PDB-8brs: Crystal structure of a variant of penicillin G acylase from Bacil...

ID or keywords:

Entry

Database: PDB / ID: 8brs

Title

Crystal structure of a variant of penicillin G acylase from Bacillaceae i. s. sp. FJAT-27231 with reduced surface entropy and additionally engineered crystal contact.

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: 1.2 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	Feb 7, 2024	Group: Data collection / Category: chem_comp_atom / chem_comp_bond

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	8brs.cif.gz	446.5 KB	Display	PDBx/mmCIF format
PDB format	pdb8brs.ent.gz	367.5 KB	Display	PDB format
PDBx/mmJSON format	8brs.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	8brs_validation.pdf.gz	453.6 KB	Display	wwPDB validaton report
Full document	8brs_full_validation.pdf.gz	454.6 KB	Display
Data in XML	8brs_validation.xml.gz	34.4 KB	Display
Data in CIF	8brs_validation.cif.gz	54.6 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/br/8brs ftp://data.pdbj.org/pub/pdb/validation_reports/br/8brs	HTTPS FTP

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Related structure data

Related structure data	8brqC 8brrC 8brtC 6nvxS S: Starting model for refinement 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)	61.322, 101.627, 139.435
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: penicillin G acylase, alpha-subunit, Surface entropy reduction variant (variant hypho_4) 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: 61386.867 Da / Num. of mol.: 1 / Mutation: K135A, E136A, K137A, K403A, E404A, E405A, K408L Source method: isolated from a genetically manipulated source Details: penicillin G acylase, beta-subunit, Surface entropy reduction variant (variant hypho_4) 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: penicillin G acylase, alpha-subunit, Surface entropy reduction variant (variant hypho_4)
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: 61386.867 Da / Num. of mol.: 1 / Mutation: K135A, E136A, K137A, K403A, E404A, E405A, K408L
Source method: isolated from a genetically manipulated source
Details: penicillin G acylase, beta-subunit, Surface entropy reduction variant (variant hypho_4)
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, 848 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: obtained synthetically / Formula: Ca
#6: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 842 / 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: 2.51 Å³/Da / Density % sol: 51.07 %
Crystal grow	Temperature: 293 K / Method: microbatch / pH: 7.5 Details: 100 mM HEPES pH 7.5 30 mg/ml PEG 8000 Microbatch under paraffin oil Cryoprotection: 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 EIGER X 16M / Detector: PIXEL / Date: Sep 8, 2021
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.0332 Å / Relative weight: 1
Reflection	Resolution: 1.2→82.13 Å / Num. obs: 261459 / % possible obs: 96.3 % / Redundancy: 11.6 % / CC_1/2: 0.999 / R_merge(I) obs: 0.054 / R_pim(I) all: 0.016 / R_rim(I) all: 0.056 / Χ²: 0.87 / Net I/σ(I): 21.9 / Num. measured all: 3042104
Reflection shell	Resolution: 1.2→1.26 Å / % possible obs: 79.1 % / Redundancy: 6.5 % / R_merge(I) obs: 0.536 / Num. measured all: 202112 / Num. unique obs: 31090 / CC_1/2: 0.874 / R_pim(I) all: 0.221 / R_rim(I) all: 0.583 / Χ²: 0.53 / 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
Starting model: 6NVX

6nvx

Resolution: 1.2→46.04 Å / SU ML: 0.09 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 14.69 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1559	13202	5.05 %
R_work	0.1442	-	-
obs	0.1448	261330	96.25 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 1.2→46.04 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5807	0	35	842	6684

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.008	6115
X-RAY DIFFRACTION	f_angle_d	0.995	8277
X-RAY DIFFRACTION	f_dihedral_angle_d	6.162	824
X-RAY DIFFRACTION	f_chiral_restr	0.079	845
X-RAY DIFFRACTION	f_plane_restr	0.012	1076

