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- PDB-8ay1: Crystal structure of the C. elegans POFUT2 (CePoFUT2) triple muta... -

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Basic information

Entry
Database: PDB / ID: 8ay1
TitleCrystal structure of the C. elegans POFUT2 (CePoFUT2) triple mutant (R298K-R299K-A418C) in complex with the Rattus norvegicus TSR4 single mutant (E10C) from F-spondin
ComponentsGDP-fucose protein O-fucosyltransferase 2,Spondin-1
KeywordsTRANSFERASE / PoFUT2 / TSR / water molecules / Protein O-fucosylation
Function / homology
Function and homology information


O-glycosylation of TSR domain-containing proteins / protein O-linked fucosylation / peptide-O-fucosyltransferase / peptide-O-fucosyltransferase activity / positive regulation of amyloid precursor protein catabolic process / fucose metabolic process / positive regulation of protein processing / LBD domain binding / negative regulation of amyloid-beta formation / extracellular matrix ...O-glycosylation of TSR domain-containing proteins / protein O-linked fucosylation / peptide-O-fucosyltransferase / peptide-O-fucosyltransferase activity / positive regulation of amyloid precursor protein catabolic process / fucose metabolic process / positive regulation of protein processing / LBD domain binding / negative regulation of amyloid-beta formation / extracellular matrix / protein processing / cell adhesion / endoplasmic reticulum / Golgi apparatus / extracellular region / metal ion binding
Similarity search - Function
GDP-fucose protein O-fucosyltransferase 2-like / Reeler domain / Spondin, N-terminal / Spondin, N-terminal domain superfamily / Spondin_N / Spondin domain profile. / : / Reeler domain / Reeler domain superfamily / Reelin domain profile. ...GDP-fucose protein O-fucosyltransferase 2-like / Reeler domain / Spondin, N-terminal / Spondin, N-terminal domain superfamily / Spondin_N / Spondin domain profile. / : / Reeler domain / Reeler domain superfamily / Reelin domain profile. / GDP-fucose protein O-fucosyltransferase / GDP-fucose protein O-fucosyltransferase / Rossmann fold - #11340 / Rossmann fold - #11350 / Spondin-like TSP1 domain / Spondin-like TSP1 domain / Thrombospondin type 1 domain / Thrombospondin type-1 (TSP1) repeat superfamily / Thrombospondin type-1 (TSP1) repeat profile. / Thrombospondin type 1 repeats / Thrombospondin type-1 (TSP1) repeat / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Spondin-1 / GDP-fucose protein O-fucosyltransferase 2
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.13 Å
AuthorsHurtado-Guerrero, R. / Merino, P.
Funding support Spain, 1items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and Universities Spain
CitationJournal: Angew.Chem.Int.Ed.Engl. / Year: 2022
Title: The Essential Role of Water Molecules in the Reaction Mechanism of Protein O-Fucosyltransferase 2.
Authors: Sanz-Martinez, I. / Garcia-Garcia, A. / Tejero, T. / Hurtado-Guerrero, R. / Merino, P.
History
DepositionSep 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Nov 30, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.4Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.5Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GDP-fucose protein O-fucosyltransferase 2,Spondin-1
B: GDP-fucose protein O-fucosyltransferase 2,Spondin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)109,01028
Polymers106,6402
Non-polymers2,37026
Water2,792155
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The fusion protein is monomeric.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8920 Å2
ΔGint-161 kcal/mol
Surface area35520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)156.848, 156.848, 80.451
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number173
Space group name H-MP63

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein GDP-fucose protein O-fucosyltransferase 2,Spondin-1 / Patterning defective protein 2 / Peptide-O-fucosyltransferase 2 / O-FucT-2 / F-spondin


Mass: 53320.156 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans ...Details: Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.,Note that this is a fusion protein formed by CePoFUT2 from C. elegans and RnTSR4 from F-spondin from Rattus norvegicus.
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: pad-2, K10G9.3, Spon1, rCG_39552 / Production host: Komagataella pastoris (fungus)
References: UniProt: Q8WR51, UniProt: Q3B7D6, peptide-O-fucosyltransferase
#2: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 4 types, 179 molecules

#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C2H6O2
#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 155 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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: 2.69 Å3/Da / Density % sol: 54.2 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: BIS-Tris calcium chloride dihydrate MPD ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALBA / Beamline: XALOC / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Oct 16, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.13→20 Å / Num. obs: 62973 / % possible obs: 99.6 % / Redundancy: 8.5 % / CC1/2: 0.998 / Rsym value: 0.032 / Net I/σ(I): 10.6
Reflection shellResolution: 2.13→2.24 Å / Redundancy: 8.1 % / Mean I/σ(I) obs: 1.1 / Num. unique obs: 8995 / CC1/2: 0.491 / Rpim(I) all: 0.765 / % possible all: 98.1

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Processing

Software
NameVersionClassification
REFMAC5.8.0352refinement
XDSdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5FOE

5foe


Resolution: 2.13→19.9 Å / Cor.coef. Fo:Fc: 0.968 / Cor.coef. Fo:Fc free: 0.953 / SU B: 6.769 / SU ML: 0.164 / Cross valid method: THROUGHOUT / ESU R: 0.201 / ESU R Free: 0.18 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24037 2439 3.9 %RANDOM
Rwork0.19351 ---
obs0.19531 60376 99.33 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 61.322 Å2
Baniso -1Baniso -2Baniso -3
1-0.05 Å20.02 Å2-0 Å2
2--0.05 Å20 Å2
3----0.16 Å2
Refinement stepCycle: 1 / Resolution: 2.13→19.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6931 0 138 155 7224
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0127267
X-RAY DIFFRACTIONr_bond_other_d0.0010.0166465
X-RAY DIFFRACTIONr_angle_refined_deg1.3891.6589809
X-RAY DIFFRACTIONr_angle_other_deg0.4821.5715085
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.2775829
X-RAY DIFFRACTIONr_dihedral_angle_2_deg10.939562
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.376101264
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.0680.21025
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.028134
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021540
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it4.9676.0643328
X-RAY DIFFRACTIONr_mcbond_other4.9656.0643328
X-RAY DIFFRACTIONr_mcangle_it6.689.0774144
X-RAY DIFFRACTIONr_mcangle_other6.6819.0774145
X-RAY DIFFRACTIONr_scbond_it6.0746.6613939
X-RAY DIFFRACTIONr_scbond_other5.9536.6253892
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other8.3149.7085591
X-RAY DIFFRACTIONr_long_range_B_refined10.50677.028125
X-RAY DIFFRACTIONr_long_range_B_other10.50677.0198125
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.13→2.184 Å
RfactorNum. reflection% reflection
Rfree0.327 170 -
Rwork0.328 4206 -
obs--95.48 %

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