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- PDB-8f61: Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Di... -

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Basic information

Entry
Database: PDB / ID: 8f61
TitleDihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine Quasi-Anaerobically
ComponentsDihydropyrimidine dehydrogenase [NADP(+)]
KeywordsFLAVOPROTEIN / dihydropyrimidine dehydrogenase / dehydrogenase / mutant / dihydrothymine
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 / FLAVIN MONONUCLEOTIDE / Chem-FNR / IRON/SULFUR CLUSTER / THYMINE / (5S)-5-methyl-1,3-diazinane-2,4-dione / Dihydropyrimidine dehydrogenase [NADP(+)]
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.14 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)457,45532
Polymers446,3494
Non-polymers11,10628
Water14,358797
1
A: Dihydropyrimidine dehydrogenase [NADP(+)]
B: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,72816
Polymers223,1752
Non-polymers5,55314
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31110 Å2
ΔGint-291 kcal/mol
Surface area68090 Å2
MethodPISA
2
C: Dihydropyrimidine dehydrogenase [NADP(+)]
D: Dihydropyrimidine dehydrogenase [NADP(+)]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,72816
Polymers223,1752
Non-polymers5,55314
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area31100 Å2
ΔGint-304 kcal/mol
Surface area68210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.784, 157.667, 162.564
Angle α, β, γ (deg.)90.00, 95.93, 90.00
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, 825 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-TDR / THYMINE


Mass: 126.113 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H6N2O2 / 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.: 2 / Source method: obtained synthetically / Formula: C17H23N4O9P / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-XH5 / (5S)-5-methyl-1,3-diazinane-2,4-dione


Mass: 128.129 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H8N2O2 / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 797 / 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: 47.38 %
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), 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: 2.14→161.69 Å / Num. obs: 118212 / % possible obs: 89.3 % / Redundancy: 3.7 % / Biso Wilson estimate: 26.04 Å2 / CC1/2: 0.989 / Rmerge(I) obs: 0.172 / Rpim(I) all: 0.103 / Rrim(I) all: 0.203 / Net I/σ(I): 7.2
Reflection shellResolution: 2.14→2.45 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.762 / Mean I/σ(I) obs: 2 / Num. unique obs: 21096 / CC1/2: 0.485 / Rpim(I) all: 0.459 / Rrim(I) all: 0.893 / % possible all: 55.9

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Processing

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

7m31


Resolution: 2.14→161.69 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 25.35 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2298 5647 4.78 %
Rwork0.187 --
obs0.1891 118135 52.71 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.14→161.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms30922 0 500 797 32219
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00332158
X-RAY DIFFRACTIONf_angle_d0.64443776
X-RAY DIFFRACTIONf_dihedral_angle_d8.2714342
X-RAY DIFFRACTIONf_chiral_restr0.0444948
X-RAY DIFFRACTIONf_plane_restr0.0065568
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.14-2.170.415560.347684X-RAY DIFFRACTION1
2.17-2.190.353370.235677X-RAY DIFFRACTION1
2.19-2.220.3131140.261211X-RAY DIFFRACTION3
2.22-2.230.227210.304398X-RAY DIFFRACTION3
2.26-2.280.3712160.359404X-RAY DIFFRACTION8
2.28-2.310.3199210.272563X-RAY DIFFRACTION8
2.31-2.340.3641380.276821X-RAY DIFFRACTION12
2.34-2.380.3458480.2831919X-RAY DIFFRACTION13
2.38-2.410.304540.27891211X-RAY DIFFRACTION17
2.41-2.450.2713710.26581360X-RAY DIFFRACTION19
2.45-2.50.3285750.27321749X-RAY DIFFRACTION24
2.5-2.540.312980.26832018X-RAY DIFFRACTION28
2.54-2.590.30561100.27362270X-RAY DIFFRACTION32
2.59-2.640.35391250.26052748X-RAY DIFFRACTION38
2.64-2.70.2811610.26013176X-RAY DIFFRACTION45
2.7-2.760.30561970.24683716X-RAY DIFFRACTION53
2.76-2.830.29492210.24984381X-RAY DIFFRACTION61
2.83-2.910.28992750.24864965X-RAY DIFFRACTION70
2.91-2.990.28732930.23995732X-RAY DIFFRACTION81
2.99-3.090.28483410.22816384X-RAY DIFFRACTION90
3.09-3.20.27933540.21536991X-RAY DIFFRACTION98
3.2-3.330.25063600.19957067X-RAY DIFFRACTION99
3.33-3.480.24733610.197058X-RAY DIFFRACTION99
3.48-3.640.23692810.186245X-RAY DIFFRACTION98
3.67-3.890.22813150.16447058X-RAY DIFFRACTION99
3.89-4.190.16973850.15197033X-RAY DIFFRACTION99
4.19-4.620.17133300.14057097X-RAY DIFFRACTION99
4.62-5.280.20453670.1517017X-RAY DIFFRACTION98
5.28-6.660.19933730.176883X-RAY DIFFRACTION96
6.66-161.690.16963490.15297152X-RAY DIFFRACTION98

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