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- EMDB-0037: Structure of activated transcription complex Pol II-DSIF-PAF-SPT6... -

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Entry
Database: EMDB / ID: EMD-0037
TitleStructure of activated transcription complex Pol II-DSIF-PAF-SPT6, CTR9 selected particles (Map H)
Map dataGlobal refinement of CTR9 selected EC* particles (Map H).
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: 3.34 Å
AuthorsVos SM / Farnung L / Boehning M / Linden A / Wigge C / Urlaub H / Cramer P
Funding support Germany, 4 items
OrganizationGrant numberCountry
European Research Council693023 Germany
Volkswagen Foundation Germany
European Molecular Biology OrganizationALTF-725-2014 Germany
German Research FoundationDFG SFB860 Germany
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-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0037.png

Downloads & links

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Map

FileDownload / File: emd_0037.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobal refinement of CTR9 selected EC* particles (Map H).
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.049 Å
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Contour LevelBy AUTHOR: 0.00667
Minimum - Maximum-0.018458545 - 0.053401053
Average (Standard dev.)0.0001379251 (±0.0021176182)
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_0037_msk_1.map
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Mask #2

Fileemd_0037_msk_2.map
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Mask #3

Fileemd_0037_msk_3.map
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Mask #4

Fileemd_0037_msk_4.map
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Additional map: Focused refinement of CTR9 selected EC* particles on...

Fileemd_0037_additional_1.map
AnnotationFocused refinement of CTR9 selected EC* particles on CTR9 N-terminus (Map H).
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Additional map: Half map 1 of focus refinement for RPB...

Fileemd_0037_additional_10.map
AnnotationHalf map 1 of focus refinement for RPB 4-7 stalk from CTR9 selected particles (Map H).
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Additional map: Half map 2 of focus refinement for RPB...

Fileemd_0037_additional_11.map
AnnotationHalf map 2 of focus refinement for RPB 4-7 stalk from CTR9 selected particles (Map H).
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Additional map: Focused refinement of CTR9 selected EC* particles on...

Fileemd_0037_additional_12.map
AnnotationFocused refinement of CTR9 selected EC* particles on RPB4-7 stalk (Map H).
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Additional map: Focused refinement of CTR9 selected EC* particles on...

Fileemd_0037_additional_2.map
AnnotationFocused refinement of CTR9 selected EC* particles on CTR9 C-terminus/WDR61 (Map H).
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Additional map: Half map 1 of focus refinement for CTR9...

Fileemd_0037_additional_3.map
AnnotationHalf map 1 of focus refinement for CTR9 C-terminus/WDR61 from CTR9 selected particles (Map H).
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Additional map: Postprocessed map for focused refinement of CTR9 C-terminus/WDR61...

Fileemd_0037_additional_4.map
AnnotationPostprocessed map for focused refinement of CTR9 C-terminus/WDR61 from CTR9 selected EC* particles(Map H).
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Additional map: Half map 2 of focus refinement for CTR9...

Fileemd_0037_additional_5.map
AnnotationHalf map 2 of focus refinement for CTR9 C-terminus/WDR61 from CTR9 selected particles (Map H).
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Additional map: Postprocessed map for global refinement of CTR9 selected...

Fileemd_0037_additional_6.map
AnnotationPostprocessed map for global refinement of CTR9 selected EC* particles(Map H) with an applied B factor of -86.13.
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Additional map: Postprocessed map for focused refinement of RBP4-7 stalk...

Fileemd_0037_additional_7.map
AnnotationPostprocessed map for focused refinement of RBP4-7 stalk from CTR9 selected EC* particles(Map H).
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Additional map: Half map 1 of focus refinement for CTR9...

Fileemd_0037_additional_8.map
AnnotationHalf map 1 of focus refinement for CTR9 N-terminus from CTR9 selected particles (Map H).
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Additional map: Half map 2 of focus refinement for CTR9...

Fileemd_0037_additional_9.map
AnnotationHalf map 2 of focus refinement for CTR9 N-terminus from CTR9 selected particles (Map H).
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Half map: Half map 1 of global refinement of CTR9 selected particles (Map H).

Fileemd_0037_half_map_1.map
AnnotationHalf map 1 of global refinement of CTR9 selected particles (Map H).
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Half map: Half map 2 of global refinement of CTR9 selected particles (Map H).

Fileemd_0037_half_map_2.map
AnnotationHalf map 2 of global refinement of CTR9 selected particles (Map H).
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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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 107349
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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