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- EMDB-2828: Structure of Mot1 in complex with TBP, NC2 and DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-2828
TitleStructure of Mot1 in complex with TBP, NC2 and DNA
Map dataNegative Stain reconstruction of the Mot1 TBP NC2 complex
Sample
  • Sample: Mot1 in complex with TBP, NC2a, NC2b and DNA
  • Protein or peptide: Mot1
KeywordsSwi/Snf2 / chromatin remodelling / TBP / ATPase
Biological speciesEncephalitozoon cuniculi (fungus)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsButryn A / Schuller JM / Stoehr G / Runge-Wollmann P / Foerster F / Auble DT / Hopfner K-P
CitationJournal: Elife / Year: 2015
Title: Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1.
Authors: Agata Butryn / Jan M Schuller / Gabriele Stoehr / Petra Runge-Wollmann / Friedrich Förster / David T Auble / Karl-Peter Hopfner /
Abstract: Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent ...Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.
History
DepositionNov 26, 2014-
Header (metadata) releaseJan 21, 2015-
Map releaseAug 26, 2015-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2828.tif

image-2828.png

Downloads & links

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Map

FileDownload / File: emd_2828.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative Stain reconstruction of the Mot1 TBP NC2 complex
Voxel sizeX=Y=Z: 1.61 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.04712194 - 0.15408267
Average (Standard dev.)0.00092478 (±0.01138391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 241.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.611.611.61
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z241.500241.500241.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0470.1540.001

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Supplemental data

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Sample components

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Entire : Mot1 in complex with TBP, NC2a, NC2b and DNA

EntireName: Mot1 in complex with TBP, NC2a, NC2b and DNA
Components
  • Sample: Mot1 in complex with TBP, NC2a, NC2b and DNA
  • Protein or peptide: Mot1

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Supramolecule #1000: Mot1 in complex with TBP, NC2a, NC2b and DNA

SupramoleculeName: Mot1 in complex with TBP, NC2a, NC2b and DNA / type: sample / ID: 1000
Details: only the C-terminal part of Mot1 was used in the construct
Number unique components: 5

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Macromolecule #1: Mot1

MacromoleculeName: Mot1 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Encephalitozoon cuniculi (fungus)
Recombinant expressionRecombinant cell: Sf9

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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

Concentration0.05 mg/mL
BufferpH: 6.5 / Details: 20 mM MES pH, 60 mM KCl, 5 mM MgCl2 and 2 mM DTT
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 20 seconds.
GridDetails: 200 mesh carbon support grid for negative stain
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 62000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJan 28, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 144 / Average electron dose: 25 e/Å2

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Image processing

CTF correctionDetails: Micrograph level
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: XMIPP / Number images used: 8192
DetailsThe particles were manually selected using e2boxer

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