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- PDB-3dl5: Crystal Structure of the A287F Active Site Mutant of TS-DHFR from... -

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Basic information

Entry
Database: PDB / ID: 3dl5
TitleCrystal Structure of the A287F Active Site Mutant of TS-DHFR from Cryptosporidium hominis
ComponentsDihydrofolate reductase, DHFR
KeywordsOXIDOREDUCTASE / Enzyme active site mutant / Enzyme-ligand complex
Function / homology
Function and homology information


Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID / DIHYDROFOLIC ACID / Chem-NDP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / :
Similarity search - Component
Biological speciesCryptosporidium hominis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.74 Å
AuthorsVargo, M.A. / Martucci, W.E. / Anderson, K.S.
CitationJournal: Biochemistry / Year: 2008
Title: Explaining an unusually fast parasitic enzyme: folate tail-binding residues dictate substrate positioning and catalysis in Cryptosporidium hominis thymidylate synthase.
Authors: Martucci, W.E. / Vargo, M.A. / Anderson, K.S.
History
DepositionJun 26, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Nov 13, 2019Group: Source and taxonomy / Structure summary / Category: entity_src_gen / struct_keywords
Item: _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name ..._entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name / _entity_src_gen.pdbx_host_org_strain / _struct_keywords.text
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 30, 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

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Assembly

Deposited unit
A: Dihydrofolate reductase, DHFR
B: Dihydrofolate reductase, DHFR
C: Dihydrofolate reductase, DHFR
D: Dihydrofolate reductase, DHFR
E: Dihydrofolate reductase, DHFR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,56525
Polymers301,6935
Non-polymers9,87220
Water8,017445
1
C: Dihydrofolate reductase, DHFR
D: Dihydrofolate reductase, DHFR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62610
Polymers120,6772
Non-polymers3,9498
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15970 Å2
ΔGint-48 kcal/mol
Surface area38750 Å2
MethodPISA
2
A: Dihydrofolate reductase, DHFR
B: Dihydrofolate reductase, DHFR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62610
Polymers120,6772
Non-polymers3,9498
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15990 Å2
ΔGint-47 kcal/mol
Surface area38580 Å2
MethodPISA
3
E: Dihydrofolate reductase, DHFR
hetero molecules

E: Dihydrofolate reductase, DHFR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62610
Polymers120,6772
Non-polymers3,9498
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_457-x-1,y,-z+21
Buried area15700 Å2
ΔGint-45 kcal/mol
Surface area39350 Å2
MethodPISA
Unit cell
Length a, b, c (Å)214.807, 116.542, 219.212
Angle α, β, γ (deg.)90.00, 95.21, 90.00
Int Tables number5
Space group name H-MC121

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Components

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Protein , 1 types, 5 molecules ABCDE

#1: Protein
Dihydrofolate reductase, DHFR /


Mass: 60338.613 Da / Num. of mol.: 5 / Mutation: A287F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptosporidium hominis (eukaryote) / Gene: Chro.40506 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (DE3) / References: UniProt: Q5CGA3, dihydrofolate reductase

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Non-polymers , 5 types, 465 molecules

#2: Chemical
ChemComp-UMP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / DUMP / Deoxyuridine monophosphate


Mass: 308.182 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C9H13N2O8P
#3: Chemical
ChemComp-CB3 / 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID


Mass: 477.469 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C24H23N5O6
#4: Chemical
ChemComp-DHF / DIHYDROFOLIC ACID / Dihydrofolic acid


Mass: 443.413 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C19H21N7O6
#5: Chemical
ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 445 / 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: 4.55 Å3/Da / Density % sol: 72.94 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.1mM ammonium sulfate, 0.2mM, lithium sulfate, 0.1mM Tris, 12% PEG-6000, flash frozen in 25% ethylene glycol, pH 8.0, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X29A / Wavelength: 1.08 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 16, 2006
RadiationMonochromator: Si (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.08 Å / Relative weight: 1
ReflectionResolution: 2.74→45.4 Å / Num. obs: 141384 / % possible obs: 99.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.6 % / Biso Wilson estimate: 68 Å2 / Rmerge(I) obs: 0.123 / Net I/σ(I): 9.5
Reflection shellResolution: 2.74→2.91 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.682 / Mean I/σ(I) obs: 2.1 / Num. unique all: 22312 / % possible all: 99.8

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Processing

Software
NameVersionClassification
CBASSdata collection
AMoREphasing
CNS1.1refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1QZF

1qzf


Resolution: 2.74→45.4 Å / Isotropic thermal model: anisotropic / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.245 7082 -random
Rwork0.214 ---
all0.214 141384 --
obs0.214 141384 99.8 %-
Refine analyze
FreeObs
Luzzati coordinate error0.41 Å0.35 Å
Luzzati d res low-5 Å
Luzzati sigma a0.53 Å0.45 Å
Refinement stepCycle: LAST / Resolution: 2.74→45.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20673 0 675 445 21793
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d25.3
X-RAY DIFFRACTIONc_improper_angle_d2.3
LS refinement shellResolution: 2.74→2.91 Å / Rfactor Rfree error: 0.01
RfactorNum. reflection% reflection
Rfree0.359 1181 -
Rwork0.324 --
obs-22312 99.8 %

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