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- EMDB-26804: Cryo-EM structure of the SAGA core module -

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Basic information

Entry
Database: EMDB / ID: EMD-26804
TitleCryo-EM structure of the SAGA core module
Map data
Sample
  • Complex: SAGA
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsVasyliuk D / Yip CK
Funding support Canada, 1 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-03951 Canada
CitationJournal: Sci Rep / Year: 2022
Title: Conformational landscape of the yeast SAGA complex as revealed by cryo-EM.
Authors: Diana Vasyliuk / Joeseph Felt / Ellen D Zhong / Bonnie Berger / Joseph H Davis / Calvin K Yip /
Abstract: Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by ...Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.
History
DepositionApr 28, 2022-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26804.png

Downloads & links

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Map

FileDownload / File: emd_26804.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.332 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-1.8905694 - 5.973297
Average (Standard dev.)-0.0068778843 (±0.076246984)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 599.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26804_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26804_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SAGA

EntireName: SAGA
Components
  • Complex: SAGA

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Supramolecule #1: SAGA

SupramoleculeName: SAGA / type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303a

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationFormulaName
40.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
0.01 %C58H114O26Tween-20
GridModel: EMS Lacey Carbon / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2s blot time, -5 blot force..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsPNCC Krios 4
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 17955 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1175683
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2)
Startup modelType of model: NONE / Details: CryoSPARC ab initio reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 542311
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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