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- PDB-8rep: Crystal structure of oxidized ThyX-Y91F mutant -

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Basic information

Entry
Database: PDB / ID: 8rep
TitleCrystal structure of oxidized ThyX-Y91F mutant
ComponentsFlavin-dependent thymidylate synthase
KeywordsTRANSFERASE / FLAVIN-DEPENDEN THYMIDYLATE SYNTHASE / METHYLENETETRAHYDROFOLATE
Function / homology
Function and homology information


thymidylate synthase (FAD) / thymidylate synthase (FAD) activity / thymidylate synthase activity / dTMP biosynthetic process / dTTP biosynthetic process / NADPH binding / flavin adenine dinucleotide binding / methylation
Similarity search - Function
Thymidylate synthase ThyX / Thymidylate synthase ThyX superfamily / Thymidylate synthase complementing protein / Flavin-dependent thymidylate synthase (thyX) domain profile.
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Flavin-dependent thymidylate synthase
Similarity search - Component
Biological speciesThermotoga maritima (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.201 Å
AuthorsPecqueur, L. / Hamdane, D.
Funding support France, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-15-CE11-0004-01 France
Agence Nationale de la Recherche (ANR)ANR-11-LABX-0011-01 France
Agence Nationale de la Recherche (ANR)ANR-11-IDEX-0004-02 France
CitationJournal: Biomolecules / Year: 2025
Title: Structural Plasticity of Flavin-Dependent Thymidylate Synthase Controlled by the Enzyme Redox State.
Authors: Pecqueur, L. / Lombard, M. / Hamdane, D.
History
DepositionDec 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Apr 9, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Flavin-dependent thymidylate synthase
B: Flavin-dependent thymidylate synthase
C: Flavin-dependent thymidylate synthase
D: Flavin-dependent thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,59518
Polymers109,9514
Non-polymers4,64414
Water1,78399
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23750 Å2
ΔGint-27 kcal/mol
Surface area30380 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.919, 116.801, 142.754
Angle α, β, γ (deg.)90, 90, 90
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Flavin-dependent thymidylate synthase


Mass: 27487.680 Da / Num. of mol.: 4 / Mutation: Y91F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (bacteria) / Gene: thyX / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9WYT0

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Non-polymers , 5 types, 113 molecules

