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- PDB-5g5p: Structure of the Saccharomyces cerevisiae TREX-2 complex -

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Basic information

Entry
Database: PDB / ID: 5g5p
TitleStructure of the Saccharomyces cerevisiae TREX-2 complex
Components
  • (NUCLEAR MRNA EXPORT PROTEIN SAC3) x 2
  • 26S PROTEASOME COMPLEX SUBUNIT SEM1Proteasome
  • NUCLEAR MRNA EXPORT PROTEIN THP1
KeywordsTRANSPORT PROTEIN / MRNA / MRNA 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 CEREVISIAE (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsAibara, S. / Bai, X.C. / 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, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 23, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2016Group: Data collection
Revision 1.2Dec 14, 2016Group: Other
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: NUCLEAR MRNA EXPORT PROTEIN SAC3
B: NUCLEAR MRNA EXPORT PROTEIN THP1
C: 26S PROTEASOME COMPLEX SUBUNIT SEM1
D: NUCLEAR MRNA EXPORT PROTEIN SAC3
E: NUCLEAR MRNA EXPORT PROTEIN SAC3
F: NUCLEAR MRNA EXPORT PROTEIN SAC3


Theoretical massNumber of molelcules
Total (without water)361,9276
Polymers361,9276
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein NUCLEAR MRNA EXPORT PROTEIN SAC3 / LEUCINE PERMEASE TRANSCRIPTIONAL REGULATOR / SAC3


Mass: 93213.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PACEBACRZ / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P46674
#2: Protein NUCLEAR MRNA EXPORT PROTEIN THP1 / BUD SITE SELECTION PROTEIN 29 / THP1


Mass: 52734.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PACEBACRZ / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: Q08231
#3: Protein 26S PROTEASOME COMPLEX SUBUNIT SEM1 / Proteasome / SEM1


Mass: 10397.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PACEBACRZ / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: O94742
#4: Protein NUCLEAR MRNA EXPORT PROTEIN SAC3 / LEUCINE PERMEASE TRANSCRIPTIONAL REGULATOR / SAC3


Mass: 68527.133 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: CHAINS D, F AND E ARE MODELLED AS POLYALA
Source: (gene. exp.) SACCHAROMYCES CEREVISIAE (brewer's yeast)
Plasmid: PACEBACRZ / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm)
Sequence detailsCHAINS D, E AND F ARE BUILT AS POLYALA DUE TO RESOLUTION DOES NOT ALLOW SIDE CHAIN BUILDING

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Sac3 in complex with Thp1 and Sem1 / Type: COMPLEX / Oligomeric details: One heterotrimer / Source: MULTIPLE SOURCES
Molecular weightValue: 0.19 MDa / Experimental value: NO
Buffer solutionName: 20 MM HEPES, 300 MM NACL, 5 MM DTT / pH: 8 / Details: 20 mM HEPES, 300 mM NaCl, 5 mM DTT
SpecimenConc.: 0.015 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: OTHER
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 85 K / Humidity: 100 % / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Nov 3, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 4500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderTemperature: 85 K / Temperature (max): 90 K / Temperature (min): 80 K
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
Image scansNum. digital images: 496

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Processing

EM softwareName: RELION / Category: 3D reconstruction
Particle selectionDetails: The particles were processed using Relion
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81559 / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
RefinementHighest resolution: 5.3 Å
Refinement stepCycle: LAST / Highest resolution: 5.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6830 0 0 0 6830

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