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- PDB-7ud6: Designed Enzyme SH3-588 (Catechol O-methyltransferase catalytic d... -

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Basic information

Entry
Database: PDB / ID: 7ud6
TitleDesigned Enzyme SH3-588 (Catechol O-methyltransferase catalytic domain and Src homology 3 binding domain fusion)
ComponentsTyrosine-protein kinase Fyn,Catechol O-methyltransferase
KeywordsTRANSFERASE / Designed enzyme / COMT / SH3 / fusion protein
Function / homology
Function and homology information


Enzymatic degradation of dopamine by COMT / Enzymatic degradation of Dopamine by monoamine oxidase / positive regulation of homocysteine metabolic process / Methylation / norepinephrine secretion / response to dopamine / mastication / catechol-containing compound metabolic process / catecholamine catabolic process / catechol O-methyltransferase activity ...Enzymatic degradation of dopamine by COMT / Enzymatic degradation of Dopamine by monoamine oxidase / positive regulation of homocysteine metabolic process / Methylation / norepinephrine secretion / response to dopamine / mastication / catechol-containing compound metabolic process / catecholamine catabolic process / catechol O-methyltransferase activity / renal sodium excretion / : / : / S-adenosylhomocysteine metabolic process / catechol O-methyltransferase / renal filtration / developmental process / renin secretion into blood stream / dopamine secretion / negative regulation of dopamine metabolic process / renal albumin absorption / catecholamine metabolic process / habituation / artery development / response to salt / short-term memory / S-adenosylmethionine metabolic process / cerebellar cortex morphogenesis / dopamine catabolic process / norepinephrine metabolic process / cellular response to phosphate starvation / glomerulus development / fear response / multicellular organismal reproductive process / synaptic transmission, dopaminergic / response to angiotensin / cellular response to cocaine / estrogen metabolic process / exploration behavior / response to food / cholesterol efflux / response to temperature stimulus / response to pain / response to corticosterone / prostaglandin metabolic process / glycogen metabolic process / dopamine metabolic process / startle response / detection of temperature stimulus involved in sensory perception of pain / : / behavioral fear response / multicellular organismal response to stress / response to amphetamine / : / learning / response to cytokine / kidney development / female pregnancy / negative regulation of smooth muscle cell proliferation / visual learning / multicellular organism growth / response to organic cyclic compound / response to toxic substance / memory / cognition / regulation of blood pressure / response to wounding / response to estrogen / gene expression / cell body / postsynapse / methylation / postsynaptic membrane / vesicle / response to oxidative stress / response to lipopolysaccharide / dendritic spine / learning or memory / response to hypoxia / response to xenobiotic stimulus / axon / glutamatergic synapse / dendrite / magnesium ion binding / membrane / plasma membrane / cytosol
Similarity search - Function
Fyn/Yrk, SH3 domain / Catechol O-methyltransferase, eukaryotic / O-methyltransferase / Class I-like SAM-dependent O-methyltransferase / SAM-dependent O-methyltransferase class I-type profile. / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain ...Fyn/Yrk, SH3 domain / Catechol O-methyltransferase, eukaryotic / O-methyltransferase / Class I-like SAM-dependent O-methyltransferase / SAM-dependent O-methyltransferase class I-type profile. / SH3 domain / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
: / S-ADENOSYL-L-HOMOCYSTEINE / Tyrosine-protein kinase Fyn / Catechol O-methyltransferase
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsOngpipattanakul, C. / Nair, S.K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat.Chem.Biol. / Year: 2023
Title: Designer installation of a substrate recruitment domain to tailor enzyme specificity.
Authors: Park, R. / Ongpipattanakul, C. / Nair, S.K. / Bowers, A.A. / Kuhlman, B.
History
DepositionMar 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
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

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Assembly

Deposited unit
A: Tyrosine-protein kinase Fyn,Catechol O-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,0583
Polymers32,6341
Non-polymers4242
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)68.883, 68.883, 302.697
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number178
Space group name H-MP6122

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Components

#1: Protein Tyrosine-protein kinase Fyn,Catechol O-methyltransferase


Mass: 32634.008 Da / Num. of mol.: 1
Mutation: Q26V,N29R,E37L,R39V,R73E,Y74F,Q77L,N78V,T93Q,Q97Y,K98R
Source method: isolated from a genetically manipulated source
Details: The enzyme is a fusion between COMT from Rattus norvegicus and the SH3 domain from Homo sapiens.
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Rattus norvegicus (Norway rat)
Gene: FYN, Comt / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: E5RFS5, UniProt: P22734, catechol O-methyltransferase
#2: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Sequence detailsCOMT domain in 7UD6 was designed using PDB ID: 5P9V as a template. 5P9V contains two humanizing ...COMT domain in 7UD6 was designed using PDB ID: 5P9V as a template. 5P9V contains two humanizing mutations, Ile153 and Cys157 (7UD6 numbering), which are not present in Uniprot ID: P22734. Additional engineered residues present in 7UD6 were computationally designed to enhance interactions between the COMT and SH3 domains

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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: 3.41 Å3/Da / Density % sol: 63.93 %
Crystal growTemperature: 297 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 0.7 M magnesium formate, 0.1 M bis-tris propone pH 7.0.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.97872 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Apr 12, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 2.59→59.654 Å / Num. obs: 14097 / % possible obs: 99.6 % / Redundancy: 27.5 % / CC1/2: 0.999 / Rmerge(I) obs: 0.107 / Rpim(I) all: 0.021 / Rrim(I) all: 0.11 / Net I/σ(I): 26.3
Reflection shellResolution: 2.594→2.602 Å / Redundancy: 21.2 % / Rmerge(I) obs: 1.36 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 140 / CC1/2: 0.923 / Rpim(I) all: 0.297 / Rrim(I) all: 1.394 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.18.2_3874: ???)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1M27, 4PYN

1m27

4pyn


Resolution: 2.59→55.5 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 34.42 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2779 671 4.78 %
Rwork0.2466 --
obs0.2483 14038 99.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.59→55.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2095 0 1 2 2098
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0092138
X-RAY DIFFRACTIONf_angle_d1.4592901
X-RAY DIFFRACTIONf_dihedral_angle_d14.563299
X-RAY DIFFRACTIONf_chiral_restr0.071327
X-RAY DIFFRACTIONf_plane_restr0.012368
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.59-2.790.38741110.33522593X-RAY DIFFRACTION99
2.79-3.070.3991400.30612590X-RAY DIFFRACTION100
3.08-3.520.33141470.28012619X-RAY DIFFRACTION99
3.52-4.430.22371250.23582655X-RAY DIFFRACTION98
4.43-55.50.25681480.21972910X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: -14.1947 Å / Origin y: 30.3494 Å / Origin z: -27.0127 Å
111213212223313233
T1.008 Å2-0.2398 Å20.2008 Å2-0.4704 Å2-0.0006 Å2--0.6154 Å2
L0.4692 °2-0.0732 °2-0.5851 °2-1.5495 °2-1.1233 °2--1.9904 °2
S0.4842 Å °0.2346 Å °0.0031 Å °0.6519 Å °0.0546 Å °0.1711 Å °-1.2402 Å °0.3535 Å °0.2779 Å °
Refinement TLS groupSelection details: all

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