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

ID or keywords:

Entry

Database: PDB / ID: 8brt

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: 1.31 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	8brt.cif.gz	442.7 KB	Display	PDBx/mmCIF format
PDB format	pdb8brt.ent.gz	364.6 KB	Display	PDB format
PDBx/mmJSON format	8brt.json.gz		Tree view	PDBx/mmJSON format
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Validation report

Summary document	8brt_validation.pdf.gz	452 KB	Display	wwPDB validaton report
Full document	8brt_full_validation.pdf.gz	452.9 KB	Display
Data in XML	8brt_validation.xml.gz	34.1 KB	Display
Data in CIF	8brt_validation.cif.gz	53.9 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/br/8brt ftp://data.pdbj.org/pub/pdb/validation_reports/br/8brt	HTTPS FTP

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Related structure data

Related structure data	8brqC 8brrC 8brsC 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

86.6 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)	61.314, 101.221, 139.493
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: 24681.822 Da / Num. of mol.: 1 / Mutation: T167V, K201A, K202A, E203A Source method: isolated from a genetically manipulated source Details: penicillin G acylase, alpha-subunit, Surface entropy reduction variant (variant hypho_7) 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: 61416.914 Da / Num. of mol.: 1 / Mutation: G14A, K135A, E136A, K137A, K403A, E404A, E405A Source method: isolated from a genetically manipulated source Details: penicillin G acylase, beta-subunit, Surface entropy reduction variant (variant hypho_7) 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: 24681.822 Da / Num. of mol.: 1 / Mutation: T167V, K201A, K202A, E203A
Source method: isolated from a genetically manipulated source
Details: penicillin G acylase, alpha-subunit, Surface entropy reduction variant (variant hypho_7)
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: 61416.914 Da / Num. of mol.: 1 / Mutation: G14A, K135A, E136A, K137A, K403A, E404A, E405A
Source method: isolated from a genetically manipulated source
Details: penicillin G acylase, beta-subunit, Surface entropy reduction variant (variant hypho_7)
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, 822 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.: 2 / 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.: 817 / 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 PEG 8000 Microbatch under paraffin oil

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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: 1.31→81.92 Å / Num. obs: 208981 / % possible obs: 99.8 % / Redundancy: 10 % / CC_1/2: 0.999 / R_merge(I) obs: 0.05 / R_pim(I) all: 0.017 / R_rim(I) all: 0.052 / Χ²: 0.84 / Net I/σ(I): 19.8 / Num. measured all: 2081190
Reflection shell	Resolution: 1.31→1.38 Å / % possible obs: 99.9 % / Redundancy: 10 % / R_merge(I) obs: 0.713 / Num. measured all: 303662 / Num. unique obs: 30239 / CC_1/2: 0.859 / R_pim(I) all: 0.233 / R_rim(I) all: 0.751 / Χ²: 0.56 / Net I/σ(I) obs: 2.8

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Processing

Software

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

Refinement

Method to determine structure:

MOLECULAR REPLACEMENT / Resolution: 1.31→46.05 Å / SU ML: 0.11 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 15.23 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.1611	10360	4.96 %
R_work	0.1485	-	-
obs	0.1492	208869	99.76 %

Solvent computation

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

Refinement step

Cycle: LAST / Resolution: 1.31→46.05 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	5829	0	29	817	6675

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.008	6077
X-RAY DIFFRACTION	f_angle_d	0.952	8215
X-RAY DIFFRACTION	f_dihedral_angle_d	13.715	2236
X-RAY DIFFRACTION	f_chiral_restr	0.075	837
X-RAY DIFFRACTION	f_plane_restr	0.01	1064

