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- PDB-4p08: Engineered thermostable dimeric cocaine esterase -

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Basic information

Entry
Database: PDB / ID: 4p08
TitleEngineered thermostable dimeric cocaine esterase
ComponentsCocaine esterase
KeywordsHYDROLASE / esterase / disulfide-linked dimer / cocaine / mutant
Function / homology
Function and homology information


cocaine esterase / cocaine catabolic process / carboxylic ester hydrolase activity / dipeptidyl-peptidase activity / cytoplasm
Similarity search - Function
alpha-amino acid ester hydrolase ( Helical cap domain) / alpha-amino acid ester hydrolase ( Helical cap domain) / CocE/Serine esterase / Xaa-Pro dipeptidyl-peptidase, C-terminal / X-Pro dipeptidyl-peptidase C-terminal non-catalytic domain / X-Pro dipeptidyl-peptidase C-terminal non-catalytic domain / Xaa-Pro dipeptidyl-peptidase-like domain / X-Pro dipeptidyl-peptidase (S15 family) / Galactose-binding domain-like / Galactose-binding-like domain superfamily ...alpha-amino acid ester hydrolase ( Helical cap domain) / alpha-amino acid ester hydrolase ( Helical cap domain) / CocE/Serine esterase / Xaa-Pro dipeptidyl-peptidase, C-terminal / X-Pro dipeptidyl-peptidase C-terminal non-catalytic domain / X-Pro dipeptidyl-peptidase C-terminal non-catalytic domain / Xaa-Pro dipeptidyl-peptidase-like domain / X-Pro dipeptidyl-peptidase (S15 family) / Galactose-binding domain-like / Galactose-binding-like domain superfamily / Alpha/Beta hydrolase fold, catalytic domain / Alpha/Beta hydrolase fold / Jelly Rolls / Sandwich / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Biological speciesRhodococcus sp. (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.341 Å
AuthorsRodgers, D.W. / Chow, K.-M. / Fang, L. / Zhan, C.-G.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA035552 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA032910 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA013930 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)DA025100 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103486 United States
CitationJournal: Acs Chem.Biol. / Year: 2014
Title: Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.
Authors: Fang, L. / Chow, K.M. / Hou, S. / Xue, L. / Chen, X. / Rodgers, D.W. / Zheng, F. / Zhan, C.G.
History
DepositionFeb 20, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 16, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 1, 2014Group: Database references
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Derived calculations ...Author supporting evidence / Derived calculations / Other / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_audit_support ...entity_src_gen / pdbx_audit_support / pdbx_database_status / pdbx_struct_assembly / pdbx_struct_oper_list / software
Item: _entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization ..._entity_src_gen.pdbx_alt_source_flag / _pdbx_audit_support.funding_organization / _pdbx_database_status.pdb_format_compatible / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_oper_list.symmetry_operation / _software.classification
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 27, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine_hist
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine_hist.pdbx_number_atoms_nucleic_acid / _refine_hist.pdbx_number_atoms_protein

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cocaine esterase


Theoretical massNumber of molelcules
Total (without water)61,9451
Polymers61,9451
Non-polymers00
Water7,728429
1
A: Cocaine esterase

A: Cocaine esterase


Theoretical massNumber of molelcules
Total (without water)123,8902
Polymers123,8902
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation12_554x,x-y,-z-1/61
Buried area2380 Å2
ΔGint-13 kcal/mol
Surface area38600 Å2
MethodPISA
Unit cell
Length a, b, c (Å)106.647, 106.647, 220.532
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Components on special symmetry positions
IDModelComponents
11A-694-

HOH

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Components

#1: Protein Cocaine esterase /


Mass: 61945.078 Da / Num. of mol.: 1 / Mutation: T172R, G173Q, L196C, I301C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodococcus sp. (bacteria) / Strain: MB1 / Gene: cocE / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) / References: UniProt: Q9L9D7, cocaine esterase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 429 / Source method: isolated from a natural source / Formula: H2O

