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- PDB-1k3t: Structure of Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase ... -

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Basic information

Entry
Database: PDB / ID: 1k3t
TitleStructure of Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi Complexed with Chalepin, a Coumarin Derivative Inhibitor
ComponentsGlyceraldehyde-3-phosphate dehydrogenase
KeywordsOXIDOREDUCTASE / Apo-Protein / gGAPDH-Chalepin complex / glycosome / Trypanosoma cruzi
Function / homology
Function and homology information


glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / glycosome / glycolytic process / glucose metabolic process / NAD binding / NADP binding / cytosol
Similarity search - Function
Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Dihydrodipicolinate Reductase; domain 2 ...Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Dihydrodipicolinate Reductase; domain 2 / Dihydrodipicolinate Reductase; domain 2 / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-BRZ / Glyceraldehyde-3-phosphate dehydrogenase, glycosomal
Similarity search - Component
Biological speciesTrypanosoma cruzi (eukaryote)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.95 Å
AuthorsPavao, F.
Citation
Journal: FEBS Lett. / Year: 2002
Title: Structure of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase complexed with chalepin, a natural product inhibitor, at 1.95 A resolution.
Authors: Pavao, F. / Castilho, M.S. / Pupo, M.T. / Dias, R.L. / Correa, A.G. / Fernandes, J.B. / da Silva, M.F. / Mafezoli, J. / Vieira, P.C. / Oliva, G.
#1: Journal: Thesis / Year: 2001
Title: Estudos Cristalograficos e Planejamento Racional de Inibidores Especificos da Enzima Gliceraldeido-3-fosfato Desidrogenase Glicossomal (gGAPDH) de Trypanosoma cruzi
Authors: Pavao, F.
#2: Journal: Pure Appl.Chem. / Year: 2001
Title: Strategies for the Isolation and Identification of Trypanocidal Compounds from the Rutales
Authors: Vieira, P.C. / Mafezoli, J. / Pupo, M.T. / Fernandes, J.B. / Silva, M.F.G.F. / Albuquerque, S. / Oliva, G. / Pavao, F.
History
DepositionOct 4, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Apr 3, 2024Group: 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 / pdbx_initial_refinement_model / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glyceraldehyde-3-phosphate dehydrogenase
B: Glyceraldehyde-3-phosphate dehydrogenase
C: Glyceraldehyde-3-phosphate dehydrogenase
D: Glyceraldehyde-3-phosphate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,7655
Polymers156,4504
Non-polymers3141
Water16,880937
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area16020 Å2
ΔGint-103 kcal/mol
Surface area49010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.080, 84.970, 105.240
Angle α, β, γ (deg.)90.00, 96.32, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Glyceraldehyde-3-phosphate dehydrogenase / GAPDH


Mass: 39112.539 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Trypanosoma cruzi (eukaryote) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)gap-
References: UniProt: P22513, glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)
#2: Chemical ChemComp-BRZ / 6-(1,1-DIMETHYLALLYL)-2-(1-HYDROXY-1-METHYLETHYL)-2,3-DIHYDRO-7H-FURO[3,2-G]CHROMEN-7-ONE


Mass: 314.376 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H22O4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 937 / 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: 2.338 Å3/Da / Density % sol: 47.45 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.1
Details: 16% PEG 8000, 0.1M calcium acetate, 0.1M sodium cacodylate, pH 7.1, VAPOR DIFFUSION, HANGING DROP, temperature 295K
Crystal grow
*PLUS
Temperature: 22 ℃ / pH: 7.6 / Method: vapor diffusion
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
18 mg/mlprotein1drop
20.1 MTEA1droppH7.6
31 mMNAD+1drop
41 mMdithiothreitol1drop
51 mMPMSF1drop
61 mMEDTA1drop
70.001 mMleupeptin1drop
80.001 mMpepstatin pre-incubated1drop
95 mMinhibitor1drop
1010 %(v/v)acetonitrile1drop
110.1 Msodium cacodylate1reservoirpH6.9-7.1
120.1 Mcalcium acetate1reservoir
1316-18 %PEG80001reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.54 Å
DetectorType: RIGAKU RAXIS IIC / Detector: IMAGE PLATE / Date: Jul 30, 1998
RadiationMonochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.95→44.72 Å / Num. all: 81630 / Num. obs: 81630 / % possible obs: 77.9 % / Redundancy: 2.4 % / Biso Wilson estimate: 22.48 Å2 / Rmerge(I) obs: 0.107 / Net I/σ(I): 6.78
Reflection shellResolution: 1.95→2.02 Å / Redundancy: 1.6 % / Rmerge(I) obs: 0.483 / Mean I/σ(I) obs: 1.1 / Num. unique all: 3980 / % possible all: 38.1
Reflection
*PLUS
Num. measured all: 195798 / Rmerge(I) obs: 0.107
Reflection shell
*PLUS
% possible obs: 38.1 % / Rmerge(I) obs: 0.483

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Processing

Software
NameClassification
AMoREphasing
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Holo-D-Glyceraldehyde-3-phosphate dehydrogenase from T. cruzi solved at 2.8 A resolution by Souza et al., FEBS Letters, 424 (1998), 131-135.

