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- EMDB-23038: CryoEM map of SAGA-like SLIK transcription coactivator -

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Basic information

Entry
Database: EMDB / ID: EMD-23038
TitleCryoEM map of SAGA-like SLIK transcription coactivator
Map data
Sample
  • Complex: Transcription coactivator
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.5 Å
AuthorsAdamus K / Reboul C / Elmlund H / Boudes M / Elmlund D
Funding support Australia, 2 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP170101850 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1125909 Australia
CitationJournal: J Biol Chem / Year: 2021
Title: SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent.
Authors: Klaudia Adamus / Cyril Reboul / Jarrod Voss / Cheng Huang / Ralf B Schittenhelm / Sarah N Le / Andrew M Ellisdon / Hans Elmlund / Marion Boudes / Dominika Elmlund /
Abstract: The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8, one ...The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear. To investigate, we assayed the chromatin modifying functions of SAGA and SLIK, revealing identical kinetics on minimal substrates in vitro. We also examined the binding of SAGA and SLIK to TBP and concluded that interestingly, both protein complexes have similar affinity for TBP. Additionally, despite the loss of Spt8 and C-terminus of Spt7 in SLIK, TBP prebound to SLIK is not released in the presence of TATA-box DNA, just like TBP prebound to SAGA. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (K = 10-17 nM), and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of the Spt8 subunit in SLIK neither drive any major conformational differences in its structure compared with SAGA, nor significantly affect HAT, DUB, or DNA-binding activities in vitro.
History
DepositionNov 25, 2020-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateJul 14, 2021-
Current statusJul 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0082
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0082
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23038.map.gz / Format: CCP4 / Size: 184 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 364 pix.
= 495.04 Å
1.36 Å/pix.
x 364 pix.
= 495.04 Å
1.36 Å/pix.
x 364 pix.
= 495.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.0082 / Movie #1: 0.0082
Minimum - Maximum-0.009191512 - 0.028513148
Average (Standard dev.)0.0002424705 (±0.0018257939)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions364364364
Spacing364364364
CellA=B=C: 495.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z364364364
origin x/y/z0.0000.0000.000
length x/y/z495.040495.040495.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS364364364
D min/max/mean-0.0090.0290.000

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Supplemental data

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Sample components

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Entire : Transcription coactivator

EntireName: Transcription coactivator
Components
  • Complex: Transcription coactivator

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Supramolecule #1: Transcription coactivator

SupramoleculeName: Transcription coactivator / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: 6.8
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Details: 10 mAmps
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 11.2 sec. / 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
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: 323000
CTF correctionSoftware - Name: SIMPLE (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 16623
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SIMPLE (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 14700 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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