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Entry
Database: PDB / ID: 2vem
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 / glycolytic process / gluconeogenesis / cytoplasm / cytosol
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-bromo-2-oxo-propoxy)phosphonic acid / TERTIARY-BUTYL ALCOHOL / Triosephosphate isomerase, glycosomal
Similarity search - Component
Biological speciesTRYPANOSOMA BRUCEI BRUCEI (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 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 25, 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 1.2Jul 24, 2019Group: Data collection / Derived calculations / Category: diffrn_source / struct_conn
Item: _diffrn_source.pdbx_synchrotron_site / _struct_conn.pdbx_leaving_atom_flag
Revision 1.3Dec 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id
Revision 1.4Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification
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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)51,8595
Polymers51,3192
Non-polymers5403
Water1,76598
1
A: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,8922
Polymers25,6591
Non-polymers2331
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
2
B: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,9663
Polymers25,6591
Non-polymers3072
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS
Unit cell
Length a, b, c (Å)45.800, 86.900, 56.400
Angle α, β, γ (deg.)90.00, 97.20, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Ens-ID: 1 / Refine code: 2

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ILEILEASNASNAA7 - 116 - 10
21ILEILEASNASNBB7 - 116 - 10
12GLNGLNALAALAAA38 - 4236 - 40
22GLNGLNALAALABB38 - 4236 - 40
13VALVALALAALAAA61 - 6459 - 62
23VALVALALAALABB61 - 6459 - 62
14TRPTRPLEULEUAA90 - 9381 - 84
24TRPTRPLEULEUBB90 - 9381 - 84
15METMETILEILEAA122 - 127113 - 118
25METMETILEILEBB122 - 127113 - 118
16VALVALTYRTYRAA163 - 166154 - 157
26VALVALTYRTYRBB163 - 166154 - 157
17ARGARGTYRTYRAA207 - 210198 - 201
27ARGARGTYRTYRBB207 - 210198 - 201
18GLYGLYALAALAAA230 - 233221 - 224
28GLYGLYALAALABB230 - 233221 - 224

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Components

#1: Protein GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE / 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-BBR / (3-bromo-2-oxo-propoxy)phosphonic acid


Mass: 232.955 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H6BrO5P
#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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 98 / 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
Has protein modificationY
Nonpolymer detailsBROMO HYDROXY ACETONE PHOSPHATE (BBR): BROMO HYDROXY ACETONE PHOSPHATE IS COVALENTLY ATTACHED TO ...BROMO HYDROXY ACETONE PHOSPHATE (BBR): BROMO HYDROXY ACETONE PHOSPHATE IS COVALENTLY ATTACHED TO THE CATALYTIC GLU167. BROMIDE LEAVES IN THIS REACTION.

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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: 2.23 Å3/Da / Density % sol: 44.8 % / 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: 2.2→25 Å / Num. obs: 22262 / % possible obs: 99.8 % / Observed criterion σ(I): 3 / Redundancy: 7.6 % / Rmerge(I) obs: 0.14 / Net I/σ(I): 14.36
Reflection shellResolution: 2.2→2.3 Å / Redundancy: 7.6 % / Rmerge(I) obs: 0.62 / Mean I/σ(I) obs: 3.63 / % 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: 2.2→19.66 Å / Cor.coef. Fo:Fc: 0.935 / Cor.coef. Fo:Fc free: 0.884 / SU B: 14.279 / SU ML: 0.18 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / ESU R: 0.337 / ESU R Free: 0.233 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.247 1114 5 %RANDOM
Rwork0.193 ---
obs0.196 21148 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: 22.51 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å20 Å20.15 Å2
2--0.47 Å20 Å2
3----0.26 Å2
Refinement stepCycle: LAST / Resolution: 2.2→19.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3593 0 23 98 3714
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0223711
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.4051.9415041
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3035471
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.77824.57151
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.85715626
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.8131518
X-RAY DIFFRACTIONr_chiral_restr0.0890.2584
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022744
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.2090.21707
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.30.22529
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1620.2175
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1610.233
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.0770.23
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.651.52419
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.10323777
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.64631492
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.5854.51263
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 / Refine-ID: X-RAY DIFFRACTION

NumberTypeRms dev position (Å)Weight position
144tight positional0.050.05
124medium positional0.060.5
144tight thermal0.150.5
124medium thermal0.192
LS refinement shellResolution: 2.2→2.26 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.323 80
Rwork0.228 1520
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)
11.78310.05220.12392.2952-1.30712.1782-0.02190.1909-0.0006-0.2245-0.0077-0.08840.10540.1490.0296-0.0632-0.0220.0149-0.0719-0.0026-0.096316.113-0.4922.805
22.17750.5286-0.46151.4585-0.57351.1060.0496-0.12680.01670.1128-0.0070.0151-0.05630.0062-0.0427-0.08650.0149-0.0042-0.08920.0029-0.10229.69-17.03528.904
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 250
2X-RAY DIFFRACTION2B2 - 250

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