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- PDB-7m32: Dihydropyrimidine Dehydrogenase (DPD) C671A Mutant Soaked with Ur... -

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Basic information

Entry
Database: PDB / ID: 7m32
TitleDihydropyrimidine Dehydrogenase (DPD) C671A Mutant Soaked with Uracil and NADPH Anaerobically
ComponentsDihydropyrimidine dehydrogenase [NADP(+)]
KeywordsFLAVOPROTEIN / Oxidoreductase / Dihydropyrimidine Dehydrogenase / DPD / mutant / uracil / flavin / soaking / ligand / FMN / FAD
Function / homology
Function and homology information


dihydropyrimidine dehydrogenase (NADP+) / thymidine catabolic process / dihydropyrimidine dehydrogenase (NADP+) activity / uracil binding / beta-alanine biosynthetic process / thymine catabolic process / uracil catabolic process / FMN binding / NADP binding / flavin adenine dinucleotide binding ...dihydropyrimidine dehydrogenase (NADP+) / thymidine catabolic process / dihydropyrimidine dehydrogenase (NADP+) activity / uracil binding / beta-alanine biosynthetic process / thymine catabolic process / uracil catabolic process / FMN binding / NADP binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding / protein homodimerization activity / metal ion binding / cytoplasm / cytosol
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
ALANINE / FLAVIN-ADENINE DINUCLEOTIDE / Chem-FNR / LEUCINE / PROLINE / IRON/SULFUR CLUSTER / URACIL / Dihydropyrimidine dehydrogenase [NADP(+)]
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.82 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)457,89137
Polymers446,2854
Non-polymers11,60633
Water47,3792630
1
A: Dihydropyrimidine dehydrogenase [NADP(+)]
B: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,88818
Polymers223,1432
Non-polymers5,74516
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31350 Å2
ΔGint-294 kcal/mol
Surface area68260 Å2
MethodPISA
2
C: Dihydropyrimidine dehydrogenase [NADP(+)]
D: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,00319
Polymers223,1432
Non-polymers5,86117
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31480 Å2
ΔGint-286 kcal/mol
Surface area68760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.870, 158.730, 162.700
Angle α, β, γ (deg.)90.000, 95.890, 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: 111571.281 Da / Num. of mol.: 4 / Mutation: C671A
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 , 8 types, 2663 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-URA / URACIL


Mass: 112.087 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H4N2O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-FNR / 1-DEOXY-1-(7,8-DIMETHYL-2,4-DIOXO-3,4-DIHYDRO-2H-BENZO[G]PTERIDIN-1-ID-10(5H)-YL)-5-O-PHOSPHONATO-D-RIBITOL / TWO ELECTRON REDUCED FLAVIN MONONUCLEOTIDE


Mass: 458.360 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H23N4O9P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ALA / ALANINE


Type: L-peptide linking / Mass: 89.093 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H7NO2 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-LEU / LEUCINE


Type: L-peptide linking / Mass: 131.173 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H13NO2 / Feature type: SUBJECT OF INVESTIGATION
#8: 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
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2630 / 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: 2.36 Å3/Da / Density % sol: 47.8 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: ~4.5 mg/mL (~40 uM) of C671A DPD 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 ...Details: ~4.5 mg/mL (~40 uM) of C671A DPD 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%. C671A 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 uracil, 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: 1.82→51.69 Å / Num. obs: 354924 / % possible obs: 96.5 % / Redundancy: 4.8 % / Biso Wilson estimate: 25.22 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.147 / Rpim(I) all: 0.074 / Rrim(I) all: 0.165 / Net I/σ(I): 7.4 / Num. measured all: 1705022 / Scaling rejects: 41
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
1.82-1.874.91.946127495261460.3410.9692.1790.996.2
8.14-51.694.60.0411915841650.9980.020.04627.598.6

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Processing

Software
NameVersionClassification
Aimless0.7.4data scaling
PHENIX1.19_4092refinement
PDB_EXTRACT3.27data extraction
autoPROCdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1H7W

1h7w


Resolution: 1.82→47.26 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 21.29 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2019 17911 5.05 %
Rwork0.1689 336851 -
obs0.1706 354762 96.29 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 131.41 Å2 / Biso mean: 34.5565 Å2 / Biso min: 13.88 Å2
Refinement stepCycle: final / Resolution: 1.82→47.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms30658 0 529 2630 33817
Biso mean--27.17 39.66 -
Num. residues----4028
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 (%)
1.82-1.840.36735930.3481111871178096
1.84-1.860.36486190.3382111011172096
1.86-1.890.35465980.3187111791177796
1.89-1.910.31896030.2939111541175796
1.91-1.930.30815870.2795112321181996
1.93-1.960.3075960.2658112071180396
1.96-1.990.29546090.2456111831179296
1.99-2.020.26765830.2352111471173096
2.02-2.050.24936490.2208111461179596
2.05-2.080.2616230.2189110631168696
2.08-2.120.22696590.2032108091146894
2.12-2.160.24515660.192898161038284
2.16-2.20.2135750.1858111101168595
2.2-2.240.22196740.1803112821195698
2.24-2.290.2046100.1705114091201998
2.29-2.350.19426300.1663113451197598
2.35-2.410.21595930.1645114411203498
2.41-2.470.2075780.1588113971197598
2.47-2.540.19246280.1493114181204698
2.54-2.620.19485450.1563115171206298
2.62-2.720.19065370.1517114871202498
2.72-2.830.20525860.158114681205498
2.83-2.960.20365860.1604114821206898
2.96-3.110.19685850.161114421202798
3.11-3.310.19635940.1596114311202598
3.31-3.560.18625430.158114221196597
3.56-3.920.18665020.1419105211102390
3.92-4.490.15025590.127110531161294
4.49-5.650.15046650.13731164912314100
5.65-47.260.16576360.1459117531238999

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