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- EMDB-1883: Three-dimensional structure of the RNA polymerase II-Iwr1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-1883
TitleThree-dimensional structure of the RNA polymerase II-Iwr1 complex
Map dataThis is a map of the RNA Polymerase II-Iwr1 complex.
Sample
  • Sample: RNA polymerase II-Iwr1
  • Protein or peptide: DNA-directed RNA polymerasePolymerase
  • Protein or peptide: Import adaptor
Keywordstranscription / Pol II / nuclear import
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.9 Å
AuthorsCzeko E / Seizl M / Augsberger C / Mielke T / Cramer P
CitationJournal: Mol Cell / Year: 2011
Title: Iwr1 directs RNA polymerase II nuclear import.
Authors: Elmar Czeko / Martin Seizl / Christian Augsberger / Thorsten Mielke / Patrick Cramer /
Abstract: RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we ...RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitating or sensing complete Pol II assembly in the cytoplasm. Iwr1 then uses an N-terminal bipartite nuclear localization signal that is recognized by karyopherin α to direct Pol II nuclear import. In the nucleus, Iwr1 is displaced from Pol II by transcription initiation factors and nucleic acids, enabling its export and recycling. Iwr1 function is Pol II specific, transcription independent, and apparently conserved from yeast to human.
History
DepositionFeb 22, 2011-
Header (metadata) releaseMar 18, 2011-
Map releaseApr 21, 2011-
UpdateApr 21, 2011-
Current statusApr 21, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1883.jpg

Downloads & links

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Map

FileDownload / File: emd_1883.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of the RNA Polymerase II-Iwr1 complex.
Voxel sizeX=Y=Z: 3.71 Å
Density
Contour LevelBy AUTHOR: 0.0005 / Movie #1: 0.0005
Minimum - Maximum-0.0012936 - 0.00285934
Average (Standard dev.)0.0000121694 (±0.000265325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 259.7 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.713.713.71
M x/y/z707070
origin x/y/z-0.000-0.000-0.000
length x/y/z259.700259.700259.700
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-0.0010.0030.000

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Supplemental data

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Sample components

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Entire : RNA polymerase II-Iwr1

EntireName: RNA polymerase II-Iwr1
Components
  • Sample: RNA polymerase II-Iwr1
  • Protein or peptide: DNA-directed RNA polymerasePolymerase
  • Protein or peptide: Import adaptor

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Supramolecule #1000: RNA polymerase II-Iwr1

SupramoleculeName: RNA polymerase II-Iwr1 / type: sample / ID: 1000 / Oligomeric state: Monomeric / Number unique components: 2
Molecular weightTheoretical: 550 KDa

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Macromolecule #1: DNA-directed RNA polymerase

MacromoleculeName: DNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Name.synonym: Pol II / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Organelle: Nucleus
Molecular weightTheoretical: 510 KDa

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Macromolecule #2: Import adaptor

MacromoleculeName: Import adaptor / type: protein_or_peptide / ID: 2 / Name.synonym: Iwr1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21b

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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

Concentration0.1 mg/mL
BufferpH: 7.25
Details: 5 mM HEPES, 40 mM Ammonium sulfate, 10 microM Zinc chloride, 10 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 80 K / Instrument: OTHER / Details: Vitrification instrument: FEI Vitrobot / Method: Blot for 10 s before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 67000
Sample stageSpecimen holder: Multispecimen holder / Specimen holder model: OTHER
TemperatureAverage: 80 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Number real images: 35 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Defocus groups
Final angle assignmentDetails: SPIDER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 25206
DetailsParticles were selected using Signature and thereafter checked manually

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Atomic model buiding 1

Initial modelPDB ID:

1wcm

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Least-square

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