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- PDB-4pcf: Structure-based protein engineering of a monomeric triosephosphat... -

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Basic information

Entry
Database: PDB / ID: 4pcf
TitleStructure-based protein engineering of a monomeric triosephosphate isomerase towards changing substrate specificity
ComponentsMa18-TIM
KeywordsISOMERASE / TRIOSEPHOSPHATE ISOMERASE / TIM BARREL / PROTEIN ENGINEERING / SUBSTRATE SPECIFICITY
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
Triosephosphate isomerase, glycosomal
Similarity search - Component
Biological speciesTrypanosoma brucei brucei (eukaryote)
MethodX-RAY DIFFRACTION / Resolution: 2.71 Å
AuthorsKrause, M. / Neubauer, P. / Wierenga, R.K.
Funding support Finland, Germany, 3items
OrganizationGrant numberCountry
Academy of Finland117874 Finland
EMBO Shortterm FellowshipASTF 188-2011 Germany
FEBS Shortterm Fellowship Germany
CitationJournal: Acta Crystallogr.,Sect.F / Year: 2016
Title: Crystal structures of two monomeric triosephosphate isomerase variants identified via a directed-evolution protocol selecting for L-arabinose isomerase activity.
Authors: Krause, M. / Kiema, T.R. / Neubauer, P. / Wierenga, R.K.
History
DepositionApr 15, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 22, 2015Provider: repository / Type: Initial release
Revision 1.1Jun 29, 2016Group: Data collection / Database references
Revision 1.2Dec 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / diffrn_radiation_wavelength / pdbx_database_related / pdbx_struct_oper_list / 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_related.db_id / _pdbx_database_related.details / _pdbx_struct_oper_list.symmetry_operation / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ma18-TIM
B: Ma18-TIM
C: Ma18-TIM


Theoretical massNumber of molelcules
Total (without water)77,5103
Polymers77,5103
Non-polymers00
Water2,648147
1
A: Ma18-TIM


Theoretical massNumber of molelcules
Total (without water)25,8371
Polymers25,8371
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Ma18-TIM


Theoretical massNumber of molelcules
Total (without water)25,8371
Polymers25,8371
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Ma18-TIM


Theoretical massNumber of molelcules
Total (without water)25,8371
Polymers25,8371
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3610 Å2
ΔGint-21 kcal/mol
Surface area28880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)92.920, 49.420, 95.510
Angle α, β, γ (deg.)90.00, 118.50, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13B
23C

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: SER / Beg label comp-ID: SER / Refine code: _

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11THRTHRAA2 - 2492 - 238
21THRTHRBB2 - 2492 - 238
12GLNGLNAA2 - 2502 - 239
22GLNGLNCC2 - 2502 - 239
13THRTHRBB2 - 2492 - 238
23THRTHRCC2 - 2492 - 238

NCS ensembles :
ID
1
2
3

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Components

#1: Protein Ma18-TIM


Mass: 25836.600 Da / Num. of mol.: 3 / Mutation: E23G, A70T, S96F, A178V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma brucei brucei (eukaryote) / Plasmid: pMK / Production host: Escherichia coli (E. coli) / Strain (production host): BL21
References: UniProt: P04789*PLUS, triose-phosphate isomerase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 147 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 2.48 Å3/Da / Density % sol: 50.07 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 8.2 / Details: 1.75M (NH4)2HPO4,

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: BRUKER AXS MICROSTAR / Wavelength: 1.542 Å
DetectorType: Bruker Platinum 135 / Detector: CCD / Date: Aug 14, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.542 Å / Relative weight: 1
ReflectionResolution: 2.71→83.94 Å / Num. obs: 19927 / % possible obs: 99.5 % / Redundancy: 6.96 % / Net I/σ(I): 12.46
Reflection shellResolution: 2.71→2.73 Å / Redundancy: 1.23 % / Mean I/σ(I) obs: 2.49 / % possible all: 56.3

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Processing

SoftwareName: REFMAC / Version: 5.8.0049 / Classification: refinement
RefinementResolution: 2.71→83.94 Å / Cor.coef. Fo:Fc: 0.868 / Cor.coef. Fo:Fc free: 0.798 / SU B: 18.335 / SU ML: 0.358 / Cross valid method: THROUGHOUT / ESU R Free: 0.413 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2832 1078 5.1 %RANDOM
Rwork0.2349 ---
obs0.23735 19927 99.41 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 17.806 Å2
Baniso -1Baniso -2Baniso -3
1-0.19 Å2-0 Å20.11 Å2
2--0.01 Å20 Å2
3----0.2 Å2
Refinement stepCycle: 1 / Resolution: 2.71→83.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5398 0 0 147 5545
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0195503
X-RAY DIFFRACTIONr_bond_other_d0.0050.025395
X-RAY DIFFRACTIONr_angle_refined_deg1.4811.9337471
X-RAY DIFFRACTIONr_angle_other_deg1.277312351
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4065703
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.98424.35223
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.71515917
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.4441527
X-RAY DIFFRACTIONr_chiral_restr0.080.2866
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.026254
X-RAY DIFFRACTIONr_gen_planes_other0.0040.021263
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.1251.7522827
X-RAY DIFFRACTIONr_mcbond_other1.1261.7522826
X-RAY DIFFRACTIONr_mcangle_it2.0232.6213525
X-RAY DIFFRACTIONr_mcangle_other2.0222.6213526
X-RAY DIFFRACTIONr_scbond_it0.8411.8542676
X-RAY DIFFRACTIONr_scbond_other0.8411.8552677
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.5482.7373947
X-RAY DIFFRACTIONr_long_range_B_refined3.64214.1516499
X-RAY DIFFRACTIONr_long_range_B_other3.63614.1646486
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A127730.17
12B127730.17
21A135040.14
22C135040.14
31B130030.16
32C130030.16
LS refinement shellResolution: 2.709→2.78 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.344 60 -
Rwork0.312 1370 -
obs--93.1 %

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