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- PDB-4hst: Crystal structure of a double mutant of a class III engineered ce... -

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Basic information

Entry
Database: PDB / ID: 4hst
TitleCrystal structure of a double mutant of a class III engineered cephalosporin acylase
Components
  • glutaryl-7-aminocephalosporanic acid acylase alpha chain
  • glutaryl-7-aminocephalosporanic acid acylase beta chain
KeywordsHYDROLASE / protein engineering / substrate specificity / transition state analogue / N-terminal hydrolase
Function / homology
Function and homology information


Penicillin Acylase III; Chain A, Domain 2 / 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 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 4-Layer Sandwich ...Penicillin Acylase III; Chain A, Domain 2 / 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 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 4-Layer Sandwich / Arc Repressor Mutant, subunit A / Roll / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
5,5-dihydroxy-L-norvaline
Similarity search - Component
Biological speciesPseudomonas (RNA similarity group I)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.571 Å
AuthorsVrielink, A. / Golden, E. / Patterson, R. / Tie, W.J. / Anandan, A. / Flematti, G. / Molla, G. / Rosini, E. / Pollegioni, L.
CitationJournal: Biochem.J. / Year: 2013
Title: Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.
Authors: Golden, E. / Paterson, R. / Tie, W.J. / Anandan, A. / Flematti, G. / Molla, G. / Rosini, E. / Pollegioni, L. / Vrielink, A.
History
DepositionOct 30, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 27, 2013Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2013Group: Database references
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.3Feb 28, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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: glutaryl-7-aminocephalosporanic acid acylase alpha chain
B: glutaryl-7-aminocephalosporanic acid acylase beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,2313
Polymers84,0822
Non-polymers1491
Water17,583976
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13640 Å2
ΔGint-95 kcal/mol
Surface area25310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.385, 77.845, 191.990
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein glutaryl-7-aminocephalosporanic acid acylase alpha chain


Mass: 24850.951 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas (RNA similarity group I) / Plasmid: pET24 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
#2: Protein glutaryl-7-aminocephalosporanic acid acylase beta chain


Mass: 59231.137 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas (RNA similarity group I) / Plasmid: pET24 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
#3: Chemical ChemComp-GLJ / 5,5-dihydroxy-L-norvaline


Type: L-peptide linking / Mass: 149.145 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 976 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsPROTEIN IS AN ENGINEERED CONSTRUCT DERIVED FROM PSEUDOMONAS PENICILLIN ACYLASE 2 (UNP P15558 ...PROTEIN IS AN ENGINEERED CONSTRUCT DERIVED FROM PSEUDOMONAS PENICILLIN ACYLASE 2 (UNP P15558 RESIDUES 1-229 FOR ALPHA CHAIN, 240-774 FOR BETA CHAIN) WITH TWO ADDITIONAL MUTATIONS (H296S, H309S) RELATIVE TO PDB ENTRY 4HSR.

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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: 3.04 Å3/Da / Density % sol: 59.52 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 10-20% PEG8000, 10 mM Tris-HCl, pH 8.0-8.5, VAPOR DIFFUSION, HANGING DROP, temperature 291K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.95663 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Mar 28, 2009
RadiationProtocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95663 Å / Relative weight: 1
ReflectionResolution: 1.571→95.995 Å / Num. all: 133919 / Num. obs: 133919 / % possible obs: 93.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 12 % / Biso Wilson estimate: 15.188 Å2 / Rmerge(I) obs: 0.095 / Net I/σ(I): 16.1

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Processing

Software
NameVersionClassification
Blu-IceIcedata collection
SHELXSphasing
REFMAC5.6.0117refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MAD / Resolution: 1.571→95.995 Å / Cor.coef. Fo:Fc: 0.977 / Cor.coef. Fo:Fc free: 0.963 / SU B: 1.949 / SU ML: 0.032 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.063 / ESU R Free: 0.062 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : RESIDUAL ONLY
RfactorNum. reflection% reflectionSelection details
Rfree0.16225 6723 5 %RANDOM
Rwork0.11931 ---
all0.12145 127103 --
obs0.12145 127103 93.4 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 15.178 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å20 Å2-0 Å2
2--0.19 Å20 Å2
3----0.02 Å2
Refinement stepCycle: LAST / Resolution: 1.571→95.995 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5751 0 10 976 6737
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0270.0196069
X-RAY DIFFRACTIONr_angle_refined_deg2.221.9478321
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1245801
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.65622.174276
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.56615902
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.7711565
X-RAY DIFFRACTIONr_chiral_restr0.2980.2895
X-RAY DIFFRACTIONr_gen_planes_refined0.0150.0214807
X-RAY DIFFRACTIONr_rigid_bond_restr7.34336069
X-RAY DIFFRACTIONr_sphericity_free43.0135289
X-RAY DIFFRACTIONr_sphericity_bonded14.14556569
LS refinement shellResolution: 1.571→1.612 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.245 411 -
Rwork0.178 8052 -
obs--83.81 %
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)
10.3171-0.03520.00020.46920.1090.45260.00390.03780.0599-0.05120.0065-0.0236-0.05790.0277-0.01040.0274-0.00320.00310.02870.00840.012420.948164.3837123.2173
20.32620.00780.01680.3362-0.01420.4120.01560.0151-0.0134-0.02420.0038-0.01510.03360.0129-0.01940.0146-0.0013-0.00330.01850.00290.003619.096450.771123.9174
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A14 - 229
2X-RAY DIFFRACTION2B2 - 601

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