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- EMDB-0032: Structure of activated transcription complex Pol II-DSIF-PAF-SPT6... -

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Basic information

Entry
Database: EMDB / ID: EMD-0032
TitleStructure of activated transcription complex Pol II-DSIF-PAF-SPT6, KOW1 selected particles (Map C)
Map dataGlobal refinement of KOW1 selected EC* particles.
Sample
  • Complex: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)
    • Complex: RNA Polymerase II
    • Complex: associated proteins
    • Complex: Nucleic acids
Biological speciesSus scrofa (pig) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsVos SM / Farnung L / Boehning M / Linden A / Wigge C / Urlaub H / Cramer P
CitationJournal: Nature / Year: 2018
Title: Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.
Authors: Seychelle M Vos / Lucas Farnung / Marc Boehning / Christoph Wigge / Andreas Linden / Henning Urlaub / Patrick Cramer /
Abstract: Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here ...Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
History
DepositionMay 26, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseSep 5, 2018-
UpdateSep 12, 2018-
Current statusSep 12, 2018Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0032.png

Downloads & links

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Map

FileDownload / File: emd_0032.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobal refinement of KOW1 selected EC* particles.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 360 pix.
= 377.64 Å		1.05 Å/pix.
x 360 pix.
= 377.64 Å		1.05 Å/pix.
x 360 pix.
= 377.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.049 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00743
Minimum - Maximum-0.016864154 - 0.049601495
Average (Standard dev.)0.00019487434 (±0.0020386248)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_0032_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Postprocessed map of global refinement for KOW1 selected...

Fileemd_0032_additional.map
AnnotationPostprocessed map of global refinement for KOW1 selected particles with an applied B factor of -90.57(Map C).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of global refinement for KOW1 selected particles (Map C).

Fileemd_0032_half_map_1.map
AnnotationHalf map 2 of global refinement for KOW1 selected particles (Map C).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of global refinement for KOW1 selected particles (Map C).

Fileemd_0032_half_map_2.map
AnnotationHalf map 1 of global refinement for KOW1 selected particles (Map C).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)

EntireName: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)
Components
  • Complex: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)
    • Complex: RNA Polymerase II
    • Complex: associated proteins
    • Complex: Nucleic acids

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Supramolecule #1: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)

SupramoleculeName: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#21, #23
Molecular weightTheoretical: 1.257 MDa

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Supramolecule #2: RNA Polymerase II

SupramoleculeName: RNA Polymerase II / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#9, #11, #10, #12
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: associated proteins

SupramoleculeName: associated proteins / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #13, #16, #18, #23, #19-#22
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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Supramolecule #4: Nucleic acids

SupramoleculeName: Nucleic acids / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #14-#15, #17
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 10s wait prior to blotting, blotting 8.5s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 69279
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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