Resolution: 1.95→15 Å / SU B: 6.158 / SU ML: 0.172 / Cross valid method: THROUGHOUT / ESU R: 0.267 / ESU R Free: 0.233
Stereochemistry target values: John Priestle's with added co-factors from York.
Details: REFINEMENT PERFORMED WITHOUT NON-CRYSTALLOGRAPHIC SYMMETRY. FROM THE INITIAL CYCLES OF THE REFINEMENT, IT WAS EVIDENT THAT THE INHIBITOR WAS CLOSELY PACKED AGAINST THE ACTIVE SITE RESIDUE ...Details: REFINEMENT PERFORMED WITHOUT NON-CRYSTALLOGRAPHIC SYMMETRY. FROM THE INITIAL CYCLES OF THE REFINEMENT, IT WAS EVIDENT THAT THE INHIBITOR WAS CLOSELY PACKED AGAINST THE ACTIVE SITE RESIDUE CYS166, INDUCING A CONFORMATIONAL CHANGE OF THE -SH GROUP OF THIS AMINOACID THROUGH A ROTATION OF c1 (Chi1 angle), IN ORDER TO ACCOMMODATE THE POSITIONING OF THE INHIBITOR AT THE ACTIVE SITE. THE FINAL ELECTRON DENSITY MAPS SHOW TWO ALTERNATIVE CONFORMATIONS OF CYS166, ONE OF THEM APPARENTLY CONNECTING TO THE INHIBITOR. THIS IS AN ARTIFACT, SINCE THE SIDE CHAIN OF THIS RESIDUE CAN ONLY BE AT THE NATIVE CONFORMATION IF THE INHIBITOR IS ABSENT. THEREFORE, THE EVIDENT TWO CONFORMATIONS OF THE CYS166 SIDE CHAIN GUARANTEES THAT THE INHIBITOR HAS TO HAVE PARTIAL OCCUPANCY. THE FIRST 160 RESIDUES FROM N-TERM AND THE LAST 7 RESIDUES FROM C-TERM OF MONOMER C SHOWS HIGH DEGREE OF STATIC DISORDER DUE TO PARTIAL OCCUPANCY OF INHIBITOR (85%), RESULTING IN A LACK OF ELECTRON DENSITY TO THE SEVERAL SIDE CHAINS AND TO SOME BACKBONE ATOMS. IN SPITE OF THIS, THE COORDINATES OF RESPECTIVES SIDE CHAINS ARE KEPT.
RfactorNum. reflection% reflectionSelection details
Rfree0.28 2478 3 %RANDOM
Rwork0.201 ---
all-81471 --
obs-81471 100 %-
Displacement parametersBiso mean: 31.81 Å2
Refinement stepCycle: LAST / Resolution: 1.95→15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10982 0 23 937 11942
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.150.02
X-RAY DIFFRACTIONo_bond_d_na
X-RAY DIFFRACTIONo_bond_d_prot
X-RAY DIFFRACTIONp_angle_d0.040.04
X-RAY DIFFRACTIONp_planar_d0.0410.05
X-RAY DIFFRACTIONo_angle_d_na
X-RAY DIFFRACTIONo_angle_d_prot
X-RAY DIFFRACTIONo_angle_deg
X-RAY DIFFRACTIONo_angle_deg_na
X-RAY DIFFRACTIONo_angle_deg_prot
X-RAY DIFFRACTIONo_dihedral_angle_d
X-RAY DIFFRACTIONo_dihedral_angle_d_na
X-RAY DIFFRACTIONo_dihedral_angle_d_prot
X-RAY DIFFRACTIONo_improper_angle_d
X-RAY DIFFRACTIONo_improper_angle_d_na
X-RAY DIFFRACTIONo_improper_angle_d_prot
X-RAY DIFFRACTIONp_mcbond_it1.82
X-RAY DIFFRACTIONp_mcangle_it2.63
X-RAY DIFFRACTIONp_scbond_it2.12
X-RAY DIFFRACTIONp_scangle_it33
Refinement
*PLUS
Lowest resolution: 15 Å / % reflection Rfree: 3 % / Rfactor Rfree: 0.28 / Rfactor Rwork: 0.201
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_angle_d
X-RAY DIFFRACTIONp_angle_deg3.1

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