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- EMDB-10481: Structure of complete, activated transcription complex Pol II-DSI... -

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Basic information

Entry
Database: EMDB / ID: EMD-10481
TitleStructure of complete, activated transcription complex Pol II-DSIF-PAF-SPT6 uncovers allosteric elongation activation by RTF1 (Map 2)
Map dataPostprocessed map with an applied B factor of -20
Sample
  • Complex: Complete EC*
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsVos SM / Cramer P / Farnung L
Funding support Germany, 4 items
OrganizationGrant numberCountry
European Research Council693023 Germany
German Research FoundationSFB860 Germany
German Research FoundationSFB860 Germany
Volkswagen Foundation Germany
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Structure of complete Pol II-DSIF-PAF-SPT6 transcription complex reveals RTF1 allosteric activation.
Authors: Seychelle M Vos / Lucas Farnung / Andreas Linden / Henning Urlaub / Patrick Cramer /
Abstract: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation ...Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.
History
DepositionNov 11, 2019-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10481.png

Downloads & links

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Map

FileDownload / File: emd_10481.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map with an applied B factor of -20
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: 0.0118 / Movie #1: 0.014
Minimum - Maximum-0.018671148 - 0.06299646
Average (Standard dev.)0.00031911727 (±0.0029787312)
SymmetrySpace group: 1
Details

EMDB XML:

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

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0491.0491.049
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z377.640377.640377.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0190.0630.000

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Supplemental data

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Mask #1

Fileemd_10481_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_10481_msk_2.map
Projections & Slices
AxesZYX

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Histogram

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Half map: half map 1 of refined particles

Fileemd_10481_half_map_1.map
Annotationhalf map 1 of refined particles
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 of refined particles

Fileemd_10481_half_map_2.map
Annotationhalf map 2 of refined particles
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Sample components

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Entire : Complete EC*

EntireName: Complete EC*
Components
  • Complex: Complete EC*

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Supramolecule #1: Complete EC*

SupramoleculeName: Complete EC* / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#24
Molecular weightTheoretical: 1.34 MDa

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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
Component:
ConcentrationFormulaName
100.0 mMNaCl
20.0 mMNa-HEPES
1.0 mMDTT
20.0 mMTRIS-HCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 2 microliters applied to both sides of grid. Sample incubated on grid for 10s prior to blotting. Blotting for 8.5s..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 13679 / 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

Particle selectionNumber selected: 611983
CTF correctionSoftware - Name: Warp
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 64977
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6gmh

,

4l1u

,

6afo

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 115

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