#2: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#3: Chemical
ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O4
#4: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#5: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 99 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: 2.08 Å3/Da / Density % sol: 40.92 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 1 uL protein at 5-7 mg/ml in Tris 25 mM pH 8 NaCl 150 mM with 1 ul 42-46% w/v PEG 200 in Tris 0.1M pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.980113 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Nov 16, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.980113 Å / Relative weight: 1
ReflectionResolution: 2.201→90.398 Å / Num. obs: 47316 / % possible obs: 99.7 % / Redundancy: 6.55 %
Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last ...Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last updated 2020-04-13: http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v50.dic/Index/) and the actual quantities provided by MRFANA (https://github.com/githubgphl/MRFANA) from the autoPROC package (https://www.globalphasing.com/autoproc/). In general, the mmCIF categories here should provide items that are currently used in the PDB archive. If there are alternatives, the one recommended by the PDB developers has been selected. The distinction between *_all and *_obs quantities is not always clear: often only one version is actively used within the PDB archive (or is the one recommended by PDB developers). The intention of distinguishing between classes of reflections before and after some kind of observation criterion was applied, can in principle be useful - but such criteria change in various ways throughout the data processing steps (rejection of overloaded or too partial reflections, outlier/misfit rejections during scaling etc) and there is no retrospect computation of data scaling/merging statistics for the reflections used in the final refinement (where another observation criterion might have been applied). Typical data processing will usually only provide one version of statistics at various stages and these are given in the recommended item here, irrespective of the "_all" and "_obs" connotation, see e.g. the use of _reflns.pdbx_Rmerge_I_obs, _reflns.pdbx_Rrim_I_all and _reflns.pdbx_Rpim_I_all. Please note that all statistics related to "merged intensities" (or "merging") are based on inverse-variance weighting of the individual measurements making up a symmetry-unique reflection. This is standard for several decades now, even if some of the dictionary definitions seem to suggest that a simple "mean" or "average" intensity is being used instead. R-values are always given for all symmetry-equivalent reflections following Friedel's law, i.e. Bijvoet pairs are not treated separately (since we want to describe the overall mean intensity and not the mean I(+) and I(-) here). The Rrim metric is identical to the Rmeas R-value and only differs in name. _reflns.pdbx_number_measured_all is the number of measured intensities just before the final merging step (at which point no additional rejection takes place). _reflns.number_obs is the number of symmetry-unique observations, i.e. the result of merging those measurements via inverse-variance weighting. _reflns.pdbx_netI_over_sigmaI is based on the merged intensities (_reflns.number_obs) as expected. _reflns.pdbx_redundancy is synonymous with "multiplicity". The per-shell item _reflns_shell.number_measured_all corresponds to the overall value _reflns.pdbx_number_measured_all. The per-shell item _reflns_shell.number_unique_all corresponds to the overall value _reflns.number_obs. The per-shell item _reflns_shell.percent_possible_all corresponds to the overall value _reflns.percent_possible_obs. The per-shell item _reflns_shell.meanI_over_sigI_obs corresponds to the overall value given as _reflns.pdbx_netI_over_sigmaI. But be aware of the incorrect definition of the former in the current dictionary!
CC1/2: 0.99 / CC1/2 anomalous: -0.167 / Rmerge(I) obs: 0.2325 / Rpim(I) all: 0.0984 / Rrim(I) all: 0.2532 / AbsDiff over sigma anomalous: 0.734 / Net I/σ(I): 5.57 / Num. measured all: 309734 / % possible anomalous: 99.4 / Redundancy anomalous: 3.47
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. measured obsNum. unique allNum. unique obsCC1/2CC1/2 anomalousRpim(I) allRrim(I) allAbsDiff over sigma anomalous% possible anomalousRedundancy anomalous% possible all
5.973-90.3986.510.080713.51645716457252725270.995-0.3010.03380.08780.5491003.7397.9
4.741-5.9736.260.10111.921511215112241524150.991-0.2760.04290.11010.63698.13.4498.6
4.141-4.7416.670.10712.851598415984239823980.99-0.3110.04450.11620.59999.53.5999.4
3.763-4.1416.130.127111458814588237923790.987-0.3020.05480.13880.70398.33.399.7
3.493-3.7636.350.15839.631513215132238423840.981-0.270.06760.17280.72299.13.3999.7
3.287-3.4936.580.19228.311572815728239223920.973-0.1890.08140.20950.74699.73.599.8
3.122-3.2876.690.24317.011576115761235623560.966-0.1330.10250.26470.75999.23.5599.8
2.987-3.1226.750.30945.911596315963236423640.9410.0020.13040.33680.79599.53.58100
2.872-2.9876.810.40684.721590715907233623360.935-0.0280.16930.44170.76299.83.59100
2.772-2.8726.810.49894.061620116201237823780.911-0.0480.20820.54210.781003.58100
2.686-2.7726.860.58773.611613016130235223520.877-0.020.24320.63760.77599.83.61100
2.609-2.6866.60.68073.241544915449234023400.82-0.0360.28770.74110.77499.63.4699.9
2.54-2.6096.120.8452.591432014320234123410.733-0.0560.37070.92560.76599.83.1999.9
2.478-2.546.451.01062.311522715227235923590.721-0.0190.43061.10160.75999.33.39100
2.422-2.4786.61.20622.021546715467234523450.652-0.0070.51011.3130.7699.43.46100
2.37-2.4226.691.44531.791555215552232423240.530.0660.60841.57220.7999.43.5100
2.323-2.376.731.60431.661575415754234123410.525-0.0750.67151.74370.74799.53.52100
2.279-2.3236.741.80451.571567315673232523250.3960.0350.75471.96110.75499.63.52100
2.238-2.2796.512.26981.241536715367235923590.403-0.0590.96492.47310.73199.53.4100
2.201-2.2386.072.40271.141396213962230123010.3470.0081.06672.63740.73999.23.15100

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Processing

Software
NameVersionClassification
autoPROC1.0.5 (20230222)data processing
XDSJan 10, 2022data reduction
Aimless0.7.9data scaling
TRUNCATE8.0.011data processing
BUSTER2.10.4refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.201→43.53 Å / Cor.coef. Fo:Fc: 0.927 / Cor.coef. Fo:Fc free: 0.918 / SU R Cruickshank DPI: 0.328 / Cross valid method: THROUGHOUT / SU R Blow DPI: 0.319 / SU Rfree Blow DPI: 0.209 / SU Rfree Cruickshank DPI: 0.214
RfactorNum. reflection% reflectionSelection details
Rfree0.2413 2310 -RANDOM
Rwork0.2218 ---
obs0.2228 46240 97.5 %-
Displacement parametersBiso mean: 41.99 Å2
Baniso -1Baniso -2Baniso -3
1--9.466 Å20 Å20 Å2
2--5.8022 Å20 Å2
3---3.6637 Å2
Refine analyzeLuzzati coordinate error obs: 0.31 Å
Refinement stepCycle: LAST / Resolution: 2.201→43.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6987 0 312 99 7398
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0077503HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.8810188HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2551SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes1206HARMONIC5
X-RAY DIFFRACTIONt_it7503HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion957SEMIHARMONIC5
X-RAY DIFFRACTIONt_ideal_dist_contact5850SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion3.2
X-RAY DIFFRACTIONt_other_torsion17.15
LS refinement shellResolution: 2.201→2.22 Å
RfactorNum. reflection% reflection
Rfree0.3153 49 -
Rwork0.3254 --
obs--84.39 %

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