Natural Sciences and Engineering Research Council (NSERC, Canada)
RGPIN-2018-03951
Canada
Citation
Journal: 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.
Model: EMS Lacey Carbon / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen 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
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Details
PNCC Krios 4
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 17955 / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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