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- PDB-4e57: Crystal Structure of spacer 6aa-shortened cephalosporin acylase mutant -

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Basic information

Entry
Database: PDB / ID: 4.0E+57
TitleCrystal Structure of spacer 6aa-shortened cephalosporin acylase mutant
ComponentsCephalosporin acylase
KeywordsHYDROLASE / cephalosporin acylase / spacer / autocleavage / glutaryl-7-aminocephalosporanic-acid acylase / N-terminal nucleophile (Ntn) hydrolase superfamily
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
Penicillin amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL acylase / Penicillin/GL-7-ACA/AHL/aculeacin-A acylase / Penicillin amidase type, domain 1 / Penicillin amidase type, N-terminal domain, B-knob ...Penicillin amidase (Acylase) alpha subunit, N-terminal domain / Aminohydrolase, alpha-helical knob region / Penicillin Amidohydrolase, domain 1 / Penicillin Amidohydrolase; domain 1 / Penicillin G acylase, beta-roll domain / Aminohydrolase, N-terminal nucleophile (Ntn) domain, beta-sheet knob region / Penicillin/GL-7-ACA/AHL acylase / Penicillin/GL-7-ACA/AHL/aculeacin-A acylase / Penicillin amidase type, domain 1 / Penicillin amidase type, N-terminal domain, B-knob / Penicillin amidase type, A-knob / Penicillin amidase / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Roll / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
: / Cephalosporin acylase
Similarity search - Component
Biological speciesPseudomonas sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2 Å
AuthorsYin, J. / Zhang, Z. / Wu, G. / Huang, X.
CitationJournal: To be Published
Title: spacer shortened Cephalosporin acylase
Authors: Yin, J. / Zhang, Z. / Wu, G. / Huang, X.
History
DepositionMar 14, 2012Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Mar 20, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ncs_dom_lim / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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 / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cephalosporin acylase
B: Cephalosporin acylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,02835
Polymers155,3192
Non-polymers3,71033
Water23,8521324
1
A: Cephalosporin acylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,79520
Polymers77,6591
Non-polymers2,13619
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Cephalosporin acylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,23315
Polymers77,6591
Non-polymers1,57414
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)73.599, 73.599, 383.834
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number76
Space group name H-MP41
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: PHE / End label comp-ID: PHE / Refine code: 4 / Auth seq-ID: 10 - 600 / Label seq-ID: 10 - 600

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

#1: Protein Cephalosporin acylase


Mass: 77659.281 Da / Num. of mol.: 2 / Fragment: UNP residues 31-719 / Mutation: DELETION, S166A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas sp. (bacteria) / Strain: C130 / Gene: CA, ca1 / Plasmid: pET28 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: O86089, glutaryl-7-aminocephalosporanic-acid acylase
#2: Chemical...
ChemComp-CD / CADMIUM ION


Mass: 112.411 Da / Num. of mol.: 33 / Source method: obtained synthetically / Formula: Cd
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1324 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHE SEQUENCE CONFLICT IS LIKELY TO BE A SPONTANEOUS MUTATION.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.35 Å3/Da / Density % sol: 63.25 % / Mosaicity: 0.114 °
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: Sodium acetate trihydrate, pH 7.5, VAPOR DIFFUSION, HANGING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U / Wavelength: 0.97916 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 15, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97916 Å / Relative weight: 1
ReflectionResolution: 2→50 Å / Num. obs: 136743 / % possible obs: 100 % / Redundancy: 7.6 % / Rmerge(I) obs: 0.122 / Χ2: 0.974 / Net I/σ(I): 6.8
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
2-2.077.40.515136100.8161100
2.07-2.157.70.381136980.9091100
2.15-2.257.70.288136511.0031100
2.25-2.377.70.24137091.0091100
2.37-2.527.70.195135941.0021100
2.52-2.717.70.156136720.9991100
2.71-2.997.70.1251367411100
2.99-3.427.70.105136590.9961100
3.42-4.317.20.097137331.0021100
4.31-507.30.081137431.001199.9

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
REFMAC5.6.0117refinement
PDB_EXTRACT3.1data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1FM2

1fm2


Resolution: 2→48.28 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.945 / Occupancy max: 1 / Occupancy min: 0.5 / SU B: 5.578 / SU ML: 0.088 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.143 / ESU R Free: 0.128 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES: WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2067 6866 5 %RANDOM
Rwork0.1825 ---
obs0.1837 136607 99.96 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 129.17 Å2 / Biso mean: 28.7359 Å2 / Biso min: 14.58 Å2
Baniso -1Baniso -2Baniso -3
1-0.68 Å20 Å20 Å2
2--0.68 Å20 Å2
3----1.36 Å2
Refinement stepCycle: LAST / Resolution: 2→48.28 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10658 0 33 1324 12015
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0210969
X-RAY DIFFRACTIONr_angle_refined_deg1.0411.93914990
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.43351352
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.00223.226561
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.075151575
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.87715100
X-RAY DIFFRACTIONr_chiral_restr0.0720.21568
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0218836
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Number: 4615 / Refine-ID: X-RAY DIFFRACTION

TypeRms dev position (Å)Weight position
MEDIUM POSITIONAL0.140.5
MEDIUM THERMAL0.342
LS refinement shellResolution: 2→2.052 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.277 548 -
Rwork0.233 9510 -
all-10058 -
obs--99.9 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°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)
10.3831-0.12020.30250.5315-0.33110.99750.05490.0182-0.02980.05990.01170.01240.0299-0.0863-0.06650.14720.0172-0.04770.0166-0.00280.065511.018226.5787-68.2601
20.37660.11040.32190.56010.36051.03930.0542-0.0163-0.03-0.0670.0145-0.0080.02180.091-0.06870.1454-0.0204-0.04880.01840.00340.063425.7874-10.2126-28.6442
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A9 - 685
2X-RAY DIFFRACTION2B9 - 685

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