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Basic information

Entry
Database: PDB / ID: 2vek
TitleStructure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
ComponentsTRIOSEPHOSPHATE ISOMERASE
KeywordsISOMERASE / TRIOSEPHOSPHATE ISOMERASE / TIM BARREL / GLYCOLYSIS / ENGINEERING / PENTOSE SHUNT / BINDING POCKET / GLUCONEOGENESIS / LIPID SYNTHESIS / SUBSTRATE SPECIFICITY / FATTY ACID BIOSYNTHESIS / TIM / ENZYME / MONOMERIC / GLYCOSOME
Function / homology
Function and homology information


glycosome / triose-phosphate isomerase / triose-phosphate isomerase activity / glycerol catabolic process / glyceraldehyde-3-phosphate biosynthetic process / gluconeogenesis / glycolytic process / cytosol / cytoplasm
Similarity search - Function
Triosephosphate isomerase, bacterial/eukaryotic / Triosephosphate isomerase, active site / Triosephosphate isomerase active site. / Triosephosphate isomerase / Triosephosphate isomerase superfamily / Triosephosphate isomerase / Triosephosphate isomerase (TIM) family profile. / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel ...Triosephosphate isomerase, bacterial/eukaryotic / Triosephosphate isomerase, active site / Triosephosphate isomerase active site. / Triosephosphate isomerase / Triosephosphate isomerase superfamily / Triosephosphate isomerase / Triosephosphate isomerase (TIM) family profile. / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
3-(BUTYLSULPHONYL)-PROPANOIC ACID / CITRIC ACID / TERTIARY-BUTYL ALCOHOL / Triosephosphate isomerase, glycosomal
Similarity search - Component
Biological speciesTRYPANOSOMA BRUCEI BRUCEI (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsAlahuhta, M. / Salin, M. / Casteleijn, M.G. / Kemmer, C. / El-Sayed, I. / Augustyns, K. / Neubauer, P. / Wierenga, R.K.
CitationJournal: Protein Eng.Des.Sel. / Year: 2008
Title: Structure-Based Protein Engineering Efforts with a Monomeric Tim Variant: The Importance of a Single Point Mutation for Generating an Active Site with Suitable Binding Properties.
Authors: Alahuhta, M. / Salin, M. / Casteleijn, M.G. / Kemmer, C. / El-Sayed, I. / Augustyns, K. / Neubauer, P. / Wierenga, R.K.
History
DepositionOct 24, 2007Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Version format compliance
Revision 1.2Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "BA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TRIOSEPHOSPHATE ISOMERASE
B: TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,9716
Polymers51,3192
Non-polymers6534
Water6,756375
1
A: TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,8512
Polymers25,6591
Non-polymers1921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,1204
Polymers25,6591
Non-polymers4603
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)45.960, 87.220, 56.480
Angle α, β, γ (deg.)90.00, 97.50, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein TRIOSEPHOSPHATE ISOMERASE / TIM / TRIOSE-PHOSPHATE ISOMERASE


Mass: 25659.289 Da / Num. of mol.: 2 / Fragment: RESIDUES 2-13,15-72,80-234,238-250 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) TRYPANOSOMA BRUCEI BRUCEI (eukaryote) / Plasmid: PET3A / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / References: UniProt: P04789, triose-phosphate isomerase
#2: Chemical ChemComp-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H8O7
#3: Chemical ChemComp-TBU / TERTIARY-BUTYL ALCOHOL / 2-METHYL-2-PROPANOL


Mass: 74.122 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O
#4: Chemical ChemComp-ASF / 3-(BUTYLSULPHONYL)-PROPANOIC ACID


Mass: 194.249 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H14O4S
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 375 / Source method: isolated from a natural source / Formula: H2O
Compound detailsENGINEERED RESIDUE IN CHAIN A,B, VAL 233 TO ALA
Sequence detailsRESIDUES 14, 72-78, 235-237(THREE RESIDUES DELETED IN LOOP8) IN CHAINS A, B AND C HAVE BEEN REMOVED ...RESIDUES 14, 72-78, 235-237(THREE RESIDUES DELETED IN LOOP8) IN CHAINS A, B AND C HAVE BEEN REMOVED RESULTING IN A DELETION MUTATION OF UNP P04789

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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: 2.24 Å3/Da / Density % sol: 45.24 % / Description: NONE
Crystal growpH: 5.5
Details: 20% PEG6000, 2,5% T-BUTANOL, 0.1 M CITRIC ACID PH 5,5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-2 / Wavelength: 0.873
DetectorType: MARRESEARCH / Detector: CCD / Date: Dec 14, 2005
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.873 Å / Relative weight: 1
ReflectionResolution: 1.6→25 Å / Num. obs: 56766 / % possible obs: 97.6 % / Observed criterion σ(I): 3 / Redundancy: 4.2 % / Rmerge(I) obs: 0.12 / Net I/σ(I): 9.99
Reflection shellResolution: 1.6→1.7 Å / Redundancy: 4.3 % / Rmerge(I) obs: 0.41 / Mean I/σ(I) obs: 3.27 / % possible all: 96.9

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Processing

Software
NameVersionClassification
REFMAC5.3.0028refinement
XDSdata reduction
XDSdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1DKW

1dkw


Resolution: 1.6→19.74 Å / Cor.coef. Fo:Fc: 0.961 / Cor.coef. Fo:Fc free: 0.945 / SU B: 3.098 / SU ML: 0.055 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.09 / ESU R Free: 0.087 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.193 2839 5 %RANDOM
Rwork0.166 ---
obs0.167 53925 100 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 18.76 Å2
Baniso -1Baniso -2Baniso -3
1-0.42 Å20 Å20.51 Å2
2--0.18 Å20 Å2
3----0.47 Å2
Refinement stepCycle: LAST / Resolution: 1.6→19.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3609 0 43 375 4027
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0223891
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.4371.9485338
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.9215534
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.58324.379153
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.75615665
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.5121520
X-RAY DIFFRACTIONr_chiral_restr0.1060.2629
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.022910
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.210.21817
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.3050.22728
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1230.2315
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2070.250
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.2650.210
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.9041.52520
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.38923979
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it2.37631571
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it3.7274.51327
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.6→1.64 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.23 207
Rwork0.196 3935
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)
11.05810.2587-0.05331.0889-0.57251.4073-0.01510.01610.0105-0.07610.0061-0.01070.04010.08380.009-0.0669-0.00670.0099-0.0933-0.0033-0.091716.096-0.4342.699
21.2560.2058-0.1820.9688-0.11870.88860.0110.0023-0.02020.00630.00640.0161-0.0160.0136-0.0174-0.09210.0051-0.006-0.1020.0075-0.10859.389-17.09728.846
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 250
2X-RAY DIFFRACTION2B2 - 250

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