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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 dataNegative-stain EM 3D reconstruction of UGGT with the Fab of monoclonal antibody
Sample
  • Complex: UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
Biological speciesThermomyces dupontii (fungus)
Methodsingle particle reconstruction / negative staining / 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
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  • 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

emd_30386.png

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)
AnnotationNegative-stain EM 3D reconstruction of UGGT with the Fab of monoclonal antibody
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.
Components
  • Complex: UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.

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Supramolecule #1: UGGT with the Fab of monoclonal antibody directed against the Trx...

SupramoleculeName: UGGT with the Fab of monoclonal antibody directed against the Trx4 domain.
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Thermomyces dupontii (fungus)
Recombinant expressionOrganism: Tobacco etch virus

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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

BufferpH: 7.7
StainingType: NEGATIVE / Material: uranyl acetate

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Electron microscopy

MicroscopeJEOL 2200FS
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 22.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2251
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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