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- EMDB-8291: EM map of the Elp123 subcomplex of yeast Elongator -

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Basic information

Entry
Database: EMDB / ID: EMD-8291
TitleEM map of the Elp123 subcomplex of yeast Elongator
Map dataElp123 subcomplex of yeast Elongator
Sample
  • Complex: Elp123 subcomplex of yeast Elongator
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 24.1 Å
AuthorsSetiaputra D / Cheng DTH / Hansen JM / Yip CK
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
DepositionJul 16, 2016-
Header (metadata) releaseJul 20, 2016-
Map releaseDec 7, 2016-
UpdateFeb 14, 2018-
Current statusFeb 14, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.109
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.109
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8291.png

Downloads & links

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Map

FileDownload / File: emd_8291.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationElp123 subcomplex of yeast Elongator
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7 Å/pix.
x 100 pix.
= 700. Å		7 Å/pix.
x 100 pix.
= 700. Å		7 Å/pix.
x 100 pix.
= 700. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.109 / Movie #1: 0.109
Minimum - Maximum-0.19143131 - 0.5995339
Average (Standard dev.)0.0004064846 (±0.016235426)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 700.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z777
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z700.000700.000700.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.1910.6000.000

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Supplemental data

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Sample components

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Entire : Elp123 subcomplex of yeast Elongator

EntireName: Elp123 subcomplex of yeast Elongator
Components
  • Complex: Elp123 subcomplex of yeast Elongator

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Supramolecule #1: Elp123 subcomplex of yeast Elongator

SupramoleculeName: Elp123 subcomplex of yeast Elongator / type: complex / ID: 1 / Parent: 0 / Details: Endogenously purified yeast Elp123 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
300.0 mMNaClsodium chloride
1.0 mMC10H16N2O8EDTA
10.0 %C3H8O3Glycerol

Details: The final purification buffer was used for size exclusion chromatography of the complex.
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 - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
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: 106 / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 49000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 28121
Details: 200 particles were manually picked to generate 5 class averages. These were used as templates for autopicking using the RELION software.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 3)CTFFIND3, launched from the SPIDER interface, was used to determine CTF parameters.
SPIDER (ver. 22.10)SPIDER was used to generate and apply the phase flip correction images to the raw micrographs.

Details: CTF parameters were calculated on entire micrographs prior to any data processing using CTFFIND within SPIDER. Phase-flip correction was done in SPIDER.
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 81 / Random conical tilt - Tilt angle: 65 degrees
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.1 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: 16362
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 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 3D classificationNumber classes: 4 / Avg.num./class: 4091 / 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.
FSC plot (resolution estimation)

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