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- EMDB-8239: EM map of the intact yeast Elongator complex -

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Basic information

Entry
Database: EMDB / ID: EMD-8239
TitleEM map of the intact yeast Elongator complex
Map dataEndogenously purified yeast Elongator complex
Sample
  • Complex: Intact yeast Elongator complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.2 Å
AuthorsSetiaputra D / Cheng DTH / Hansen JM / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (Canada)418157-2012 Canada
CitationJournal: EMBO Rep / Year: 2017
Title: Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.
Authors: Dheva T Setiaputra / Derrick Th Cheng / Shan Lu / Jesse M Hansen / Udit Dalwadi / Cindy Hy Lam / Jeffrey L To / Meng-Qiu Dong / Calvin K Yip /
Abstract: Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique ...Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.
History
DepositionJun 8, 2016-
Header (metadata) releaseJul 20, 2016-
Map releaseDec 7, 2016-
UpdateSep 20, 2023-
Current statusSep 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8239.png

Downloads & links

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Map

FileDownload / File: emd_8239.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEndogenously purified yeast Elongator complex
Voxel sizeX=Y=Z: 7 Å
Density
Contour LevelBy AUTHOR: 0.128 / Movie #1: 0.128
Minimum - Maximum-0.3160276 - 0.60536766
Average (Standard dev.)0.0021289485 (±0.039052565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 504.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z777
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z504.000504.000504.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS727272
D min/max/mean-0.3160.6050.002

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Supplemental data

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Sample components

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Entire : Intact yeast Elongator complex

EntireName: Intact yeast Elongator complex
Components
  • Complex: Intact yeast Elongator complex

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Supramolecule #1: Intact yeast Elongator complex

SupramoleculeName: Intact yeast Elongator complex / type: complex / ID: 1 / Parent: 0 / Details: Endogenously purified yeast Elongator complex
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ1991
Molecular weightTheoretical: 850 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

BufferpH: 7.4
Component:
ConcentrationFormulaName
40.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid

Details: The final purification buffer was part of a glycerol gradient containing crosslinker as per the GraFix technique (Stark 2010): 40 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, ...Details: The final purification buffer was part of a glycerol gradient containing crosslinker as per the GraFix technique (Stark 2010): 40 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 1 mM EDTA, ~20% glycerol, ~0.02% glutaraldehyde. Fractions containing the sample were concentrated and the buffer was exchanged for the final buffer: 40 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA.
StainingType: NEGATIVE / Material: Uranyl formate
Details: Sample was applied to a glow-discharged copper grid overlaid with carbon (carbon evaporator and amyl acetate) and stained with uranyl formate.
GridModel: Ted Pella Gilder Grids / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 100 / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 17074
Details: 200 particles were manually picked to generate 5 class averages. These were used as templates for autopicking using the RELION software.
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 81 / Random conical tilt - Tilt angle: 65 degrees
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Angular sampling: 7.5 degrees
Software - Name: RELION (ver. 1.3)
Software - details: Initial angular assignment was done using the 3D Classification function to generate a suitable model and particle set for refinement.
Details: 3D Classification step in RELION
Final 3D classificationNumber classes: 5 / Avg.num./class: 1993 / Software - Name: RELION (ver. 1.3)
Software - details: THe 3D classification function in RELION was used to characterize heterogeneity in 3D.
Details: 2D classification was used to get rid of bad particles, and showed a high degree of preferred orientation. 3D classification was used to clean up the dataset. Most of the classes were ...Details: 2D classification was used to get rid of bad particles, and showed a high degree of preferred orientation. 3D classification was used to clean up the dataset. Most of the classes were similar and were therefore merged.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Software - details: Final refinement was done using the 3D auto-refine function.
Details: 3D auto-refine step in RELION, which automatically increments the angular sampling
Final reconstructionNumber classes used: 4 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 25.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3)
Software - details: Output from RELION auto-refine was masked using the post-processing function to generate the final reconstruction.
Details: The final model from RELION's auto-refine was further processed using the post-processing function in RELION to calculate the final masked map, with no map sharpening applied.
Number images used: 8190
FSC plot (resolution estimation)

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