin G ... , 2 types, 2 molecules AB

#1: Protein	Penicillin G acylase Mass: 24683.795 Da / Num. of mol.: 1 / Mutation: K201A, K202A, E203A Source method: isolated from a genetically manipulated source Details: Surface entropy reduction variant of penicillin G acylase, alpha-subunit (base variant) Source: (gene. exp.) Bacillus sp. FJAT-27231 (bacteria) / Gene: AC623_04440 / Production host: Priestia megaterium (bacteria) / References: UniProt: A0A0K9H482
#2: Protein	Penicillin G acylase Mass: 61402.891 Da / Num. of mol.: 1 / Mutation: K135A, E136A, K403A, E404A, E405A Source method: isolated from a genetically manipulated source Details: Surface entropy reduction variant of penicillin G acylase, beta-subunit (base variant) Source: (gene. exp.) Bacillus sp. FJAT-27231 (bacteria) / Gene: AC623_04440 / Production host: Priestia megaterium (bacteria) / References: UniProt: A0A0K9H482

#1: Protein

Penicillin G acylase

Mass: 24683.795 Da / Num. of mol.: 1 / Mutation: K201A, K202A, E203A
Source method: isolated from a genetically manipulated source
Details: Surface entropy reduction variant of penicillin G acylase, alpha-subunit (base variant)
Source: (gene. exp.)

Bacillus sp. FJAT-27231 (bacteria) / Gene: AC623_04440 / Production host:

Priestia megaterium (bacteria) / References: UniProt: A0A0K9H482

#2: Protein

Penicillin G acylase

Mass: 61402.891 Da / Num. of mol.: 1 / Mutation: K135A, E136A, K403A, E404A, E405A
Source method: isolated from a genetically manipulated source
Details: Surface entropy reduction variant of penicillin G acylase, beta-subunit (base variant)
Source: (gene. exp.)

Bacillus sp. FJAT-27231 (bacteria) / Gene: AC623_04440 / Production host:

Priestia megaterium (bacteria) / References: UniProt: A0A0K9H482

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Non-polymers , 4 types, 866 molecules

#3: Chemical	ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₈H₁₈N₂O₄S / Comment: pH buffer*YM
#4: Chemical	ChemComp-BU3 / (R,R)-2,3-BUTANEDIOL Mass: 90.121 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C₄H₁₀O₂
#5: Chemical	ChemComp-CA / CALCIUM ION Mass: 40.078 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Ca
#6: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 860 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has ligand of interest	N

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Experiment

Experiment	Method: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

Crystal	Density Matthews: 2.54 Å³/Da / Density % sol: 51.65 %
Crystal grow	Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5 Details: 100 mM HEPES pH 7.5 8 % (w/v) PEG 8000 Cryo-protection: 10 % (v/v) 2,3-(R,R)-butanediol

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
Detector	Type: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Jul 20, 2020
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.0332 Å / Relative weight: 1
Reflection	Resolution: 1.63→82.51 Å / Num. obs: 109974 / % possible obs: 99.5 % / Redundancy: 8.6 % / CC_1/2: 0.999 / R_merge(I) obs: 0.085 / R_pim(I) all: 0.031 / R_rim(I) all: 0.091 / Χ²: 0.91 / Net I/σ(I): 15.4 / Num. measured all: 940666
Reflection shell	Resolution: 1.63→1.72 Å / % possible obs: 98.5 % / Redundancy: 8.2 % / R_merge(I) obs: 0.901 / Num. measured all: 127907 / Num. unique obs: 15686 / CC_1/2: 0.805 / R_pim(I) all: 0.33 / R_rim(I) all: 0.962 / Χ²: 0.76 / Net I/σ(I) obs: 2.7

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Processing

Software

Name	Version	Classification
PHENIX	1.20-4459	refinement
autoPROC		data processing
Aimless		data scaling
XDS		data reduction
PHASER		phasing

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.63→52.53 Å / SU ML: 0.13 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 15.67 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1678	5451	4.96 %
R_work	0.143	-	-
obs	0.1442	109877	99.37 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL

