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- EMDB-0040: Structure of paused transcription complex Pol II-DSIF-NELF (Map 3) -

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Basic information

Entry
Database: EMDB / ID: EMD-0040
TitleStructure of paused transcription complex Pol II-DSIF-NELF (Map 3)
Map dataGlobally refined overall NELF-B selected particles PEC map (Map 3).
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: 3.4 Å
AuthorsVos SM / Farnung L / Urlaub H / Cramer P
CitationJournal: Nature / Year: 2018
Title: Structure of paused transcription complex Pol II-DSIF-NELF.
Authors: Seychelle M Vos / Lucas Farnung / Henning Urlaub / Patrick Cramer /
Abstract: Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor ...Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
History
DepositionMay 27, 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_0040.png

Downloads & links

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Map

FileDownload / File: emd_0040.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally refined overall NELF-B selected particles PEC map (Map 3).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 256 pix.
= 314.291 Å		1.23 Å/pix.
x 256 pix.
= 314.291 Å		1.23 Å/pix.
x 256 pix.
= 314.291 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2277 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0233
Minimum - Maximum-0.024119103 - 0.10176039
Average (Standard dev.)0.00069555914 (±0.0065150736)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 314.2912 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_0040_msk_1.map
Projections & Slices
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Mask #2

Fileemd_0040_msk_2.map
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Mask #3

Fileemd_0040_msk_3.map
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Additional map: Postprocessed/B-factor sharpened NELF-B selected particles PEC map (Map...

Fileemd_0040_additional_1.map
AnnotationPostprocessed/B-factor sharpened NELF-B selected particles PEC map (Map 3). Applied B-factor of -103.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Additional map: Focused refinement on NELF-ABC.

Fileemd_0040_additional_2.map
AnnotationFocused refinement on NELF-ABC.
Projections & Slices
AxesZYX

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Additional map: Half map 1 for focused refined PEC NELF-B selected particles (Map 3).

Fileemd_0040_additional_3.map
AnnotationHalf map 1 for focused refined PEC NELF-B selected particles (Map 3).
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Additional map: Half map 2 for focused refined PEC NELF-B selected particles (Map 3).

Fileemd_0040_additional_4.map
AnnotationHalf map 2 for focused refined PEC NELF-B selected particles (Map 3).
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Postprocessed/B-factor sharpened NELF-B selected particles focused refined PEC...

Fileemd_0040_additional_5.map
AnnotationPostprocessed/B-factor sharpened NELF-B selected particles focused refined PEC map (Map 3).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 for globally refined PEC NELF-B...

Fileemd_0040_half_map_1.map
AnnotationHalf map 1 for globally refined PEC NELF-B selected particles (Map 3).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 for globally refined PEC NELF-B...

Fileemd_0040_half_map_2.map
AnnotationHalf map 2 for globally refined PEC NELF-B selected particles (Map 3).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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: 927.1 KDa

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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

Concentration0.2 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number real images: 11740 / Average exposure time: 9.0 sec. / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal magnification: 165000
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

Startup modelType of model: EMDB MAP
EMDB ID:

3819

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 40960
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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