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 (%)
1.31-1.32	0.2404	336	0.2248	6490	X-RAY DIFFRACTION	100
1.32-1.34	0.2388	337	0.2193	6636	X-RAY DIFFRACTION	100
1.34-1.35	0.2186	345	0.2104	6546	X-RAY DIFFRACTION	100
1.35-1.37	0.2348	309	0.2046	6572	X-RAY DIFFRACTION	100
1.37-1.39	0.2207	347	0.1972	6595	X-RAY DIFFRACTION	100
1.39-1.41	0.2188	326	0.1927	6594	X-RAY DIFFRACTION	100
1.41-1.43	0.1916	325	0.1814	6605	X-RAY DIFFRACTION	100
1.43-1.45	0.1972	334	0.1812	6523	X-RAY DIFFRACTION	100
1.45-1.47	0.1852	329	0.1757	6576	X-RAY DIFFRACTION	100
1.47-1.5	0.1933	352	0.1679	6581	X-RAY DIFFRACTION	100
1.5-1.52	0.1789	335	0.1656	6567	X-RAY DIFFRACTION	100
1.52-1.55	0.1712	346	0.1611	6566	X-RAY DIFFRACTION	100
1.55-1.58	0.1791	369	0.1591	6586	X-RAY DIFFRACTION	100
1.58-1.61	0.1725	345	0.1623	6580	X-RAY DIFFRACTION	100
1.61-1.65	0.1785	362	0.1546	6592	X-RAY DIFFRACTION	100
1.65-1.69	0.1575	351	0.1527	6567	X-RAY DIFFRACTION	100
1.69-1.73	0.1625	358	0.1513	6587	X-RAY DIFFRACTION	100
1.73-1.77	0.1749	391	0.1551	6573	X-RAY DIFFRACTION	100
1.77-1.83	0.1688	333	0.1528	6609	X-RAY DIFFRACTION	100
1.83-1.89	0.1611	324	0.1516	6602	X-RAY DIFFRACTION	100
1.89-1.95	0.1679	345	0.1587	6630	X-RAY DIFFRACTION	100
1.95-2.03	0.1589	360	0.1508	6630	X-RAY DIFFRACTION	100
2.03-2.12	0.1569	357	0.147	6618	X-RAY DIFFRACTION	100
2.12-2.24	0.1577	309	0.1463	6675	X-RAY DIFFRACTION	100
2.24-2.38	0.1629	381	0.1407	6653	X-RAY DIFFRACTION	100
2.38-2.56	0.1622	366	0.1446	6655	X-RAY DIFFRACTION	100
2.56-2.82	0.1498	334	0.1454	6732	X-RAY DIFFRACTION	100
2.82-3.22	0.1674	327	0.145	6745	X-RAY DIFFRACTION	100
3.22-4.06	0.1453	375	0.1349	6732	X-RAY DIFFRACTION	99
4.06-46.05	0.1465	352	0.1381	6892	X-RAY DIFFRACTION	98

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	0.0122	0.0076	0.0046	0.0037	-0.0055	0.0251	0.0068	-0.1394	-0.0331	0.0513	0.0109	-0.0393	-0.1053	0.2038	0.0007	0.2004	-0.0226	-0.0485	0.2604	0.0262	0.1982	16.9002	-4.3008	44.1013
2	0.1392	0.1009	-0.0747	0.1853	0.132	0.2887	0.015	-0.0896	-0.0388	0.0661	-0.0466	0.0499	-0.0029	0.0042	-0	0.179	0.007	0.0097	0.16	0.02	0.1469	-4.0139	-10.3326	46.9466
3	0.0146	0.0779	0.0938	0.1812	-0.0743	0.1464	-0.0154	0.0039	-0.098	-0.0003	0.0487	0.0052	0.039	-0.0152	0	0.1332	-0.0076	0.0049	0.1757	-0.0201	0.1728	-15.5449	-17.4096	16.8451
4	0.1138	-0.0937	0.0896	0.2044	0.028	0.3192	-0.0272	0.029	-0.0337	0.0369	0.0207	-0.0151	-0.0074	0.0639	-0	0.1288	0.002	0.011	0.1586	-0.0013	0.1573	4.9324	-11.5293	26.784
5	0.0962	-0.0036	0.0138	-0.0022	0.0016	0.0095	-0.1205	-0.3271	-0.0592	0.1563	-0.119	0.0599	0.1375	-0.1487	-0.3662	0.2168	-0.324	0.3656	0.1697	-0.0016	0.4192	-26.9765	-23.8189	40.7857
6	0.0752	0.131	0.1439	0.4045	0.1082	0.3178	-0.0198	-0.0305	-0.1274	0.031	0.0019	0.0158	0.114	0.0417	-0.0003	0.1475	0.0052	0.0293	0.1307	-0.0014	0.2132	-5.2417	-25.1052	25.3246
7	0.1335	-0.1319	0.2065	0.1153	-0.0016	0.2485	-0.0266	0.1093	-0.0537	-0.0633	0.002	-0.006	-0.0497	0.0833	-0.0015	0.1557	-0.0191	0.016	0.1762	-0.0159	0.145	-0.769	-7.2653	8.3619
8	0.0915	-0.0695	0.0131	0.0586	0.0036	0.0308	-0.1429	0.0337	0.2185	-0.0099	-0.0241	-0.1014	-0.2513	0.0915	0.0008	0.3386	-0.0427	-0.0592	0.17	0.0347	0.176	-6.439	13.8438	2.3792
9	0.1579	0.0914	0.1154	0.2718	0.0227	0.5036	-0.087	-0.0236	0.0007	-0.0298	0.0281	0.0362	-0.1788	-0.0808	-0.0011	0.187	0.0161	-0.0142	0.156	-0.0136	0.1378	-13.7782	2.5254	11.4604
10	0.0568	0.0829	-0.017	0.0757	-0.014	0.09	-0.0128	-0.0966	0.0123	0.0407	0.0222	-0.0103	-0.2019	0.2002	-0.0018	0.2061	-0.0372	-0.0001	0.2174	-0.0043	0.1733	11.347	3.3408	33.8039

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 133 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 134 through 198 )
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 313 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 314 through 345 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 346 through 473 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 474 through 527 )

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

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Non-polymers , 4 types, 822 molecules

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