Refinement step

Cycle: LAST / Resolution: 1.63→52.53 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5837	0	35	860	6732

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.009	6175
X-RAY DIFFRACTION	f_angle_d	0.976	8357
X-RAY DIFFRACTION	f_dihedral_angle_d	14.378	2292
X-RAY DIFFRACTION	f_chiral_restr	0.056	851
X-RAY DIFFRACTION	f_plane_restr	0.011	1088

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 (%)
1.63-1.65	0.2756	183	0.2356	3323	X-RAY DIFFRACTION	97
1.65-1.67	0.2451	169	0.2147	3425	X-RAY DIFFRACTION	99
1.67-1.69	0.2289	185	0.1973	3418	X-RAY DIFFRACTION	100
1.69-1.71	0.2072	151	0.1835	3448	X-RAY DIFFRACTION	98
1.71-1.73	0.2133	177	0.1757	3429	X-RAY DIFFRACTION	100
1.73-1.75	0.1923	179	0.1713	3436	X-RAY DIFFRACTION	98
1.75-1.78	0.1973	193	0.1659	3414	X-RAY DIFFRACTION	100
1.78-1.81	0.1921	171	0.1669	3444	X-RAY DIFFRACTION	98
1.81-1.83	0.1811	202	0.1637	3423	X-RAY DIFFRACTION	100
1.83-1.86	0.1927	205	0.1637	3432	X-RAY DIFFRACTION	99
1.86-1.9	0.2134	157	0.1633	3474	X-RAY DIFFRACTION	100
1.9-1.93	0.1819	164	0.1705	3423	X-RAY DIFFRACTION	99
1.93-1.97	0.1897	191	0.1535	3482	X-RAY DIFFRACTION	100
1.97-2.01	0.1861	189	0.1502	3408	X-RAY DIFFRACTION	99
2.01-2.05	0.1839	181	0.1438	3475	X-RAY DIFFRACTION	99
2.05-2.1	0.1503	179	0.1371	3506	X-RAY DIFFRACTION	100
2.1-2.15	0.166	178	0.1354	3438	X-RAY DIFFRACTION	99
2.15-2.21	0.15	168	0.1356	3504	X-RAY DIFFRACTION	100
2.21-2.27	0.1686	164	0.1322	3511	X-RAY DIFFRACTION	100
2.27-2.35	0.1626	174	0.1296	3498	X-RAY DIFFRACTION	100
2.35-2.43	0.1403	181	0.1283	3481	X-RAY DIFFRACTION	99
2.43-2.53	0.1501	191	0.1324	3473	X-RAY DIFFRACTION	100
2.53-2.64	0.1654	165	0.1327	3531	X-RAY DIFFRACTION	100
2.64-2.78	0.1704	178	0.137	3533	X-RAY DIFFRACTION	100
2.78-2.96	0.1782	190	0.141	3506	X-RAY DIFFRACTION	100
2.96-3.19	0.1504	195	0.1434	3523	X-RAY DIFFRACTION	100
3.19-3.51	0.1576	179	0.1332	3564	X-RAY DIFFRACTION	100
3.51-4.01	0.1581	208	0.13	3545	X-RAY DIFFRACTION	100
4.01-5.06	0.1339	209	0.1175	3586	X-RAY DIFFRACTION	100
5.06-52.53	0.1872	195	0.1625	3773	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	3.5273	-2.615	1.5733	2.4557	-2.2652	3.1678	-0.3735	-0.4517	0.2251	0.7914	0.0973	-0.5244	-0.6205	0.3819	0.2327	0.2947	-0.0164	-0.0906	0.3198	-0.0189	0.2339	17.0859	-3.6062	45.6206
2	1.1547	0.1371	-0.1129	1.0648	0.4184	1.1054	-0.0027	-0.1526	-0.033	0.1275	-0.0159	0.07	-0.0004	-0.0182	0.0199	0.1967	0.0106	0.0055	0.1908	0.0216	0.1391	-4.5175	-10.7737	46.04
3	4.297	0.7382	0.3276	0.7984	-0.1003	1.4829	-0.0447	0.2464	-0.2798	-0.0889	0.0481	0.0273	0.0827	-0.0806	-0.0091	0.1719	-0.0038	-0.0224	0.0922	-0.0243	0.1716	-19.1508	-18.0965	10.5937
4	0.683	-0.1601	-0.0197	0.5489	-0.0485	0.8028	-0.0261	-0.0317	-0.0083	0.0451	0.0159	-0.0292	-0.0332	0.0993	0.0134	0.136	0.0139	-0.0031	0.1291	-0.0003	0.1285	5.1424	-11.2397	27.893