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 (%)
1.2-1.21	0.2158	298	0.2354	5911	X-RAY DIFFRACTION	69
1.21-1.23	0.2527	319	0.2201	6484	X-RAY DIFFRACTION	76
1.23-1.24	0.228	371	0.213	6892	X-RAY DIFFRACTION	81
1.24-1.26	0.2034	396	0.2052	7345	X-RAY DIFFRACTION	86
1.26-1.27	0.205	413	0.1909	7697	X-RAY DIFFRACTION	90
1.27-1.29	0.207	440	0.1813	7923	X-RAY DIFFRACTION	94
1.29-1.31	0.1823	457	0.1681	8224	X-RAY DIFFRACTION	97
1.31-1.33	0.182	457	0.1608	8540	X-RAY DIFFRACTION	100
1.33-1.35	0.1749	484	0.1573	8474	X-RAY DIFFRACTION	100
1.35-1.37	0.1726	489	0.1541	8535	X-RAY DIFFRACTION	100
1.37-1.4	0.1497	433	0.1557	8606	X-RAY DIFFRACTION	100
1.4-1.42	0.181	432	0.1518	8501	X-RAY DIFFRACTION	100
1.42-1.45	0.1697	447	0.1526	8580	X-RAY DIFFRACTION	100
1.45-1.48	0.1603	425	0.1483	8541	X-RAY DIFFRACTION	100
1.48-1.51	0.1639	445	0.1435	8541	X-RAY DIFFRACTION	100
1.51-1.55	0.1514	471	0.141	8575	X-RAY DIFFRACTION	100
1.55-1.58	0.1436	475	0.1367	8517	X-RAY DIFFRACTION	100
1.58-1.63	0.1505	427	0.1397	8600	X-RAY DIFFRACTION	100
1.63-1.68	0.1491	460	0.1363	8554	X-RAY DIFFRACTION	100
1.68-1.73	0.1573	452	0.1395	8576	X-RAY DIFFRACTION	100
1.73-1.79	0.1597	459	0.1455	8601	X-RAY DIFFRACTION	100
1.79-1.86	0.1472	488	0.1405	8566	X-RAY DIFFRACTION	100
1.86-1.95	0.1611	432	0.1458	8643	X-RAY DIFFRACTION	100
1.95-2.05	0.1501	465	0.1408	8595	X-RAY DIFFRACTION	100
2.05-2.18	0.1523	438	0.1392	8655	X-RAY DIFFRACTION	100
2.18-2.35	0.1561	441	0.1326	8690	X-RAY DIFFRACTION	100
2.35-2.58	0.1341	458	0.1396	8681	X-RAY DIFFRACTION	100
2.58-2.96	0.1523	490	0.1418	8685	X-RAY DIFFRACTION	100
2.96-3.73	0.1497	464	0.1396	8790	X-RAY DIFFRACTION	100
3.73-46.04	0.1595	476	0.1453	8606	X-RAY DIFFRACTION	95