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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: 2.92 Å3/Da / Density % sol: 57.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.2
Details: 0.1 M phosphate-citrate (pH 4.2), 1.6 M sodium dihydrogen phosphate, and 0.4 M di-potassium hydrogen phosphate (JCSG IV # 94)

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 22-ID / Wavelength: 1 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Mar 5, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.34→50 Å / Num. obs: 32017 / % possible obs: 99.7 % / Redundancy: 17.5 % / Biso Wilson estimate: 28.84 Å2 / Rmerge(I) obs: 0.161 / Χ2: 1.107 / Net I/av σ(I): 19.611 / Net I/σ(I): 6.1 / Num. measured all: 561710
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Num. unique allΧ2% possible allRmerge(I) obs
2.34-2.429.930561.08797.4
2.42-2.5215.631241.06799.80.845
2.52-2.6418.431421.091000.704
2.64-2.7718.731401.141000.506
2.77-2.9518.631761.1721000.354
2.95-3.1818.631661.1731000.235
3.18-3.518.531991.1071000.144
3.5-418.632241.0651000.098
4-5.0418.932951.0621000.08
5.04-5019.134951.0999.50.058

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.8 Å33.28 Å
Translation2.8 Å33.28 Å

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHASERphasing
PHENIX(phenix.refine: 1.8.1_1168)refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.341→33.28 Å / SU ML: 0.2 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 20.99 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2208 1525 5 %random
Rwork0.1769 28978 --
obs0.1791 30503 95.24 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 76 Å2 / Biso mean: 27.7686 Å2 / Biso min: 7.54 Å2
Refinement stepCycle: final / Resolution: 2.341→33.28 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4364 0 0 429 4793
Biso mean----99999 -
Num. residues----571
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0054473
X-RAY DIFFRACTIONf_angle_d0.8936114
X-RAY DIFFRACTIONf_chiral_restr0.062684
X-RAY DIFFRACTIONf_plane_restr0.004807
X-RAY DIFFRACTIONf_dihedral_angle_d11.9911583
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 11

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.3414-2.4170.33221250.24982282240784
2.417-2.50330.29611270.23272407253489
2.5033-2.60350.29661290.21642505263492
2.6035-2.72190.25971260.19682544267093
2.7219-2.86540.26151400.18992586272696
2.8654-3.04480.22081410.19342649279097
3.0448-3.27970.2021420.19162689283198
3.2797-3.60940.23281420.16762727286999
3.6094-4.13080.20461470.15152764291199
4.1308-5.20120.18261470.13682812295999
5.2012-33.28380.18571590.177130133172100
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)
10.01030.00670.0040.00930.00730.02710.0359-0.013-0.0478-0.01990.1014-0.0973-0.02490.17680.125-0.0378-0.2085-0.09910.0163-0.25740.040759.693133.8643-4.9778
20.0864-0.0092-0.04840.10350.0570.26590.1391-0.1358-0.1621-0.00050.1402-0.1603-0.01290.13230.47080.0675-0.0016-0.09360.0289-0.0550.247948.761221.79510.1091
30.27560.01450.15110.30140.150.37930.127-0.1831-0.01760.0078-0.0575-0.1085-0.0362-0.21680.28040.0928-0.02-0.0070.12350.02370.062228.4227.57180.3155
40.0065-0.00560.02070.016-0.01720.02570.0993-0.2065-0.10630.1443-0.103-0.02620.0757-0.1741-0.04870.2252-0.0923-0.05040.2930.0850.218421.286417.218710.3504
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 4 through 103 )A0
2X-RAY DIFFRACTION2chain 'A' and (resid 104 through 213 )A0
3X-RAY DIFFRACTION3chain 'A' and (resid 214 through 535 )A0
4X-RAY DIFFRACTION4chain 'A' and (resid 536 through 574 )A0

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