5	1.8298	2.4725	1.5344	4.0959	1.8539	1.5365	0.1905	-0.5712	0.1171	0.8249	-0.8182	1.0393	0.5741	-0.7094	0.5365	0.3815	-0.1249	0.1665	0.4203	-0.0401	0.4456	-26.8043	-23.5277	42.1146
6	1.6078	0.2743	0.2066	0.786	-0.0133	0.6456	0.0139	-0.0769	-0.2118	0.0032	-0.0032	0.0974	0.1175	-0.0517	-0.0156	0.1586	0.0071	0.0046	0.117	0.0059	0.1848	-5.1149	-25.0204	26.4647
7	1.4556	0.2561	0.6365	1.3542	-0.312	0.5721	0.0182	0.1189	-0.0958	-0.0782	-0.0041	0.0519	0.0516	0.0518	-0.0142	0.1653	0.0107	0.0097	0.1621	-0.032	0.1374	-1.8334	-17.0859	13.2231
8	2.1501	-1.0087	1.4272	1.1838	-0.4763	1.4188	0.0506	0.3708	0.0954	-0.1577	-0.1038	-0.082	-0.0701	0.2383	0.0488	0.1567	0.0003	0.0173	0.1597	0.0199	0.1092	0.1393	-0.1644	6.3034
9	2.2866	-0.2062	1.6733	1.693	0.4928	4.7437	-0.2088	0.2711	0.3314	-0.3744	-0.0973	-0.0744	-0.5777	0.0797	0.3087	0.2951	-0.0045	-0.007	0.1409	0.0397	0.2003	-6.5718	13.464	2.6675
10	1.1831	0.6339	-0.0438	1.527	-0.6174	0.7804	-0.0009	0.0538	0.0113	-0.038	0.0367	0.1696	-0.0618	-0.1208	-0.031	0.1558	0.0208	-0.0202	0.1797	-0.0079	0.149	-19.4843	-4.0274	6.3551
11	1.1784	0.0744	1.4638	0.7968	0.5335	2.4681	-0.1558	-0.0753	0.2687	-0.1643	-0.051	0.1016	-0.4852	-0.2962	0.2064	0.3046	0.0511	-0.0339	0.2193	-0.0031	0.2287	-15.2933	12.7189	7.2648
12	0.6575	0.239	-0.5286	0.498	-0.2574	1.77	-0.0489	-0.0895	0.0713	0.0894	0.0387	0.0608	-0.2353	-0.1803	0.0054	0.1974	0.008	-0.0206	0.1823	-0.027	0.1872	-1.3359	2.1223	29.6336
13	0.9037	-0.3544	-1.0538	1.0759	0.6763	1.8135	-0.0571	-0.2379	0.1428	0.124	0.1019	-0.1209	-0.1323	0.2787	-0.0525	0.2124	-0.0332	-0.0323	0.2149	-0.0219	0.2042	11.1876	3.8375	34.5528

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 2 through 25 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 26 through 152 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 153 through 197 )
4	X-RAY DIFFRACTION	4	chain 'B' and (resid 1 through 81 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 82 through 124 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 125 through 229 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 230 through 265 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 266 through 313 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 314 through 345 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 346 through 402 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 403 through 442 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 443 through 473 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 474 through 527 )

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Non-polymers , 4 types, 866 molecules

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Penicillin G ... , 2 types, 2 molecules AB

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