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Basic information

Entry
Database: PDB / ID: 2vel
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.
ComponentsGLYCOSOMAL TRIOSEPHOSPHATE 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 / glyceraldehyde-3-phosphate biosynthetic process / glycerol catabolic 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
2-PHOSPHOGLYCOLIC ACID / Triosephosphate isomerase, glycosomal
Similarity search - Component
Biological speciesTRYPANOSOMA BRUCEI BRUCEI (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jul 24, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.4Dec 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
Revision 1.5May 1, 2024Group: Structure summary / Category: struct / Item: _struct.title
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. 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: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
B: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,7026
Polymers51,3192
Non-polymers3834
Water6,413356
1
A: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,8513
Polymers25,6591
Non-polymers1912
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,8513
Polymers25,6591
Non-polymers1912
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)44.820, 85.600, 55.270
Angle α, β, γ (deg.)90.00, 98.23, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A7 - 11
2111B7 - 11
1211A38 - 42
2211B38 - 42
1311A61 - 64
2311B61 - 64
1411A90 - 93
2411B90 - 93
1511A122 - 127
2511B122 - 127
1611A163 - 166
2611B163 - 166
1711A207 - 210
2711B207 - 210
1811A230 - 233
2811B230 - 233
1911A18 - 31
2911B18 - 31
11011A47 - 54
21011B47 - 54
11111A79 - 87
21111B79 - 87
11211A105 - 120
21211B105 - 120
11311A140 - 151
21311B140 - 151
11411A179 - 196
21411B179 - 196
11511A219 - 224
21511B219 - 224
11611A241 - 249
21611B241 - 249

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Components

#1: Protein GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE / TIM / TRIOSE-PHOSPHATE ISOMERASE / TRIOSEPHOSPHATE 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-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-PGA / 2-PHOSPHOGLYCOLIC ACID


Mass: 156.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C2H5O6P
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 356 / Source method: isolated from a natural source / Formula: H2O
Compound detailsENGINEERED RESIDUE IN CHAIN A, VAL 233 TO ALA ENGINEERED RESIDUE IN CHAIN B, VAL 233 TO ALA

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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: 2.1 Å3/Da / Density % sol: 41.43 % / 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: EMBL/DESY, HAMBURG / Beamline: X13 / Wavelength: 0.8063
DetectorType: MARRESEARCH / Detector: CCD
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8063 Å / Relative weight: 1
ReflectionResolution: 2.3→25 Å / Num. obs: 18376 / % possible obs: 99.7 % / Observed criterion σ(I): 2 / Redundancy: 4.3 % / Rmerge(I) obs: 0.15 / Net I/σ(I): 10.71
Reflection shellResolution: 2.3→2.4 Å / Redundancy: 4.3 % / Rmerge(I) obs: 0.54 / Mean I/σ(I) obs: 3 / % possible all: 100

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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: 2.3→19.93 Å / Cor.coef. Fo:Fc: 0.932 / Cor.coef. Fo:Fc free: 0.866 / SU B: 8.802 / SU ML: 0.216 / Cross valid method: THROUGHOUT / ESU R: 0.795 / ESU R Free: 0.29 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.257 919 5 %RANDOM
Rwork0.18 ---
obs0.184 17455 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: 18.66 Å2
Baniso -1Baniso -2Baniso -3
1--0.55 Å20 Å20.42 Å2
2--0.96 Å20 Å2
3----0.29 Å2
Refinement stepCycle: LAST / Resolution: 2.3→19.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3618 0 20 356 3994
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0223788
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.4271.9415162
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4635497
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.94124.615156
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.04515636
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.5391519
X-RAY DIFFRACTIONr_chiral_restr0.0960.2591
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022849
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.2170.22241
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.3040.22652
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1750.2409
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2340.276
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1760.220
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.5281.52447
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it0.91923874
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.46231512
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.2684.51280
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
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Number: 947 / Refine-ID: X-RAY DIFFRACTION

TypeRms dev position (Å)Weight position
tight positional0.080.05
tight thermal0.140.5
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.311 67
Rwork0.2 1280

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