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Basic information

Entry
Database: PDB / ID: 2ven
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 / TIM BARREL / GLYCOLYSIS / PENTOSE SHUNT / GLUCONEOGENESIS / LIPID SYNTHESIS / FATTY ACID BIOSYNTHESIS / 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
CITRIC ACID / Triosephosphate isomerase, glycosomal
Similarity search - Component
Biological speciesTRYPANOSOMA BRUCEI BRUCEI (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 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: Refinement description / Version format compliance
Revision 1.2Nov 28, 2012Group: Database references / Derived calculations / Refinement description
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 / 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
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)51,5113
Polymers51,3192
Non-polymers1921
Water6,143341
1
A: GLYCOSOMAL TRIOSEPHOSPHATE ISOMERASE


Theoretical massNumber of molelcules
Total (without water)25,6591
Polymers25,6591
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: GLYCOSOMAL 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
MethodPISA
Unit cell
Length a, b, c (Å)46.100, 88.300, 56.500
Angle α, β, γ (deg.)90.00, 97.00, 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

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDRefine codeAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ILEILEASNASN1AA7 - 116 - 10
21ILEILEASNASN1BB7 - 116 - 10
12GLNGLNALAALA1AA38 - 4236 - 40
22GLNGLNALAALA1BB38 - 4236 - 40
13VALVALALAALA1AA61 - 6459 - 62
23VALVALALAALA1BB61 - 6459 - 62
14TRPTRPLEULEU1AA90 - 9381 - 84
24TRPTRPLEULEU1BB90 - 9381 - 84
15METMETILEILE1AA122 - 127113 - 118
25METMETILEILE1BB122 - 127113 - 118
16VALVALTYRTYR1AA163 - 166154 - 157
26VALVALTYRTYR1BB163 - 166154 - 157
17ARGARGTYRTYR1AA207 - 210198 - 201
27ARGARGTYRTYR1BB207 - 210198 - 201
18GLYGLYALAALA4AA230 - 233221 - 224
28GLYGLYALAALA4BB230 - 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-CIT / CITRIC ACID


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 341 / 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: 2.28 Å3/Da / Density % sol: 46.15 % / 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→25 Å / Num. obs: 30031 / % possible obs: 98.8 % / Observed criterion σ(I): 3 / Redundancy: 5.3 % / Rmerge(I) obs: 0.17 / Net I/σ(I): 11.51
Reflection shellResolution: 2→2.1 Å / Redundancy: 5.4 % / Rmerge(I) obs: 0.64 / Mean I/σ(I) obs: 3.47 / % possible all: 98.4

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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→19.88 Å / Cor.coef. Fo:Fc: 0.94 / Cor.coef. Fo:Fc free: 0.896 / SU B: 8.765 / SU ML: 0.13 / Cross valid method: THROUGHOUT / ESU R: 0.213 / ESU R Free: 0.181 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.237 1502 5 %RANDOM
Rwork0.186 ---
obs0.188 28528 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: 9.75 Å2
Baniso -1Baniso -2Baniso -3
1-0.06 Å20 Å20.38 Å2
2--0.55 Å20 Å2
3----0.52 Å2
Refinement stepCycle: LAST / Resolution: 2→19.88 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3559 0 13 341 3913
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0223716
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.451.9365049
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9945468
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.77924.423156
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.34715633
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.2221520
X-RAY DIFFRACTIONr_chiral_restr0.1080.2583
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.022764
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.1970.21537
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.2940.22474
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1520.2234
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1790.235
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1060.212
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.6491.52430
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.07823764
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.73731527
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.6494.51284
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

Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

Dom-IDAuth asym-IDNumberTypeRms dev position (Å)Weight position
1A240tight positional0.110.05
2B240tight positional0.110.05
1A28medium positional0.130.5
2B28medium positional0.130.5
1A240tight thermal0.690.5
2B240tight thermal0.690.5
1A28medium thermal0.612
2B28medium thermal0.612
LS refinement shellResolution: 2→2.05 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.265 108
Rwork0.217 2052
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)
11.266-0.16220.06641.3124-0.71231.73090.0260.10420.0581-0.0648-0.0225-0.0956-0.02130.0995-0.0034-0.0742-0.01840.0121-0.0615-0.0024-0.10616.175-0.4632.707
21.50520.2229-0.48390.9943-0.20180.938-0.0111-0.051-0.06550.0373-0.0142-0.03960.0320.04620.0253-0.08850.0065-0.003-0.08080.0091-0.130410.084-17.28329.04
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 250
2X-RAY DIFFRACTION2B2 - 250

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