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- EMDB-3440: Structure of the Saccharomyces cerevisiae TREX-2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3440
TitleStructure of the Saccharomyces cerevisiae TREX-2 complex
Map dataPostprocessed sharpened map, masked
Sample
  • Sample: Sac3 in complex with Thp1 and Sem1
  • Protein or peptide: Sac3
  • Protein or peptide: Thp1
  • Protein or peptide: Sem1
KeywordsmRNA export
Function / homology
Function and homology information


actin filament-based process / cellular bud site selection / SAGA complex localization to transcription regulatory region / transcription export complex 2 / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / nuclear mRNA surveillance / maintenance of DNA trinucleotide repeats / filamentous growth / mRNA 3'-end processing ...actin filament-based process / cellular bud site selection / SAGA complex localization to transcription regulatory region / transcription export complex 2 / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / nuclear mRNA surveillance / maintenance of DNA trinucleotide repeats / filamentous growth / mRNA 3'-end processing / proteasome regulatory particle, lid subcomplex / poly(A)+ mRNA export from nucleus / proteasome storage granule / transcription-coupled nucleotide-excision repair / proteasome assembly / mRNA export from nucleus / protein folding chaperone / protein export from nucleus / proteasome complex / transcription elongation by RNA polymerase II / double-strand break repair via homologous recombination / ribosomal small subunit biogenesis / mitotic cell cycle / nuclear envelope / ubiquitin-dependent protein catabolic process / double-stranded DNA binding / proteasome-mediated ubiquitin-dependent protein catabolic process / molecular adaptor activity / regulation of cell cycle / protein-containing complex binding / positive regulation of transcription by RNA polymerase II / RNA binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Csn12 family / Nuclear mRNA export protein Sac3 / SAC3 helical domain / Leucine permease transcriptional regulator helical domain / SAC3/GANP/THP3 / SAC3/GANP/THP3, conserved domain / SAC3/GANP family / DSS1/SEM1 / DSS1/SEM1 family / DSS1_SEM1 ...Csn12 family / Nuclear mRNA export protein Sac3 / SAC3 helical domain / Leucine permease transcriptional regulator helical domain / SAC3/GANP/THP3 / SAC3/GANP/THP3, conserved domain / SAC3/GANP family / DSS1/SEM1 / DSS1/SEM1 family / DSS1_SEM1 / PCI/PINT associated module / PCI domain / Proteasome component (PCI) domain / PCI domain profile.
Similarity search - Domain/homology
26S proteasome complex subunit SEM1 / Nuclear mRNA export protein SAC3 / Nuclear mRNA export protein THP1
Similarity search - Component
Biological speciesSaccharomyces cerevisae
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsAibara S / Bai XC / Stewart M
CitationJournal: J Struct Biol / Year: 2016
Title: The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region.
Authors: Shintaro Aibara / Xiao-Chen Bai / Murray Stewart /
Abstract: Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export- ...Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Sem1 bind to the Sac3(M) region (residues ∼100-551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3(CID) region (residues ∼710-805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3(M) region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3(CID):Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100kDa, a 5.3Å resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative α-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9Å resolution structure obtained by X-ray crystallography.
SUMMARY STATEMENT: We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than ...SUMMARY STATEMENT: We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation.
History
DepositionMay 26, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateNov 23, 2016-
Current statusNov 23, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5g5p
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-3440.png

Downloads & links

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Map

FileDownload / File: emd_3440.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed sharpened map, masked
Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.035
Minimum - Maximum-0.0884256 - 0.24476513
Average (Standard dev.)0.00069024 (±0.00647952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 200.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z200.200200.200200.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0880.2450.001

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Supplemental data

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Segmentation: Automask used for postprocessing

AnnotationAutomask used for postprocessing
Fileemd_3440_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: run1 half1 class001 unfil.map

Filerun1_half1_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: run1 half2 class001 unfil 1.map

Filerun1_half2_class001_unfil_1.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 : Sac3 in complex with Thp1 and Sem1

EntireName: Sac3 in complex with Thp1 and Sem1
Components
  • Sample: Sac3 in complex with Thp1 and Sem1
  • Protein or peptide: Sac3
  • Protein or peptide: Thp1
  • Protein or peptide: Sem1

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Supramolecule #1000: Sac3 in complex with Thp1 and Sem1

SupramoleculeName: Sac3 in complex with Thp1 and Sem1 / type: sample / ID: 1000 / Details: Sample is monodisperse / Oligomeric state: One heterotrimer / Number unique components: 3
Molecular weightExperimental: 190 KDa / Theoretical: 190 KDa / Method: SAXS

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Macromolecule #1: Sac3

MacromoleculeName: Sac3 / type: protein_or_peptide / ID: 1 / Details: Construct had residues 1-805 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisae / synonym: Yeast
Molecular weightTheoretical: 149.5916 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9 / Recombinant plasmid: pAceBacRZ
SequenceUniProtKB: Nuclear mRNA export protein SAC3

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Macromolecule #2: Thp1

MacromoleculeName: Thp1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisae / synonym: Yeast
Molecular weightTheoretical: 52.6857 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9 / Recombinant plasmid: pAceBacRZ
SequenceUniProtKB: Nuclear mRNA export protein THP1

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Macromolecule #3: Sem1

MacromoleculeName: Sem1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisae / synonym: Yeast
Molecular weightTheoretical: 10.372 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: sf9 / Recombinant plasmid: pAceBacRZ
SequenceUniProtKB: 26S proteasome complex subunit SEM1

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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

Concentration0.015 mg/mL
BufferpH: 8 / Details: 20 mM HEPES, 300 mM NaCl, 5 mM DTT
GridDetails: Graphene oxide treated 300 mesh gold quantifoil R2/2 grids.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 7 seconds before plunging from one side

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 35714 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: Gatan Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
DateNov 3, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 40 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 81559
DetailsThe particles were processed using relion.

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