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- EMDB-2267: negative stain single-particle reconstruction of conformation XX ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2267
Titlenegative stain single-particle reconstruction of conformation XX of the Ltn1 E3 ubiquitin Ligase
Map dataconformational snapshot XX of Ltn1
Sample
  • Sample: snapshot XX of Ltn1 conformer
  • Protein or peptide: Ltn1
Keywordsconformational heterogeneity / Ltn1/Listerin / RING E3 ubiquitin ligase / translational surveillance / neurodegenerative disease
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 42.7 Å
AuthorsLyumkis D / Doamekpor SK / Bengtson MH / Lee JW / Toro TB / Petroski MD / Lima CD / Potter CS / Carragher B / Joazeiro CAP
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase.
Authors: Dmitry Lyumkis / Selom K Doamekpor / Mario H Bengtson / Joong-Won Lee / Tasha B Toro / Matthew D Petroski / Christopher D Lima / Clinton S Potter / Bridget Carragher / Claudio A P Joazeiro /
Abstract: Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its ...Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.
History
DepositionDec 25, 2012-
Header (metadata) releaseJan 16, 2013-
Map releaseJan 16, 2013-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • 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_2267.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationconformational snapshot XX of Ltn1
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-1.39687073 - 2.72588944
Average (Standard dev.)-0.00932584 (±0.15276833)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 348.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z348.800348.800348.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-1.3972.726-0.009

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Supplemental data

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Sample components

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Entire : snapshot XX of Ltn1 conformer

EntireName: snapshot XX of Ltn1 conformer
Components
  • Sample: snapshot XX of Ltn1 conformer
  • Protein or peptide: Ltn1

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Supramolecule #1000: snapshot XX of Ltn1 conformer

SupramoleculeName: snapshot XX of Ltn1 conformer / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 180 MDa / Theoretical: 180 MDa / Method: SDS-PAGE

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Macromolecule #1: Ltn1

MacromoleculeName: Ltn1 / type: protein_or_peptide / ID: 1 / Name.synonym: Listerin / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 180 MDa / Theoretical: 180 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.01 mg/mL
BufferpH: 8 / Details: 190 mM NaCl, 20 mM Tris, 1mM BME
StainingType: NEGATIVE
Details: 3 microliters of sample at a concentration of 0.01 mg/mL was applied to a C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon. The specimen was stained with 2% ...Details: 3 microliters of sample at a concentration of 0.01 mg/mL was applied to a C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon. The specimen was stained with 2% uranyl formate 3 times, then let air-dry.
GridDetails: c-flat grids overlaid with thin (~1.5 nm) carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: single-tilt room temperature / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -55
TemperatureAverage: 298 K
DateMar 23, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 489 / Average electron dose: 15 e/Å2
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each micrograph
Final two d classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, Appion / Number images used: 722
Detailsrandom-conical tilt reconstruction

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