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- EMDB-30386: Negative-stain EM 3D reconstruction of UGGT with the Fab of monoc... -

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Basic information

Entry
Database: EMDB / ID: EMD-30386
TitleNegative-stain EM 3D reconstruction of UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
Map data
SampleUGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
Biological speciesThermomyces dupontii (fungus)
Methodsingle particle reconstruction / Resolution: 22.9 Å
AuthorsSatoh T / Song C / Murata K / Kato K
CitationJournal: Sci Rep / Year: 2017
Title: Visualisation of a flexible modular structure of the ER folding-sensor enzyme UGGT.
Authors: Tadashi Satoh / Chihong Song / Tong Zhu / Takayasu Toshimori / Kazuyoshi Murata / Yugo Hayashi / Hironari Kamikubo / Takayuki Uchihashi / Koichi Kato /
Abstract: In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates ...In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates displayed on the protein surface serve as quality tags. The ER folding-sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a gatekeeper in the ER quality control system by specifically catalysing monoglucosylation onto incompletely folded glycoproteins, thereby enabling them to interact with lectin-chaperone complexes. Here we characterise the dynamic structure of this enzyme. Our crystallographic data demonstrate that the sensor region is composed of four thioredoxin-like domains followed by a β-rich domain, which are arranged into a C-shaped structure with a large central cavity, while the C-terminal catalytic domain undergoes a ligand-dependent conformational alteration. Furthermore, small-angle X-ray scattering, cryo-electron microscopy and high-speed atomic force microscopy have demonstrated that UGGT has a flexible modular structure in which the smaller catalytic domain is tethered to the larger folding-sensor region with variable spatial arrangements. These findings provide structural insights into the working mechanism whereby UGGT operates as a folding-sensor against a variety of glycoprotein substrates through its flexible modular structure possessing extended hydrophobic surfaces for the recognition of unfolded substrates.
History
DepositionJul 17, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0425
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0425
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_30386.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 280 pix.
= 274.4 Å
0.98 Å/pix.
x 280 pix.
= 274.4 Å
0.98 Å/pix.
x 280 pix.
= 274.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.98 Å
Density
Contour LevelBy AUTHOR: 0.0425 / Movie #1: 0.0425
Minimum - Maximum-0.02121936 - 0.09164652
Average (Standard dev.)0.0014843342 (±0.007907849)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 274.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.980.980.98
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z274.400274.400274.400
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0210.0920.001

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Supplemental data

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Sample components

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Entire UGGT with the Fab of monoclonal antibody directed against the Trx...

EntireName: UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
Number of components: 1

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Component #1: protein, UGGT with the Fab of monoclonal antibody directed agains...

ProteinName: UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
Recombinant expression: No
SourceSpecies: Thermomyces dupontii (fungus)
Source (engineered)Expression System: Tobacco etch virus

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle
Sample solutionpH: 7.7
VitrificationCryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: JEOL 2200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: DIRECT ELECTRON DE-20 (5k x 3k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 2251
3D reconstructionResolution: 22.9 Å / Resolution method: FSC 0.143 CUT-OFF

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