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- PDB-7m31: Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Th... -

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Basic information

Entry
Database: PDB / ID: 7m31
TitleDihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Thymine and NADPH Anaerobically
ComponentsDihydropyrimidine dehydrogenase [NADP(+)]
KeywordsFLAVOPROTEIN / Dihydropyrimidine Dehydrogenase / DPD / mutant / uracil / flavin / soaking / ligand / FMN / FAD
Function / homology
Function and homology information


dihydropyrimidine dehydrogenase (NADP+) / uracil binding / thymidine catabolic process / dihydropyrimidine dehydrogenase (NADP+) activity / beta-alanine biosynthetic process / thymine catabolic process / uracil catabolic process / FMN binding / flavin adenine dinucleotide binding / NADP binding ...dihydropyrimidine dehydrogenase (NADP+) / uracil binding / thymidine catabolic process / dihydropyrimidine dehydrogenase (NADP+) activity / beta-alanine biosynthetic process / thymine catabolic process / uracil catabolic process / FMN binding / flavin adenine dinucleotide binding / NADP binding / 4 iron, 4 sulfur cluster binding / protein homodimerization activity / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / 4Fe-4S dicluster domain / Alpha-helical ferredoxin / Dihydroorotate dehydrogenase domain / Dihydroorotate dehydrogenase / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. ...Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / 4Fe-4S dicluster domain / Alpha-helical ferredoxin / Dihydroorotate dehydrogenase domain / Dihydroorotate dehydrogenase / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / FAD/NAD(P)-binding domain superfamily / Aldolase-type TIM barrel
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / FLAVIN MONONUCLEOTIDE / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / PROLINE / IRON/SULFUR CLUSTER / THYMINE / Dihydropyrimidine dehydrogenase [NADP(+)]
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.69 Å
AuthorsButrin, A. / Beaupre, B. / Forouzesh, D. / Liu, D. / Moran, G.
CitationJournal: Biochemistry / Year: 2021
Title: Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase.
Authors: Beaupre, B.A. / Forouzesh, D.C. / Butrin, A. / Liu, D. / Moran, G.R.
History
DepositionMar 18, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 23, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 18, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_residues
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dihydropyrimidine dehydrogenase [NADP(+)]
B: Dihydropyrimidine dehydrogenase [NADP(+)]
C: Dihydropyrimidine dehydrogenase [NADP(+)]
D: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)460,53637
Polymers446,3494
Non-polymers14,18733
Water77,5734306
1
A: Dihydropyrimidine dehydrogenase [NADP(+)]
B: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,21118
Polymers223,1752
Non-polymers7,03616
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area33860 Å2
ΔGint-291 kcal/mol
Surface area67540 Å2
MethodPISA
2
C: Dihydropyrimidine dehydrogenase [NADP(+)]
D: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,32619
Polymers223,1752
Non-polymers7,15117
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area33680 Å2
ΔGint-283 kcal/mol
Surface area68040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.040, 159.560, 162.950
Angle α, β, γ (deg.)90.000, 95.750, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Dihydropyrimidine dehydrogenase [NADP(+)] / DHPDHase / DPD / Dihydrothymine dehydrogenase / Dihydrouracil dehydrogenase


Mass: 111587.281 Da / Num. of mol.: 4 / Mutation: C671S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sus scrofa (pig) / Gene: DPYD / Production host: Escherichia coli (E. coli)
References: UniProt: Q28943, dihydropyrimidine dehydrogenase (NADP+)

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Non-polymers , 7 types, 4339 molecules

#2: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#3: 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
#4: Chemical
ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-TDR / THYMINE


Mass: 126.113 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H6N2O2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-PRO / PROLINE


Type: L-peptide linking / Mass: 115.130 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H9NO2 / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4306 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.38 Å3/Da / Density % sol: 48.26 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 4.5 mg/mL (40 uM) of DPD variants, C671S in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was prepared and diluted 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 15% PEG ...Details: 4.5 mg/mL (40 uM) of DPD variants, C671S in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was prepared and diluted 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 15% PEG 6000 at pH 4.5 to 4.9. Diffraction quality crystals with elongated morphology were obtained by the hanging-drop vapor diffusion method. Incubation was carried out in the dark to eliminate photo-degradation the quasi-labile FMN cofactor. Crystals appeared after 16 hours as both single elongated rectangular hexahedron forms (200 x 50 x 50) or urchin-like clusters. Only the single crystal form were harvested and these were placed in a Plas-Labs 830 series glove box into which a Motic binocular microscope coupled to an Accuscope 1080 P HD camera was placed. Before being placed in the glove box, the well solutions of the selected crystals were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. The glove box was then made anaerobic by flushing with pure nitrogen for approximately 10 minutes at which time the fractional dioxygen was 0.1 %, as indicated by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held below 1%. C671S DPD crystals were soaked for a minimum of 20 minutes in 25 mM HEPES, 100 mM sodium citrate, 2 mM DTT, 100 uM NADPH, 100 uM thymine, 20% PEG 6000, 20% PEG 400, pH 7.5 prior to submersion in liquid nitrogen. Frozen crystals were then removed from the anaerobic environment and stored under liquid nitrogen.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 1.1272 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Dec 9, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1272 Å / Relative weight: 1
ReflectionResolution: 1.69→45.35 Å / Num. obs: 459508 / % possible obs: 99 % / Redundancy: 4.7 % / Biso Wilson estimate: 18.44 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.19 / Rpim(I) all: 0.096 / Net I/σ(I): 6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) all% possible all
1.69-1.734.81.8510.9342490.350.94499.8
7.56-45.354.70.06620.352690.9930.03398.8

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Processing

Software
NameVersionClassification
xia2data scaling
PHENIX1.19_4092refinement
PDB_EXTRACT3.27data extraction
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1H7W

1h7w


Resolution: 1.69→36.15 Å / SU ML: 0.21 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 21.67 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2001 22716 4.95 %
Rwork0.1768 436613 -
obs0.1779 459329 98.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 98.51 Å2 / Biso mean: 26.8994 Å2 / Biso min: 10.34 Å2
Refinement stepCycle: final / Resolution: 1.69→36.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms30760 0 691 4306 35757
Biso mean--21.78 36.77 -
Num. residues----4029
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.69-1.710.35827590.3221473015489100
1.71-1.730.32947510.31251453415285100
1.73-1.750.32747660.30211465115417100
1.75-1.770.3427530.30941465415407100
1.77-1.80.31747500.2861465215402100
1.8-1.820.30637830.27261463415417100
1.82-1.850.27317480.25941457815326100
1.85-1.870.28417560.24431470215458100
1.87-1.90.2647660.23531457615342100
1.9-1.930.25597050.2282146101531599
1.93-1.970.24676940.2232146831537799
1.97-20.24588030.2129145541535799
2-2.040.22657570.2033145561531399
2.04-2.080.22297340.202146191535399
2.08-2.130.21747620.1859144621522499
2.13-2.180.21067570.1805140791483696
2.18-2.230.20496460.1784135061415291
2.23-2.290.21757770.169145961537399
2.29-2.360.19158130.16621466115474100
2.36-2.440.19227990.16651457715376100
2.44-2.520.20197970.16311466715464100
2.52-2.630.18538140.16241465915473100
2.63-2.740.20317850.16391468515470100
2.74-2.890.19797910.16591462515416100
2.89-3.070.18947220.1647146281535099
3.07-3.310.17867180.1593146351535399
3.31-3.640.17827060.1538145711527799
3.64-4.170.15017280.1331139851471395
4.17-5.250.13927890.1288146611545099
5.25-36.150.15257870.14821488315670100

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