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- EMDB-2053: Electron Microscopy of THO complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2053
TitleElectron Microscopy of THO complex
Map dataReconstruction of yeast THO complex composed of Tho2, Hpr1, Mft1, Thp2, and Tex1 subunits
Sample
  • Sample: Yeast five-subunit THO complex
  • Protein or peptide: Tho2
  • Protein or peptide: Hpr1
  • Protein or peptide: Mft1
  • Protein or peptide: Thp2
  • Protein or peptide: Tex1
KeywordsmRNA export / heterotetramer / mRNP quality control / croissant-like structure / beta-propeller / unfolded regions
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsPena A / Gewartowski K / Mroczek S / Cuellar J / Szykowska A / Prokop A / Czarnocki-Cieciura M / Piwowarski J / Tous C / Aguilera A ...Pena A / Gewartowski K / Mroczek S / Cuellar J / Szykowska A / Prokop A / Czarnocki-Cieciura M / Piwowarski J / Tous C / Aguilera A / Carrascosa JL / Valpuesta JM / Dziembowski A
CitationJournal: EMBO J / Year: 2012
Title: Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor.
Authors: Alvaro Peña / Kamil Gewartowski / Seweryn Mroczek / Jorge Cuéllar / Aleksandra Szykowska / Andrzej Prokop / Mariusz Czarnocki-Cieciura / Jan Piwowarski / Cristina Tous / Andrés Aguilera / ...Authors: Alvaro Peña / Kamil Gewartowski / Seweryn Mroczek / Jorge Cuéllar / Aleksandra Szykowska / Andrzej Prokop / Mariusz Czarnocki-Cieciura / Jan Piwowarski / Cristina Tous / Andrés Aguilera / José L Carrascosa / José María Valpuesta / Andrzej Dziembowski /
Abstract: The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In ...The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a β-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.
History
DepositionMar 15, 2012-
Header (metadata) releaseMar 22, 2012-
Map releaseMar 22, 2012-
UpdateMar 22, 2012-
Current statusMar 22, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.52
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.52
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2053.jpeg

Downloads & links

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Map

FileDownload / File: emd_2053.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast THO complex composed of Tho2, Hpr1, Mft1, Thp2, and Tex1 subunits
Voxel sizeX=Y=Z: 2.33 Å
Density
Contour LevelBy AUTHOR: 2.52 / Movie #1: 2.52
Minimum - Maximum-0.57409996 - 12.31480503
Average (Standard dev.)1.08922005 (±0.72266853)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-70-70-70
Dimensions140140140
Spacing140140140
CellA=B=C: 326.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.332.332.33
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z326.200326.200326.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-70-70-70
NC/NR/NS140140140
D min/max/mean-0.57412.3151.089

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Supplemental data

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Sample components

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Entire : Yeast five-subunit THO complex

EntireName: Yeast five-subunit THO complex
Components
  • Sample: Yeast five-subunit THO complex
  • Protein or peptide: Tho2
  • Protein or peptide: Hpr1
  • Protein or peptide: Mft1
  • Protein or peptide: Thp2
  • Protein or peptide: Tex1

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Supramolecule #1000: Yeast five-subunit THO complex

SupramoleculeName: Yeast five-subunit THO complex / type: sample / ID: 1000
Oligomeric state: One heteropentamer composed of Tho2, Hpr1, Mft1, Thp2 an Tex1 subunits
Number unique components: 5
Molecular weightExperimental: 395 MDa / Theoretical: 395 MDa / Method: Gel Filtration and Mass Spectrometry

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Macromolecule #1: Tho2

MacromoleculeName: Tho2 / type: protein_or_peptide / ID: 1 / Name.synonym: Rlr1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 180 MDa / Theoretical: 180 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #2: Hpr1

MacromoleculeName: Hpr1 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 90 MDa / Theoretical: 90 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #3: Mft1

MacromoleculeName: Mft1 / type: protein_or_peptide / ID: 3 / Name.synonym: MFT52 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 45 MDa / Theoretical: 45 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #4: Thp2

MacromoleculeName: Thp2 / type: protein_or_peptide / ID: 4 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 30 MDa / Theoretical: 30 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #5: Tex1

MacromoleculeName: Tex1 / type: protein_or_peptide / ID: 5 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 50 MDa / Theoretical: 50 MDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration0.35 mg/mL
BufferpH: 8 / Details: 10 mM Tris_HCl, 450 mM NaCl
StainingType: NEGATIVE
Details: Samples were applied onto carbon-coated copper grids and stained with 2% uranyl acetate for 1 min
GridDetails: 200 mesh carbon-coated copper grids, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1200EXII
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 5.6 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateJun 15, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Number real images: 250 / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: N. Grigorieffs CTFFIND
Final two d classificationNumber classes: 15
Final angle assignmentDetails: SPIDER protocol
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN,Spider,XMIPP / Number images used: 14115
DetailsIndividual particles were manually selected using XMIPP software package.

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