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- EMDB-8719: The Electron Microscopy map of Drosophila melanogaster UDP-glucos... -

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Basic information

Entry
Database: EMDB / ID: EMD-8719
TitleThe Electron Microscopy map of Drosophila melanogaster UDP-glucose: glycoprotein glucosyltransferase (UGGT)
Map dataThe negative stain EM map of Drosophila melanogaster UDP-glucose:glycoprotein glucosyltransferase (UGGT)
Sample
  • Complex: UDP-glucose:glycoprotein glucosyltransferase (UGGT)
Function / homology
Function and homology information


UDP-glucose:glycoprotein glucosyltransferase activity / protein N-linked glycosylation via asparagine / ERAD pathway / unfolded protein binding / endoplasmic reticulum lumen
Similarity search - Function
UDP-glucose:glycoprotein glucosyltransferase, thioredoxin-like domain 4 / UGGT, thioredoxin-like domain 3 / UGGT, thioredoxin-like domain 1 / UGGT, thioredoxin-like domain 2 / UDP-glucose:Glycoprotein Glucosyltransferase / Thioredoxin-like domain / Thioredoxin-like domain / Thioredoxin-like domain / Thioredoxin-like domain / Glucosyltransferase 24, catalytic domain ...UDP-glucose:glycoprotein glucosyltransferase, thioredoxin-like domain 4 / UGGT, thioredoxin-like domain 3 / UGGT, thioredoxin-like domain 1 / UGGT, thioredoxin-like domain 2 / UDP-glucose:Glycoprotein Glucosyltransferase / Thioredoxin-like domain / Thioredoxin-like domain / Thioredoxin-like domain / Thioredoxin-like domain / Glucosyltransferase 24, catalytic domain / Glucosyltransferase 24 / UDP-glucose:Glycoprotein Glucosyltransferase / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
UDP-glucose:glycoprotein glucosyltransferase
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / negative staining / Resolution: 17.8 Å
AuthorsVargas J / Melero R / Yang M / Calles-Garcia D / Soya N / Menade M / Ito Y / Lukacs G / Kollman J / Kozlov G / Gehring K
Funding support Canada, 1 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (Canada)RGPIN-2014-04686 Canada
CitationJournal: J Biol Chem / Year: 2017
Title: Single-particle electron microscopy structure of UDP-glucose:glycoprotein glucosyltransferase suggests a selectivity mechanism for misfolded proteins.
Authors: Daniel Calles-Garcia / Meng Yang / Naoto Soya / Roberto Melero / Marie Ménade / Yukishige Ito / Javier Vargas / Gergely L Lukacs / Justin M Kollman / Guennadi Kozlov / Kalle Gehring /
Abstract: The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to -linked glycan of misfolded proteins. As ...The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to -linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from at 18-Å resolution. Three-dimensional reconstructions revealed a cage-like structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a high-resolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGT's selectivity for misfolded glycoproteins.
History
DepositionMay 3, 2017-
Header (metadata) releaseMay 17, 2017-
Map releaseMay 24, 2017-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8719.png

Downloads & links

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Map

FileDownload / File: emd_8719.map.gz / Format: CCP4 / Size: 8.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe negative stain EM map of Drosophila melanogaster UDP-glucose:glycoprotein glucosyltransferase (UGGT)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 132 pix.
= 237.6 Å		1.8 Å/pix.
x 132 pix.
= 237.6 Å		1.8 Å/pix.
x 132 pix.
= 237.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.082936876 - 0.14943764
Average (Standard dev.)-0.00038043322 (±0.01341918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions132132132
Spacing132132132
CellA=B=C: 237.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z132132132
origin x/y/z0.0000.0000.000
length x/y/z237.600237.600237.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS132132132
D min/max/mean-0.0830.149-0.000

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Supplemental data

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Sample components

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Entire : UDP-glucose:glycoprotein glucosyltransferase (UGGT)

EntireName: UDP-glucose:glycoprotein glucosyltransferase (UGGT)
Components
  • Complex: UDP-glucose:glycoprotein glucosyltransferase (UGGT)

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Supramolecule #1: UDP-glucose:glycoprotein glucosyltransferase (UGGT)

SupramoleculeName: UDP-glucose:glycoprotein glucosyltransferase (UGGT) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF9
Molecular weightExperimental: 175 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.012 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMC4H11NO3Tris(hydroxymethyl) Aminomethane
200.0 mMNaClSodium Chloride

Details: Buffer was filtered through a 0.22um filter
StainingType: NEGATIVE / Material: uranyl formate
Details: Freshly prepared uranyl formate at 0.75% was applied on the grids for 60s and then blotted
GridModel: EMS / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus min: 2.0 µm / Nominal magnification: 62000
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 2.0)
Startup modelType of model: OTHER / Details: RANSAC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 17.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software: (Name: RELION, Scipion) / Number images used: 26624
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: Scipion / Details: Ransac method
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 2.0) / Details: Relion 2.0
Final 3D classificationNumber classes: 10 / Software: (Name: RELION, Scipion)
FSC plot (resolution estimation)

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