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- EMDB-3629: RNA polymerase II-Paf1C-TFIIS-C -

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Basic information

Entry
Database: EMDB / ID: EMD-3629
TitleRNA polymerase II-Paf1C-TFIIS-C
Map dataNon-B factor sharpened, locally filtered map
Sample
  • Complex: RNA polymerase II-Paf1C-TFIIS-C
    • Complex: Paf1C
    • Complex: RNA polymerase II
    • Complex: TFIIS
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae S288c (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsXu Y / Cramer P
Funding support Germany, 4 items
OrganizationGrant numberCountry
German Research FoundationSFB860 Germany
European Research CouncilNo 693023 Germany
German Research FoundationSPP1935 Germany
Volkswagen Foundation Germany
CitationJournal: Nat Commun / Year: 2017
Title: Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex.
Authors: Youwei Xu / Carrie Bernecky / Chung-Tien Lee / Kerstin C Maier / Björn Schwalb / Dimitry Tegunov / Jürgen M Plitzko / Henning Urlaub / Patrick Cramer /
Abstract: The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and ...The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit. The Paf1-Leo1 heterodimer and Cdc73 form opposite ends of Paf1C, whereas Ctr9 bridges between them. Consistent with the structural observations, the initiation factor TFIIF impairs Paf1C binding to Pol II, whereas the elongation factor TFIIS enhances it. We further show that Paf1C is globally required for normal mRNA transcription in yeast. These results provide a three-dimensional framework for further analysis of Paf1C function in transcription through chromatin.
History
DepositionMar 9, 2017-
Header (metadata) releaseJun 7, 2017-
Map releaseJun 7, 2017-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3629.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNon-B factor sharpened, locally filtered map
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.0133 / Movie #1: 0.017
Minimum - Maximum-0.029984547 - 0.08558542
Average (Standard dev.)-0.00007719614 (±0.0043475367)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0300.086-0.000

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Supplemental data

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Mask #1

Fileemd_3629_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1

Fileemd_3629_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2

Fileemd_3629_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : RNA polymerase II-Paf1C-TFIIS-C

EntireName: RNA polymerase II-Paf1C-TFIIS-C
Components
  • Complex: RNA polymerase II-Paf1C-TFIIS-C
    • Complex: Paf1C
    • Complex: RNA polymerase II
    • Complex: TFIIS

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Supramolecule #1: RNA polymerase II-Paf1C-TFIIS-C

SupramoleculeName: RNA polymerase II-Paf1C-TFIIS-C / type: complex / ID: 1 / Parent: 0 / Details: Elongation complex
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 35 KDa

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Supramolecule #2: Paf1C

SupramoleculeName: Paf1C / type: complex / ID: 2 / Parent: 1 / Details: Ctr9 C-terminal truncation
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 CodonPlus(DE3)RIL

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Supramolecule #3: RNA polymerase II

SupramoleculeName: RNA polymerase II / type: complex / ID: 3 / Parent: 1 / Details: 12 subunits
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464

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Supramolecule #4: TFIIS

SupramoleculeName: TFIIS / type: complex / ID: 4 / Parent: 1 / Details: TFIIS (D290A, E291A) inactive mutant
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 CodonPlus(DE3)RIL

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMNaClSodium chloridesodium chloride
2.0 mMDTTdithiothreitol
2.0 mMMgCl2magnesium chloride
0.01 mMZnCl2zinc chloride
GridModel: Quantifoil Cu R3.5/1 and Cu R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 4 micro-L of sample was placed onto Quantifoil Cu R3.5/1 and Cu R2/1 glow-discharged 200 mesh holey carbon grids, which were then blotted for 8.5 s with blot force 13 to remove the excess ...Details: 4 micro-L of sample was placed onto Quantifoil Cu R3.5/1 and Cu R2/1 glow-discharged 200 mesh holey carbon grids, which were then blotted for 8.5 s with blot force 13 to remove the excess solution before they were flash-frozen in liquid ethane..
DetailsGradient fixation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000
Specialist opticsEnergy filter - Name: GIF Quantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: SUPER-RESOLUTION / #0 - Number grids imaged: 1 / #0 - Number real images: 595 / #0 - Average exposure time: 0.4 sec. / #0 - Average electron dose: 1.22 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: SUPER-RESOLUTION / #1 - Number grids imaged: 1 / #1 - Number real images: 2146 / #1 - Average exposure time: 0.4 sec. / #1 - Average electron dose: 0.93 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 947597 / Details: total number of particles from two datasets
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

Details: bovine Pol II elongation complex
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: RELION 1.4
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4) / Details: RELION 1.4
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 43025
Image recording ID1
FSC plot (resolution estimation)

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