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- PDB-7ntf: Cryo-EM structure of unliganded O-GlcNAc transferase -

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Basic information

Entry
Database: PDB / ID: 7ntf
TitleCryo-EM structure of unliganded O-GlcNAc transferase
ComponentsIsoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
KeywordsTRANSFERASE / O-GlcNAc transferase O-linked B-n-acetylglucosamine transferase
Function / homology
Function and homology information


protein N-acetylglucosaminyltransferase complex / protein O-GlcNAc transferase / regulation of insulin receptor signaling pathway / protein O-acetylglucosaminyltransferase activity / positive regulation of transcription from RNA polymerase II promoter by glucose / acetylglucosaminyltransferase activity / regulation of necroptotic process / regulation of Rac protein signal transduction / negative regulation of stem cell population maintenance / protein O-linked glycosylation ...protein N-acetylglucosaminyltransferase complex / protein O-GlcNAc transferase / regulation of insulin receptor signaling pathway / protein O-acetylglucosaminyltransferase activity / positive regulation of transcription from RNA polymerase II promoter by glucose / acetylglucosaminyltransferase activity / regulation of necroptotic process / regulation of Rac protein signal transduction / negative regulation of stem cell population maintenance / protein O-linked glycosylation / NSL complex / regulation of glycolytic process / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / RIPK1-mediated regulated necrosis / regulation of synapse assembly / regulation of gluconeogenesis / positive regulation of stem cell population maintenance / Formation of WDR5-containing histone-modifying complexes / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of proteolysis / mitophagy / hemopoiesis / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / histone acetyltransferase complex / positive regulation of lipid biosynthetic process / negative regulation of protein ubiquitination / positive regulation of TORC1 signaling / negative regulation of cell migration / response to nutrient / cell projection / positive regulation of translation / mitochondrial membrane / cellular response to glucose stimulus / negative regulation of transforming growth factor beta receptor signaling pathway / circadian regulation of gene expression / response to insulin / Regulation of necroptotic cell death / protein processing / chromatin DNA binding / UCH proteinases / chromatin organization / positive regulation of cold-induced thermogenesis / HATs acetylate histones / glutamatergic synapse / apoptotic process / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / signal transduction / positive regulation of transcription by RNA polymerase II / protein-containing complex / nucleoplasm / nucleus / plasma membrane / cytosol
Similarity search - Function
O-GlcNAc transferase, C-terminal / Glycosyl transferase family 41 / TPR repeat / Tetratricopeptide repeat / Tetratricopeptide repeat 1 / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats ...O-GlcNAc transferase, C-terminal / Glycosyl transferase family 41 / TPR repeat / Tetratricopeptide repeat / Tetratricopeptide repeat 1 / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily
Similarity search - Domain/homology
UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.32 Å
AuthorsMeek, R.W. / Blaza, J.N. / Davies, G.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Royal SocietyRPEA180016 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT.
Authors: Richard W Meek / James N Blaza / Jil A Busmann / Matthew G Alteen / David J Vocadlo / Gideon J Davies /
Abstract: The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this ...The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
History
DepositionMar 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_CSD / _citation.journal_volume ..._citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
B: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit


Theoretical massNumber of molelcules
Total (without water)240,4522
Polymers240,4522
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit / O-GlcNAc transferase subunit p110 / O-linked N-acetylglucosamine transferase 110 kDa subunit / OGT


Mass: 120225.773 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OGT
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: O15294, protein O-GlcNAc transferase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimeric assembly of O-GlcNAc transferase / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.5
Details: 25 mM HEPES pH 7.5 150 mM NaCl 1 mM DTT 0.5 % glycerol
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot for 4 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4CTFFINDCTF correction
7Flex-EMmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74102 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
13PE3

3pe3

13PE31PDBexperimental model
21W3B

1w3b

11W3B2PDBexperimental model

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