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- PDB-8f5w: Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Di... -

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Basic information

Entry
Database: PDB / ID: 8f5w
TitleDihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine and NADPH Quasi-Anaerobically
ComponentsDihydropyrimidine dehydrogenase [NADP(+)]
KeywordsFLAVOPROTEIN / Dihydropyrimidine dehydrogenase / protein / mutant / dihyrdothymine / NADPH
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
FLAVIN-ADENINE DINUCLEOTIDE / Chem-FNR / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / IRON/SULFUR CLUSTER / THYMINE / Dihydropyrimidine dehydrogenase [NADP(+)]
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.97 Å
AuthorsKaley, N. / Smith, M. / Forouzesh, D. / Liu, D. / Moran, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1904480 United States
National Science Foundation (NSF, United States)2203593 United States
CitationJournal: Arch.Biochem.Biophys. / Year: 2023
Title: Mammalian dihydropyrimidine dehydrogenase: Added mechanistic details from transient-state analysis of charge transfer complexes.
Authors: Smith, M.M. / Forouzesh, D.C. / Kaley, N.E. / Liu, D. / Moran, G.R.
History
DepositionNov 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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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)460,42936
Polymers446,3494
Non-polymers14,08032
Water17,601977
1
A: Dihydropyrimidine dehydrogenase [NADP(+)]
B: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,21518
Polymers223,1752
Non-polymers7,04016
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area34380 Å2
ΔGint-289 kcal/mol
Surface area66720 Å2
MethodPISA
2
C: Dihydropyrimidine dehydrogenase [NADP(+)]
D: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)230,21518
Polymers223,1752
Non-polymers7,04016
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area34380 Å2
ΔGint-295 kcal/mol
Surface area67250 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.735, 158.003, 162.404
Angle α, β, γ (deg.)90.000, 96.060, 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
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 , 6 types, 1009 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-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-TDR / THYMINE


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


Mass: 18.015 Da / Num. of mol.: 977 / 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: 2.34 Å3/Da / Density % sol: 47.4 %
Description: Crystals grew as single elongated rectangular hexahedrons (200 x 50 x 50 uM).
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: DPD variant C671S (39.2 uM) in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was mixed 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 19% PEG 6000 at pH 4.7 to yield a 6 ...Details: DPD variant C671S (39.2 uM) in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was mixed 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 19% PEG 6000 at pH 4.7 to yield a 6 uL drop. Crystallization was carried out in the dark to prevent photo-degradation of the somewhat dissociable FMN cofactor. Under these conditions DPD crystals appeared within 16 hours and were left to grow for additional 24 hours. Ligands were combined with the crystals under near anaerobic conditions as follows: before being placed in a Plas-Labs 830 series glove box, the well solution beneath selected crystallization drops were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. Crystals trays were placed in the glove box that contained a Motic binocular microscope coupled to an Accuscope 1080p high-definition camera. The glove box was sealed and made quasi-anaerobic by flushing with high-purity nitrogen gas for approximately 10 minutes at which time fractional dioxygen was recorded as 0.1 %, by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held <1%. C671S DPD crystals were soaked for a minimum of 20 minutes with DHT (R,S) (200 uM) and NADPH (100 uM), each dissolved in the following cryo-solution: 20 mM sodium citrate, 0.4 mM DTT, 20% PEG 6000, 20% PEG 400, pH 7.5. The crystals were then submerged in liquid nitrogen, 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: Jun 8, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1272 Å / Relative weight: 1
ReflectionResolution: 1.97→47.23 Å / Num. obs: 283435 / % possible obs: 98.6 % / Redundancy: 4 % / Biso Wilson estimate: 26.48 Å2 / CC1/2: 0.994 / Rmerge(I) obs: 0.1301 / Rpim(I) all: 0.075 / Rrim(I) all: 0.151 / Net I/σ(I): 10
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
1.97-2.0140.90941.1136450.6330.5591.12995.2
5.35-52.294.246.4146270.0260.053100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
xia2data reduction
xia2data scaling
PHASERphasing
PHENIX1.20.1_4487refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7M31

7m31


Resolution: 1.97→47.23 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 21.12 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.217 14067 4.97 %
Rwork0.1787 269152 -
obs0.1806 283219 98.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 103.07 Å2 / Biso mean: 32.5455 Å2 / Biso min: 14.79 Å2
Refinement stepCycle: final / Resolution: 1.97→47.23 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms30850 0 692 977 32519
Biso mean--32.67 29.72 -
Num. residues----4042
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 (%)
1.97-1.990.30844240.26988593901794
1.99-2.020.30194370.25638791922897
2.02-2.040.28984480.2488950939898
2.04-2.070.28355070.23538819932698
2.07-2.10.26924870.22739018950599
2.1-2.120.26024630.21138935939899
2.12-2.150.24284850.19939033951899
2.15-2.190.23684560.19858999945599
2.19-2.220.23584610.19149103956499
2.22-2.260.23834320.19889041947399
2.26-2.30.24634700.19238943941399
2.3-2.340.2474390.19429062950199
2.34-2.380.25364710.19988988945999
2.38-2.430.24694980.18828959945799
2.43-2.480.23544600.18458994945498
2.48-2.540.24134550.18388720917596
2.54-2.610.22524830.18478591907495
2.61-2.680.23834750.18069042951799
2.68-2.750.24174720.18059066953899
2.75-2.840.21834780.18859009948799
2.84-2.950.25365120.19889010952299
2.95-3.060.24784880.19639047953599
3.06-3.20.23795080.18749018952699
3.2-3.370.22964400.189391119551100
3.37-3.580.21274450.18691249569100
3.58-3.860.20675040.164890959599100
3.86-4.250.17774790.14599059953899
4.25-4.860.16194720.14058709918195
4.86-6.120.16354690.15359094956399
6.12-47.230.15764490.13719229967899

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