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	0.0234	0.002	-0.0071	0.0129	0.0041	0.0409	0.0159	-0.193	0.0063	0.1785	-0.0028	-0.0725	-0.1503	0.1862	0.0014	0.193	-0.0144	-0.0358	0.2671	0.0202	0.1671	17.0029	-4.4405	44.5067
2	0.0286	0.0411	0.0731	0.0982	0.1266	0.1641	0.0157	-0.2099	-0.0667	0.0952	-0.038	0.0704	0.0196	-0.0006	-0	0.1743	-0.0028	0.0116	0.1909	0.0199	0.1421	-1.6028	-7.7646	45.5432
3	0.103	0.0864	0.119	0.2991	0.2954	0.2702	-0.0898	-0.12	0.0249	0.0515	-0.0896	0.0223	0.0884	-0.1623	-0.0746	0.201	0.0256	0.0185	0.2071	0.0341	0.2373	-12.797	-18.293	41.669
4	0.1063	0.0348	0.0042	0.0219	-0.0268	0.0508	0.0682	-0.2334	-0.044	0.1771	-0.0998	-0.08	0.0343	0.0128	-0.0073	0.2185	-0.0117	-0.0004	0.2222	0.046	0.1596	4.5562	-12.1898	51.4872
5	0.0119	-0.0007	-0.0026	0.0081	0.0159	0.021	0.0327	-0.087	0.0055	0.0329	-0.0184	0.0889	-0.0157	0.0013	0	0.2238	0.0039	0.038	0.1805	0.0037	0.1456	-6.5633	-2.1894	51.7768
6	0.0278	-0.0077	-0.0112	0.0121	0.007	0.0083	-0.0128	0.0502	-0.165	-0.0026	0.0079	0.0605	0.0291	-0.073	-0	0.1514	0.0001	0.013	0.1655	0.0136	0.1701	-7.6497	-15.5884	34.8687
7	0.0209	0.0164	-0.0161	0.0181	-0.0022	0.0147	-0.0573	0.0154	-0.0644	0.0044	0.0147	0.068	-0.0103	-0.0726	0.0001	0.1453	-0.0101	-0.0076	0.1946	-0.0151	0.1926	-28.7613	-16.4348	14.6607
8	0.0062	0.002	-0.0012	0.0059	-0.0095	0.0087	0.08	0.0408	-0.0608	-0.0843	-0.016	0.132	0.0476	-0.0664	-0	0.1712	-0.0177	-0.0301	0.1881	-0.0293	0.1926	-26.3626	-18.5292	5.3418
9	0.0098	0.0109	-0.0055	0.0103	0.0025	0.0292	0.009	0.0739	-0.2236	-0.0383	0.0548	-0.118	0.1553	0.1233	-0	0.1683	0.0087	0.0015	0.1887	-0.063	0.223	-4.286	-19.5526	7.3416
10	0.1601	-0.1468	0.0054	0.2124	0.0575	0.3356	-0.0217	-0.0041	-0.0361	0.0483	0.0107	-0.0032	-0.0165	0.0785	-0.0015	0.1287	0.0044	0.0052	0.1407	0.0072	0.138	5.0868	-11.3975	26.9538
11	0.2224	0.0252	0.0854	0.0083	0.0121	0.0602	-0.1884	-0.5105	-0.1354	0.1521	-0.1744	0.1901	0.2759	-0.2659	-0.2439	0.1956	-0.3008	0.3901	0.2618	-0.0555	0.4741	-26.9556	-25.19	40.3246
12	0.1251	0.0697	0.0396	0.5049	0.1084	0.4283	-0.0248	-0.039	-0.1789	0.0275	-0.0134	0.0339	0.0919	0.0269	-0.0328	0.1407	0.0026	0.0273	0.1155	0.009	0.2024	-4.907	-25.1533	25.5326
13	0.0332	0.0361	0.0215	0.0247	-0.0015	0.0385	-0.0224	0.1006	-0.1019	-0.0194	0.0037	0.0293	0.004	-0.0031	-0	0.1408	-0.0033	0.0131	0.1483	-0.0326	0.1721	-2.0713	-17.2545	12.2269
14	0.0522	-0.0879	0.0289	0.1028	0.0066	0.0928	-0.0322	0.1474	0.021	-0.1101	-0.0183	-0.0312	-0.1104	0.1091	-0	0.1785	-0.0102	-0.0001	0.1862	0.0104	0.135	0.4427	-0.2392	6.0898
15	0.0799	-0.1048	-0.0119	0.1192	-0.0104	0.0295	-0.1203	0.064	0.2209	-0.0758	-0.036	-0.0734	-0.2994	0.061	0.0089	0.3047	-0.0155	-0.0411	0.1674	0.0287	0.1675	-6.9014	13.5509	1.8157
16	0.1998	0.1066	0.0733	0.1763	-0.0456	0.1635	-0.0439	0.002	-0.0122	-0.058	0.0149	0.0987	-0.084	-0.1122	0	0.18	0.0142	-0.0202	0.1848	-0.0154	0.1502	-19.8339	-0.1747	4.0965
17	0.1351	0.0597	0.0458	0.051	0.0453	0.0347	-0.0417	-0.0533	-0.0031	0.0298	0.0186	0.0946	-0.1974	-0.14	-0.003	0.2504	0.0315	-0.0145	0.154	-0.0035	0.1675	-8.3363	10.4499	23.0377
18	0.1116	0.151	-0.115	0.1697	-0.0513	0.1664	0.005	-0.1004	-0.0259	0.0636	-0.0143	-0.0256	-0.1008	0.1438	0.0002	0.1689	-0.0222	-0.0012	0.1764	-0.0038	0.14	10.8936	2.124	31.8899

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 3 through 25 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 26 through 61 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 62 through 89 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 90 through 117 )
5	X-RAY DIFFRACTION	5	chain 'A' and (resid 118 through 133 )
6	X-RAY DIFFRACTION	6	chain 'A' and (resid 134 through 152 )
7	X-RAY DIFFRACTION	7	chain 'A' and (resid 153 through 168 )
8	X-RAY DIFFRACTION	8	chain 'A' and (resid 169 through 180 )
9	X-RAY DIFFRACTION	9	chain 'A' and (resid 181 through 197 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 1 through 81 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 82 through 124 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 125 through 229 )
13	X-RAY DIFFRACTION	13	chain 'B' and (resid 230 through 265 )
14	X-RAY DIFFRACTION	14	chain 'B' and (resid 266 through 312 )
15	X-RAY DIFFRACTION	15	chain 'B' and (resid 313 through 345 )
16	X-RAY DIFFRACTION	16	chain 'B' and (resid 346 through 424 )
17	X-RAY DIFFRACTION	17	chain 'B' and (resid 425 through 462 )
18	X-RAY DIFFRACTION	18	chain 'B' and (resid 463 through 527 )

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

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Non-polymers , 4 types, 848 molecules

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-Non-polymers , 4 types, 848 molecules

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

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Non-polymers , 4 types, 848 molecules

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Experimental details

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