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- PDB-1juj: Human Thymidylate Synthase Bound to dUMP and LY231514, a Pyrrolo(... -

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Basic information

Entry
Database: PDB / ID: 1juj
TitleHuman Thymidylate Synthase Bound to dUMP and LY231514, a Pyrrolo(2,3-d)pyrimidine-based Antifolate
ComponentsTHYMIDYLATE SYNTHASE
KeywordsTRANSFERASE / cancer / dTMP synthesis / antifolate / drug resistance
Function / homology
Function and homology information


uracil metabolic process / response to organophosphorus / intestinal epithelial cell maturation / response to folic acid / Interconversion of nucleotide di- and triphosphates / response to vitamin A / thymidylate synthase / sequence-specific mRNA binding / cartilage development / tetrahydrofolate interconversion ...uracil metabolic process / response to organophosphorus / intestinal epithelial cell maturation / response to folic acid / Interconversion of nucleotide di- and triphosphates / response to vitamin A / thymidylate synthase / sequence-specific mRNA binding / cartilage development / tetrahydrofolate interconversion / thymidylate synthase activity / folic acid binding / dTMP biosynthetic process / dTTP biosynthetic process / G1/S-Specific Transcription / DNA biosynthetic process / developmental growth / response to glucocorticoid / mRNA regulatory element binding translation repressor activity / response to progesterone / response to cytokine / liver regeneration / response to toxic substance / circadian rhythm / methylation / response to ethanol / mitochondrial inner membrane / negative regulation of translation / mitochondrial matrix / response to xenobiotic stimulus / protein homodimerization activity / mitochondrion / nucleus / cytosol / cytoplasm
Similarity search - Function
Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase superfamily / Thymidylate synthase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-LYA / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / Thymidylate synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / MIR / Resolution: 3 Å
AuthorsSayre, P.H. / Finer-Moore, J.S. / Fritz, T.A. / Biermann, D. / Gates, S.B. / MacKellar, W.C. / Patel, V.F. / Stroud, R.M.
Citation
Journal: J.Mol.Biol. / Year: 2001
Title: Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.
Authors: Sayre, P.H. / Finer-Moore, J.S. / Fritz, T.A. / Biermann, D. / Gates, S.B. / MacKellar, W.C. / Patel, V.F. / Stroud, R.M.
#1: Journal: Biochemistry / Year: 1995
Title: Crystal Structure of Human Thymidylate Synthase: A Structural Mechanism for Guiding Substrates into the Active Site
Authors: Schiffer, C.A. / Clifton, I.J. / Davisson, V.J. / Santi, D.V. / Stroud, R.M.
History
DepositionAug 24, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / 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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Apr 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Remark 295NON-CRYSTALLOGRAPHIC SYMMETRY THE TRANSFORMATIONS PRESENTED ON THE MTRIX RECORDS BELOW DESCRIBE NON- ...NON-CRYSTALLOGRAPHIC SYMMETRY THE TRANSFORMATIONS PRESENTED ON THE MTRIX RECORDS BELOW DESCRIBE NON-CRYSTALLOGRAPHIC RELATIONSHIPS AMONG ATOMS IN THIS ENTRY. APPLYING THE APPROPRIATE MTRIX TRANSFORMATION TO THE RESIDUES LISTED FIRST WILL YIELD APPROXIMATE COORDINATES FOR THE RESIDUES LISTED SECOND. CHAIN IDENTIFIERS GIVEN AS "?" REFER TO CHAINS FOR WHICH ATOMS ARE NOT FOUND IN THIS ENTRY. APPLIED TO TRANSFORMED TO TRANSFORM CHAIN RESIDUES CHAIN RESIDUES RMSD SSS M 1 A 28 .. 313 B 28 .. 313 M 2 A 28 .. 313 C 28 .. 313 M 3 A 28 .. 313 D 28 .. 313 WHERE SSS -> COLUMNS 8-10 OF MTRIX RECORDS REMARK: THE AUTHORS RESTRAINED SUBUNIT B, C, AND D AGAINST SUBUNIT A, USING A WEIGHT OF 200 KCAL/MOL-A, AND SIGB OF 2 A2.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: THYMIDYLATE SYNTHASE
B: THYMIDYLATE SYNTHASE
C: THYMIDYLATE SYNTHASE
D: THYMIDYLATE SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,88212
Polymers142,9394
Non-polymers2,9428
Water0
1
A: THYMIDYLATE SYNTHASE
B: THYMIDYLATE SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,9416
Polymers71,4702
Non-polymers1,4714
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7410 Å2
ΔGint-29 kcal/mol
Surface area22050 Å2
MethodPISA
2
C: THYMIDYLATE SYNTHASE
D: THYMIDYLATE SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,9416
Polymers71,4702
Non-polymers1,4714
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7400 Å2
ΔGint-29 kcal/mol
Surface area22130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)138.140, 98.140, 111.970
Angle α, β, γ (deg.)90.00, 110.70, 90.00
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(-0.04364, 0.52546, -0.8497), (0.53532, -0.7058, -0.46397), (-0.84352, -0.47511, -0.25049)-22.64756, -91.22199, -82.72397
2given(-0.99996, 0.00926, -0.0006), (0.00927, 0.99993, -0.00771), (0.00053, -0.00771, -0.99997)-48.72274, 4.78975, -52.76192
3given(0.04749, -0.53294, 0.84482), (0.54116, -0.69717, -0.47021), (0.83958, 0.47951, 0.2553)-27.02831, -85.96681, 30.66597
DetailsThe biological dimer is generated from the coordinates with the following noncrystallographic symmetry rotation matrix and translation vector: {* real-space rotation matrix *} {===>} ncs_matrix_2=( -0.04364 0.52546 -0.84970 ) ( 0.53532 -0.70580 -0.46397 ) ( -0.84352 -0.47511 -0.25049 ); {* real-space translation vector *} {===>} ncs_vector_2=(-22.64756 -91.22199 -82.72397); A second dimer in the asymmetric unit is generated by the following NCS symmetry operations: {* real-space rotation matrix *} {===>} ncs_matrix_3=( -0.99996 0.00926 -0.00060 ) ( 0.00927 0.99993 -0.00771 ) ( 0.00053 -0.00771 -0.99997 ); {* real-space translation vector *} {===>} ncs_vector_3=(-48.72274 4.78975 -52.76192); and {* real-space rotation matrix *} {===>} ncs_matrix_4=( 0.04749 -0.53294 0.84482 ) ( 0.54116 -0.69717 -0.47021 ) ( 0.83958 0.47951 0.25530 ); {* real-space translation vector *} {===>} ncs_vector_4=(-27.02831 -85.96681 30.66597);

