PDB-4wl2: Structure of penicillin V acylase from Pectobacterium atrosepticum

Basic information

• Entry: Database: PDB / ID: 4wl2
• Title: Structure of penicillin V acylase from Pectobacterium atrosepticum
• Components: Putative exported choloylglycine hydrolase
• Keywords: HYDROLASE / Ntn hydrolase / choloylglycine / penicillin V / periplasmic

Function / homology

Function:

penicillin amidase / periplasmic space / hydrolase activity / response to antibiotic

Domain/homology:

: / Penicillin V Acylase; Chain A / Penicillin V Acylase; Chain A / Choloylglycine hydrolase/NAAA C-terminal / Linear amide C-N hydrolases, choloylglycine hydrolase family / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta

Component:

Penicillin V acylase

• Biological species: Pectobacterium atrosepticum SCRI1043 (bacteria)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
• Authors: Ramasamy, S. / Avinash, V.S. / Pundle, A.V. / Suresh, C.G.
• Citation: Journal: J.Struct.Biol. / Year: 2016; Title: Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity; Authors: Avinash, V.S. / Panigrahi, P. / Chand, D. / Pundle, A. / Suresh, C.G. / Ramasamy, S.

History

Deposition	Oct 6, 2014	Deposition site: RCSB / Processing site: PDBJ
Revision 1.0	Oct 7, 2015	Provider: repository / Type: Initial release
Revision 1.1	Apr 20, 2016	Group: Database references
Revision 1.2	Oct 16, 2024	Group: Data collection / Database references / Derived calculations / Refinement description / Structure summary Category: chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_oper_list / struct_conn / struct_ncs_dom_lim Item: _citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.symmetry_operation / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

Assembly

Deposited unit

A: Putative exported choloylglycine hydrolase

B: Putative exported choloylglycine hydrolase

C: Putative exported choloylglycine hydrolase

D: Putative exported choloylglycine hydrolase

E: Putative exported choloylglycine hydrolase

F: Putative exported choloylglycine hydrolase

G: Putative exported choloylglycine hydrolase

H: Putative exported choloylglycine hydrolase

Summary:

• 316 kDa, 8 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	315,802	8
Polymers	315,802	8
Non-polymers	0	0
Water	829	46

1

A: Putative exported choloylglycine hydrolase

H: Putative exported choloylglycine hydrolase

Summary:

• defined by author
• 79 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	78,951	2
Polymers	78,951	2
Non-polymers	0	0
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

2

B: Putative exported choloylglycine hydrolase

C: Putative exported choloylglycine hydrolase

Summary:

• defined by author
• 79 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	78,951	2
Polymers	78,951	2
Non-polymers	0	0
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

3

D: Putative exported choloylglycine hydrolase

G: Putative exported choloylglycine hydrolase

Summary:

• defined by author
• 79 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	78,951	2
Polymers	78,951	2
Non-polymers	0	0
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

4

E: Putative exported choloylglycine hydrolase

F: Putative exported choloylglycine hydrolase

Summary:

• defined by author
• 79 kDa, 2 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	78,951	2
Polymers	78,951	2
Non-polymers	0	0
Water	36	2

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Unit cell

Length a, b, c (Å)	118.447, 150.206, 185.630
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P212121

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B
1	2	A
2	2	C
1	3	A
2	3	D
1	4	A
2	4	E
1	5	A
2	5	F
1	6	A
2	6	G
1	7	A
2	7	H
1	8	B
2	8	C
1	9	B
2	9	D
1	10	B
2	10	E
1	11	B
2	11	F
1	12	B
2	12	G
1	13	B
2	13	H
1	14	C
2	14	D
1	15	C
2	15	E
1	16	C
2	16	F
1	17	C
2	17	G
1	18	C
2	18	H
1	19	D
2	19	E
1	20	D
2	20	F
1	21	D
2	21	G
1	22	D
2	22	H
1	23	E
2	23	F
1	24	E
2	24	G
1	25	E
2	25	H
1	26	F
2	26	G
1	27	F
2	27	H
1	28	G
2	28	H

