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- PDB-5etj: Crystal structure of purine nucleoside phosphorylase (E258D, L261... -

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Basic information

Entry
Database: PDB / ID: 5etj
TitleCrystal structure of purine nucleoside phosphorylase (E258D, L261A) mutant from human complexed with DADMe-ImmG and phosphate
ComponentsPurine nucleoside phosphorylase
KeywordsTransferase/Transferase Inhibitor / phosphorylase / Transferase-Transferase Inhibitor complex
Function / homology
Function and homology information


nicotinamide riboside catabolic process / Defective PNP disrupts phosphorolysis of (deoxy)guanosine and (deoxy)inosine / purine-containing compound salvage / deoxyinosine catabolic process / purine nucleobase binding / inosine catabolic process / deoxyadenosine catabolic process / nucleotide biosynthetic process / dAMP catabolic process / urate biosynthetic process ...nicotinamide riboside catabolic process / Defective PNP disrupts phosphorolysis of (deoxy)guanosine and (deoxy)inosine / purine-containing compound salvage / deoxyinosine catabolic process / purine nucleobase binding / inosine catabolic process / deoxyadenosine catabolic process / nucleotide biosynthetic process / dAMP catabolic process / urate biosynthetic process / Ribavirin ADME / IMP catabolic process / nucleoside binding / guanosine phosphorylase activity / Purine catabolism / allantoin metabolic process / Purine salvage / purine-nucleoside phosphorylase / nucleobase-containing compound metabolic process / purine ribonucleoside salvage / purine-nucleoside phosphorylase activity / positive regulation of alpha-beta T cell differentiation / phosphate ion binding / positive regulation of T cell proliferation / positive regulation of interleukin-2 production / secretory granule lumen / ficolin-1-rich granule lumen / response to xenobiotic stimulus / immune response / Neutrophil degranulation / extracellular exosome / extracellular region / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Purine nucleoside phosphorylase I, inosine/guanosine-specific / Purine phosphorylase, family 2, conserved site / Purine and other phosphorylases family 2 signature. / Purine nucleoside phosphorylase / Nucleoside phosphorylase domain / Nucleoside phosphorylase domain / Phosphorylase superfamily / Nucleoside phosphorylase superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-IM5 / PHOSPHATE ION / Purine nucleoside phosphorylase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.3 Å
AuthorsCameron, S.A. / Suarez, J. / Schramm, V.L. / Almo, S.C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM068036 United States
CitationJournal: J.Am.Chem.Soc. / Year: 2016
Title: Modulating Enzyme Catalysis through Mutations Designed to Alter Rapid Protein Dynamics.
Authors: Zoi, I. / Suarez, J. / Antoniou, D. / Cameron, S.A. / Schramm, V.L. / Schwartz, S.D.
History
DepositionNov 17, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 14, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Sep 27, 2023Group: 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
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: Purine nucleoside phosphorylase
B: Purine nucleoside phosphorylase
C: Purine nucleoside phosphorylase
D: Purine nucleoside phosphorylase
E: Purine nucleoside phosphorylase
F: Purine nucleoside phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)219,36824
Polymers216,5526
Non-polymers2,81518
Water6,341352
1
A: Purine nucleoside phosphorylase
B: Purine nucleoside phosphorylase
C: Purine nucleoside phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)109,68412
Polymers108,2763
Non-polymers1,4089
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9880 Å2
ΔGint-93 kcal/mol
Surface area29830 Å2
MethodPISA
2
D: Purine nucleoside phosphorylase
E: Purine nucleoside phosphorylase
F: Purine nucleoside phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)109,68412
Polymers108,2763
Non-polymers1,4089
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9980 Å2
ΔGint-92 kcal/mol
Surface area30240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)268.808, 58.845, 173.155
Angle α, β, γ (deg.)90.000, 112.920, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
Purine nucleoside phosphorylase / PNP / Inosine phosphorylase / Inosine-guanosine phosphorylase


Mass: 36092.062 Da / Num. of mol.: 6 / Mutation: E258D, L261A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PNP, NP / Plasmid: pCR-T7/NT-TOPO / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS
References: UniProt: P00491, purine-nucleoside phosphorylase
#2: Chemical
ChemComp-IM5 / 2-amino-7-{[(3R,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidin-1-yl]methyl}-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one / DADMe-ImmG


Mass: 279.295 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C12H17N5O3
#3: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: PO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 352 / Source method: isolated from a natural source / Formula: H2O

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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: 2.91 Å3/Da / Density % sol: 57.77 % / Description: block
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Protein (10 mg/mL); Reservoir (0.2 M lithium sulfate, 0.1 M TRIS-HCl, pH 8.5 and 20% (w/v) PEG 4000)

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 31-ID / Wavelength: 0.97931 Å
DetectorType: RAYONIX MX225HE / Detector: CCD / Date: Nov 4, 2015
RadiationMonochromator: DIAMOND / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97931 Å / Relative weight: 1
ReflectionResolution: 2.3→50 Å / Num. all: 112272 / Num. obs: 105996 / % possible obs: 99.2 % / Redundancy: 7.47 % / Biso Wilson estimate: 46 Å2 / Rsym value: 0.095 / Net I/σ(I): 16.3
Reflection shellResolution: 2.3→2.43 Å / Redundancy: 7.51 % / Rmerge(I) obs: 0.777 / Mean I/σ(I) obs: 2.82 / % possible all: 96.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
XDSdata reduction
XDSdata scaling
MOLREP11.3.02phasing
REFMAC5.8.0123refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3PHB

3phb


Resolution: 2.3→25 Å / Cor.coef. Fo:Fc: 0.96 / Cor.coef. Fo:Fc free: 0.946 / SU B: 5.949 / SU ML: 0.139 / SU R Cruickshank DPI: 0.2189 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.219 / ESU R Free: 0.179 / SU Rfree Cruickshank DPI: 0.1788 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2109 5579 5 %RANDOM
Rwork0.1781 ---
obs0.1797 105996 99.8 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 118.12 Å2 / Biso mean: 43.583 Å2 / Biso min: 20.45 Å2
Baniso -1Baniso -2Baniso -3
1--1.45 Å2-0 Å2-0.57 Å2
2--2.4 Å2-0 Å2
3----0.34 Å2
Refinement stepCycle: final / Resolution: 2.3→25 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13365 0 180 352 13897
Biso mean--39.28 38.7 -
Num. residues----1727
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.01913857
X-RAY DIFFRACTIONr_bond_other_d0.0020.0212999
X-RAY DIFFRACTIONr_angle_refined_deg1.5271.96718799
X-RAY DIFFRACTIONr_angle_other_deg0.96329810
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.47751721
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.90223.546626
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.078152213
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.2451597
X-RAY DIFFRACTIONr_chiral_restr0.0850.22043
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.02115764
X-RAY DIFFRACTIONr_gen_planes_other0.0010.023303
X-RAY DIFFRACTIONr_mcbond_it2.5184.1756902
X-RAY DIFFRACTIONr_mcbond_other2.5184.1746901
X-RAY DIFFRACTIONr_mcangle_it3.8876.258617
LS refinement shellResolution: 2.3→2.359 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.318 406 -
Rwork0.275 7727 -
all-8133 -
obs--100 %

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