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Components

#1: Protein
THYMIDYLATE SYNTHASE / / TS


Mass: 35734.859 Da / Num. of mol.: 4 / Mutation: R46E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pGCHTS-TAA / Production host: Escherichia coli (E. coli) / Strain (production host): DH5a / References: UniProt: P04818, thymidylate synthase
#2: Chemical
ChemComp-UMP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / DUMP / Deoxyuridine monophosphate


Mass: 308.182 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H13N2O8P
#3: Chemical
ChemComp-LYA / 2-{4-[2-(2-AMINO-4-OXO-4,7-DIHYDRO-3H-PYRROLO[2,3-D]PYRIMIDIN-5-YL)-ETHYL]-BENZOYLAMINO}-PENTANEDIOIC ACID / LY231514 / Pemetrexed


Mass: 427.411 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C20H21N5O6 / Comment: medication, chemotherapy*YM

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 2

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Sample preparation

CrystalDensity Matthews: 2.48 Å3/Da / Density % sol: 50.47 %
Crystal growTemperature: 296 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: PEG-monomethyl ether 5000, sodium cacodylate, ammonium sulfate, KCL, TRIS, EDTA, DTT, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 296K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
15-10 mg/mlprotein1drop
230 %(w/v)PEG5000 MME1reservoir
30.1 Msodium cacodylate1reservoirpH6.5
40.2 Mammonium sulfate1reservoir
50.5 M1reservoirKCl

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Data collection

DiffractionMean temperature: 296 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU / Wavelength: 1.54178 Å
DetectorType: RIGAKU RAXIS II / Detector: IMAGE PLATE / Date: Jul 1, 1997
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 3→40 Å / Num. all: 23396 / Num. obs: 23396 / % possible obs: 84 % / Observed criterion σ(F): 0 / Observed criterion σ(I): -3 / Redundancy: 2.1 % / Rmerge(I) obs: 0.145 / Net I/σ(I): 5.8
Reflection shellResolution: 3→3.09 Å / Rmerge(I) obs: 0.43 / Mean I/σ(I) obs: 1.8 / % possible all: 78
Reflection
*PLUS
Highest resolution: 3 Å / Lowest resolution: 40 Å / % possible obs: 84 % / Num. measured all: 50715
Reflection shell
*PLUS
% possible obs: 78 %

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MIR
Starting model: thymidylate synthase from Leishmania major

Resolution: 3→37.96 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 131081.01 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh and Huber
Details: The asymmetric unit contains four monomers and these were refined using strict noncrystallographic symmetry constraints. The strict noncrystallographic symmetry was broken at the interface ...Details: The asymmetric unit contains four monomers and these were refined using strict noncrystallographic symmetry constraints. The strict noncrystallographic symmetry was broken at the interface between the two dimers and Arg42 at this interface in chains B and D had to be rebuilt at the end of refinement. Residues 1-27, as well as the sidechain of Arg147 are disordered in each monomer and are omitted from the structure.
RfactorNum. reflection% reflectionSelection details
Rfree0.311 2221 9.9 %RANDOM
Rwork0.268 ---
obs0.273 22378 79.5 %-
all-22378 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 30.2028 Å2 / ksol: 0.273945 e/Å3
Displacement parametersBiso mean: 25.2 Å2
Baniso -1Baniso -2Baniso -3
1--2.77 Å20 Å20.36 Å2
2---1.1 Å20 Å2
3---3.87 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.58 Å0.49 Å
Luzzati d res low-5 Å
Luzzati sigma a0.95 Å0.85 Å
Refinement stepCycle: LAST / Resolution: 3→37.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9208 0 204 0 9412
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d23.6
X-RAY DIFFRACTIONc_improper_angle_d0.86
LS refinement shellResolution: 3→3.19 Å / Rfactor Rfree error: 0.027 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.482 328 10.1 %
Rwork0.431 2926 -
obs--70.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3LY231514.PARLY231514.TOP
X-RAY DIFFRACTION4PARAMED.LIGTOPO_COVALENT.DUMP
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
σ(F): 0 / % reflection Rfree: 9.9 % / Rfactor all: 0.273 / Rfactor obs: 0.268
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 25.2 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg23.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.86
LS refinement shell
*PLUS
Rfactor Rfree: 0.482 / % reflection Rfree: 10.1 % / Rfactor Rwork: 0.431

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