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: CYS / Beg label comp-ID: CYS / End auth comp-ID: LEU / End label comp-ID: LEU / Refine code: _ / Auth seq-ID: 1 - 347 / Label seq-ID: 1 - 347

Dom-ID	Ens-ID	Auth asym-ID	Label asym-ID
1	1	A	A
2	1	B	B
1	2	A	A
2	2	C	C
1	3	A	A
2	3	D	D
1	4	A	A
2	4	E	E
1	5	A	A
2	5	F	F
1	6	A	A
2	6	G	G
1	7	A	A
2	7	H	H
1	8	B	B
2	8	C	C
1	9	B	B
2	9	D	D
1	10	B	B
2	10	E	E
1	11	B	B
2	11	F	F
1	12	B	B
2	12	G	G
1	13	B	B
2	13	H	H
1	14	C	C
2	14	D	D
1	15	C	C
2	15	E	E
1	16	C	C
2	16	F	F
1	17	C	C
2	17	G	G
1	18	C	C
2	18	H	H
1	19	D	D
2	19	E	E
1	20	D	D
2	20	F	F
1	21	D	D
2	21	G	G
1	22	D	D
2	22	H	H
1	23	E	E
2	23	F	F
1	24	E	E
2	24	G	G
1	25	E	E
2	25	H	H
1	26	F	F
2	26	G	G
1	27	F	F
2	27	H	H
1	28	G	G
2	28	H	H

NCS ensembles :

ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

Components

#1: Protein

A B C D E F G H
Putative exported choloylglycine hydrolase / PaPVA

Details:

Mass: 39475.289 Da / Num. of mol.: 8 / Fragment: UNP residues 30-376
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Gene: ECA3205 / Plasmid: pET28b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 star / References: UniProt: Q6D291, penicillin amidase

#2: Water

ChemComp-HOH / water

Details:

Mass: 18.015 Da / Num. of mol.: 46 / Source method: isolated from a natural source / Formula: H₂O

• Has protein modification: Y

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION

Sample preparation

• Crystal: Density Matthews: 2.63 ų/Da / Density % sol: 53.33 %
• Crystal grow: Temperature: 293 K / Method: vapor diffusion, hanging drop; Details: 14% PEG 3350, 0.2 M sodium potassium tartrate, 0.1 M HEPES pH 7.5; PH range: 7.0-7.5

Data collection

• Diffraction: Mean temperature: 100 K
• Diffraction source: Source: SYNCHROTRON / Site: RRCAT INDUS-2 / Beamline: PX-BL21 / Wavelength: 1.435 Å
• Detector: Type: RAYONIX MX-225 / Detector: CCD / Date: Jan 17, 2014
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 1.435 Å / Relative weight: 1
• Reflection: Resolution: 2.5→65.7 Å / Num. obs: 101210 / % possible obs: 88.5 % / Observed criterion σ(F): 488 / Redundancy: 7.26 % / R_merge(I) obs: 0.117 / R_sym value: 0.117 / Net I/av σ(I): 5.6 / Net I/σ(I): 11.7
• Reflection shell: Resolution: 2.5→2.64 Å / Redundancy: 4.99 % / R_merge(I) obs: 0.786 / Mean I/σ(I) obs: 1.8 / % possible all: 58.2

Processing

Software

Name	Version	Classification
REFMAC	5.7.0032	refinement
PHASER		phasing
MOSFLM		data processing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→63.43 Å / Cor.coef. F_o:F_c: 0.927 / Cor.coef. F_o:F_c free: 0.911 / SU B: 10.152 / SU ML: 0.222 / Cross valid method: THROUGHOUT / ESU R: 0.896 / ESU R Free: 0.314 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.24839	5077	5 %	RANDOM
Rwork	0.2183	-	-	-
obs	0.21981	96055	88.03 %	-

