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- PDB-4pa8: Crystal structure of a de novo retro-aldolase catalyzing asymmetr... -

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Basic information

Entry
Database: PDB / ID: 4pa8
TitleCrystal structure of a de novo retro-aldolase catalyzing asymmetric Michael additions, with a covalently bound product analog
Componentsretro-aldolase
KeywordsHYDROLASE / protein engineering / computer-aided design / aldolase / retro-aldolase / Michael addition / enzyme design / directed evolution / substrate specificity / de novo protein / artificial catalyst / enzyme-product analog complex / TIM-barrel fold
Function / homologyAldolase class I / TIM Barrel / Alpha-Beta Barrel / Alpha Beta / (3R)-3-(4-methoxyphenyl)-5-oxohexanenitrile
Function and homology information
Biological speciesSulfolobus solfataricus (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.2 Å
AuthorsBeck, T. / Garrabou Pi, X. / Hilvert, D.
CitationJournal: Angew.Chem.Int.Ed.Engl. / Year: 2015
Title: A Promiscuous De Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.
Authors: Garrabou, X. / Beck, T. / Hilvert, D.
History
DepositionApr 7, 2014Deposition site: RCSB / Processing site: PDBE
Revision 1.0Apr 1, 2015Provider: repository / Type: Initial release
Revision 1.1May 13, 2015Group: Database references
Revision 1.2Dec 20, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: retro-aldolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,5176
Polymers29,9231
Non-polymers5945
Water5,116284
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area740 Å2
ΔGint-30 kcal/mol
Surface area10250 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.557, 68.804, 69.341
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Detailsbiological unit is the same as asym.

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Components

#1: Protein retro-aldolase


Mass: 29923.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfolobus solfataricus (archaea) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-2K6 / (3R)-3-(4-methoxyphenyl)-5-oxohexanenitrile


Mass: 217.264 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H15NO2
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 284 / 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: 2.49 Å3/Da / Density % sol: 50.67 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: Crystals were obtained at 4 degrees C using the sitting drop vapor diffusion method by mixing 100 nL of protein solution (13.5 mg/mL) in buffer with 100 nL of reservoir solution containing 2 ...Details: Crystals were obtained at 4 degrees C using the sitting drop vapor diffusion method by mixing 100 nL of protein solution (13.5 mg/mL) in buffer with 100 nL of reservoir solution containing 2 M ammonium sulfate and 0.1 M BIS-TRIS buffer pH 5.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M-F / Detector: PIXEL / Date: Jul 10, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.2→46.45 Å / Num. obs: 94052 / % possible obs: 99.8 % / Redundancy: 6.2 % / Rmerge(I) obs: 0.031 / Rpim(I) all: 0.013 / Net I/σ(I): 24.2 / Num. measured all: 584161 / Scaling rejects: 8
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) all% possible all
1.2-1.225.40.8112.12391444660.38396.3
6.57-46.455.70.02679.538596720.01299.3

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Processing

Software
NameVersionClassification
Aimless0.1.29data scaling
SHELXLrefinement
PDB_EXTRACT3.14data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4A29

4a29


Resolution: 1.2→46.3 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflectionSelection details
Rfree0.177 4653 4.96 %random selection
Rwork0.139 ---
obs-93775 99.8 %-
Displacement parametersBiso max: 77.03 Å2 / Biso mean: 22.5577 Å2 / Biso min: 10.42 Å2
Refinement stepCycle: LAST / Resolution: 1.2→46.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1836 0 37 284 2157

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