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- EMDB-0041: Structure of paused transcription complex Pol II-DSIF-NELF, (Map 4) -

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Basic information

Entry
Database: EMDB / ID: EMD-0041
TitleStructure of paused transcription complex Pol II-DSIF-NELF, (Map 4)
Map dataGlobally refined upstream DNA, NGN, KOW1 selected particles (Map 4).
Sample
  • Complex: RNA Polymerase II-DSIF-PAF-SPT6 elongation complex (EC*)
    • Complex: RNA Polymerase II
    • Complex: associated proteins
    • Complex: Nucleic acidsNucleic acid
Biological speciesSus scrofa (pig) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 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

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0055
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0055
  • 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_0041.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobally refined upstream DNA, NGN, KOW1 selected particles (Map 4).
Voxel sizeX=Y=Z: 1.2277 Å
Density
Contour LevelBy AUTHOR: 0.0055 / Movie #1: 0.0055
Minimum - Maximum-0.008213097 - 0.023484569
Average (Standard dev.)0.000117879674 (±0.0014477392)
SymmetrySpace group: 1
Details

EMDB XML:

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

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.227699218751.227699218751.22769921875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z314.291314.291314.291
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0080.0230.000

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Supplemental data

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Mask #1

Fileemd_0041_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Postprocessed/B-factor sharpened upstream DNA, NGN, KOW1 selected particles...

Fileemd_0041_additional.map
AnnotationPostprocessed/B-factor sharpened upstream DNA, NGN, KOW1 selected particles (Map 4). Applied B-factor of -71.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 1 for globally refined upstream DNA,...

Fileemd_0041_half_map_1.map
AnnotationHalf map 1 for globally refined upstream DNA, NGN, KOW1 selected particles (Map 4).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2 for globally refined upstream DNA,...

Fileemd_0041_half_map_2.map
AnnotationHalf map 2 for globally refined upstream DNA, NGN, KOW1 selected particles (Map 4).
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 acidsNucleic acid

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 29197
FSC plot (resolution estimation)

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