• Solvent computation: Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK

Displacement parameters

B_iso mean: 42.294 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	0.37 Å2	0 Å2	-0 Å2
2-	-	-0.64 Å2	-0 Å2
3-	-	-	0.27 Å2

Refinement step

Cycle: 1 / Resolution: 2.5→63.43 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	21688	0	0	46	21734

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.013	0.019	22256
X-RAY DIFFRACTION	r_bond_other_d	0.008	0.02	20816
X-RAY DIFFRACTION	r_angle_refined_deg	1.721	1.943	30240
X-RAY DIFFRACTION	r_angle_other_deg	1.555	3	47976
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	6.812	5	2768
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	38.871	24.56	1000
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	20.286	15	3616
X-RAY DIFFRACTION	r_dihedral_angle_4_deg	21.796	15	96
X-RAY DIFFRACTION	r_chiral_restr	0.094	0.2	3264
X-RAY DIFFRACTION	r_gen_planes_refined	0.009	0.021	25480
X-RAY DIFFRACTION	r_gen_planes_other	0.007	0.02	5208
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	3.662	4.095	11096
X-RAY DIFFRACTION	r_mcbond_other	3.661	4.094	11095
X-RAY DIFFRACTION	r_mcangle_it	5.912	6.136	13856
X-RAY DIFFRACTION	r_mcangle_other	5.911	6.137	13857
X-RAY DIFFRACTION	r_scbond_it	3.445	4.274	11160
X-RAY DIFFRACTION	r_scbond_other	3.445	4.274	11161
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	5.523	6.271	16385
X-RAY DIFFRACTION	r_long_range_B_refined	9.003	31.69	24735
X-RAY DIFFRACTION	r_long_range_B_other	9.004	31.694	24734
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-ID	Dom-ID	Auth asym-ID	Number	Rms dev position (Å)
1	1	A	20341	0.1
1	2	B	20341	0.1
2	1	A	20270	0.11
2	2	C	20270	0.11
3	1	A	20385	0.11
3	2	D	20385	0.11
4	1	A	20143	0.11
4	2	E	20143	0.11
5	1	A	20306	0.11
5	2	F	20306	0.11
6	1	A	20362	0.1
6	2	G	20362	0.1
7	1	A	20066	0.11
7	2	H	20066	0.11
8	1	B	20406	0.1
8	2	C	20406	0.1
9	1	B	20647	0.09
9	2	D	20647	0.09
10	1	B	20601	0.1
10	2	E	20601	0.1
11	1	B	20480	0.1
11	2	F	20480	0.1
12	1	B	20726	0.09
12	2	G	20726	0.09
13	1	B	20356	0.11
13	2	H	20356	0.11
14	1	C	20655	0.1
14	2	D	20655	0.1
15	1	C	20393	0.1
15	2	E	20393	0.1
16	1	C	20539	0.1
16	2	F	20539	0.1
17	1	C	20591	0.1
17	2	G	20591	0.1
18	1	C	20322	0.11
18	2	H	20322	0.11
19	1	D	20497	0.1
19	2	E	20497	0.1
20	1	D	20389	0.1
20	2	F	20389	0.1
21	1	D	20594	0.09
21	2	G	20594	0.09
22	1	D	20332	0.11
22	2	H	20332	0.11
23	1	E	20415	0.11
23	2	F	20415	0.11
24	1	E	20441	0.11
24	2	G	20441	0.11
25	1	E	20216	0.11
25	2	H	20216	0.11
26	1	F	20769	0.09
26	2	G	20769	0.09
27	1	F	20228	0.11
27	2	H	20228	0.11
28	1	G	20344	0.11
28	2	H	20344	0.11

LS refinement shell

Resolution: 2.5→2.565 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.37	305	-
Rwork	0.33	5386	-
obs	-	-	67.57 %