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- EMDB-12588: Cryo-EM structure of unliganded O-GlcNAc transferase -

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Basic information

Entry
Database: EMDB / ID: EMD-12588
TitleCryo-EM structure of unliganded O-GlcNAc transferase
Map data
Sample
  • Complex: Dimeric assembly of O-GlcNAc transferase
    • Protein or peptide: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
KeywordsO-GlcNAc transferase O-linked B-n-acetylglucosamine transferase / TRANSFERASE
Function / homology
Function and homology information


negative regulation of non-canonical inflammasome complex assembly / protein N-acetylglucosaminyltransferase complex / regulation of insulin receptor signaling pathway / protein O-acetylglucosaminyltransferase activity / protein O-GlcNAc transferase / positive regulation of transcription from RNA polymerase II promoter by glucose / acetylglucosaminyltransferase activity / regulation of Rac protein signal transduction / regulation of necroptotic process / negative regulation of stem cell population maintenance ...negative regulation of non-canonical inflammasome complex assembly / protein N-acetylglucosaminyltransferase complex / regulation of insulin receptor signaling pathway / protein O-acetylglucosaminyltransferase activity / protein O-GlcNAc transferase / positive regulation of transcription from RNA polymerase II promoter by glucose / acetylglucosaminyltransferase activity / regulation of Rac protein signal transduction / regulation of necroptotic process / negative regulation of stem cell population maintenance / protein O-linked glycosylation / NSL complex / regulation of glycolytic process / RIPK1-mediated regulated necrosis / regulation of gluconeogenesis / regulation of synapse assembly / Formation of WDR5-containing histone-modifying complexes / Sin3-type complex / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / positive regulation of stem cell population maintenance / phosphatidylinositol-3,4,5-trisphosphate binding / hemopoiesis / positive regulation of proteolysis / histone acetyltransferase complex / positive regulation of lipid biosynthetic process / mitophagy / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / response to nutrient / negative regulation of protein ubiquitination / positive regulation of TORC1 signaling / negative regulation of cell migration / positive regulation of translation / cell projection / circadian regulation of gene expression / cellular response to glucose stimulus / negative regulation of transforming growth factor beta receptor signaling pathway / response to insulin / protein processing / chromatin DNA binding / Regulation of necroptotic cell death / mitochondrial membrane / UCH proteinases / positive regulation of cold-induced thermogenesis / HATs acetylate histones / chromatin organization / apoptotic process / regulation of transcription by RNA polymerase II / positive regulation of DNA-templated transcription / glutamatergic synapse / 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
UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110kDa subunit / O-GlcNAc transferase, C-terminal / Glycosyl transferase family 41 / TPR repeat / Tetratricopeptide repeat / Tetratricopeptide repeat / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats ...UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110kDa subunit / O-GlcNAc transferase, C-terminal / Glycosyl transferase family 41 / TPR repeat / Tetratricopeptide repeat / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.32 Å
AuthorsMeek RW / Blaza JN
Funding support United Kingdom, 1 items
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, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0113
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0113
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ntf
  • Surface level: 0.0113
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12588.png

Downloads & links

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Map

FileDownload / File: emd_12588.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 192 pix.
= 249.984 Å		1.3 Å/pix.
x 192 pix.
= 249.984 Å		1.3 Å/pix.
x 192 pix.
= 249.984 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.302 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0113 / Movie #1: 0.0113
Minimum - Maximum-0.03377892 - 0.10100281
Average (Standard dev.)0.00000062001254 (±0.0030219425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 249.98401 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3021.3021.302
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z249.984249.984249.984
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0340.1010.000

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Supplemental data

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Sample components

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Entire : Dimeric assembly of O-GlcNAc transferase

EntireName: Dimeric assembly of O-GlcNAc transferase
Components
  • Complex: Dimeric assembly of O-GlcNAc transferase
    • Protein or peptide: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit

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Supramolecule #1: Dimeric assembly of O-GlcNAc transferase

SupramoleculeName: Dimeric assembly of O-GlcNAc transferase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminy...

MacromoleculeName: Isoform 1 of UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: protein O-GlcNAc transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120.225773 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QASSVGNVAD STGLAELAHR EYQAGDFEAA ERHCMQLWRQ EPDNTGVLL LLSSIHFQCR RLDRSAHFST LAIKQNPLLA EAYSNLGNVY KERGQLQEAI EHYRHALRLK PDFIDGYINL A AALVAAGD ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QASSVGNVAD STGLAELAHR EYQAGDFEAA ERHCMQLWRQ EPDNTGVLL LLSSIHFQCR RLDRSAHFST LAIKQNPLLA EAYSNLGNVY KERGQLQEAI EHYRHALRLK PDFIDGYINL A AALVAAGD MEGAVQAYVS ALQYNPDLYC VRSDLGNLLK ALGRLEEAKA CYLKAIETQP NFAVAWSNLG CVFNAQGEIW LA IHHFEKA VTLDPNFLDA YINLGNVLKE ARIFDRAVAA YLRALSLSPN HAVVHGNLAC VYYEQGLIDL AIDTYRRAIE LQP HFPDAY CNLANALKEK GSVAEAEDCY NTALRLCPTH ADSLNNLANI KREQGNIEEA VRLYRKALEV FPEFAAAHSN LASV LQQQG KLQEALMHYK EAIRISPTFA DAYSNMGNTL KEMQDVQGAL QCYTRAIQIN PAFADAHSNL ASIHKDSGNI PEAIA SYRT ALKLKPDFPD AYCNLAHCLQ IVCDWTDYDE RMKKLVSIVA DQLEKNRLPS VHPHHSMLYP LSHGFRKAIA ERHGNL CLD KINVLHKPPY EHPKDLKLSD GRLRVGYVSS DFGNHPTSHL MQSIPGMHNP DKFEVFCYAL SPDDGTNFRV KVMAEAN HF IDLSQIPCNG KAADRIHQDG IHILVNMNGY TKGARNELFA LRPAPIQAMW LGYPGTSGAL FMDYIITDQE TSPAEVAE Q YSEKLAYMPH TFFIGDHANM FPHLKKKAVI DFKSNGHIYD NRIVLNGIDL KAFLDSLPDV KIVKMKCPDG GDNADSSNT ALNMPVIPMN TIAEAVIEMI NRGQIQITIN GFSISNGLAT TQINNKAATG EEVPRTIIVT TRSQYGLPED AIVYCNFNQL YKIDPSTLQ MWANILKRVP NSVLWLLRFP AVGEPNIQQY AQNMGLPQNR IIFSPVAPKE EHVRRGQLAD VCLDTPLCNG H TTGMDVLW AGTPMVTMPG ETLASRVAAS QLTCLGCLEL IAKNRQEYED IAVKLGTDLE YLKKVRGKVW KQRISSPLFN TK QYTMELE RLYLQMWEHY AAGNKPDHMI KPVEVTESA

UniProtKB: UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES pH 7.5 150 mM NaCl 1 mM DTT 0.5 % glycerol
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 1.5 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.32 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74102
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

3pe3

source_name: PDB, initial_model_type: experimental model

1w3b

source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

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

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