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- EMDB-10486: Structure of complete, activated transcription complex Pol II-DSI... -

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Basic information

Entry
Database: EMDB / ID: EMD-10486
TitleStructure of complete, activated transcription complex Pol II-DSIF-PAF-SPT6 uncovers allosteric elongation activation by RTF1 (Map 7)
Map dataPostprocessed map with an applied b factor of -50.
Sample
  • Complex: Complete EC*
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsVos SM / Farnung L / Cramer P
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 12, 2019-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0187
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0187
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10486.png

Downloads & links

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Map

FileDownload / File: emd_10486.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed map with an applied b factor of -50.
Voxel sizeX=Y=Z: 1.049 Å
Density
Contour LevelBy AUTHOR: 0.0187 / Movie #1: 0.0187
Minimum - Maximum-0.026129536 - 0.077146426
Average (Standard dev.)0.0003809813 (±0.003400596)
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-0.0260.0770.000

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Supplemental data

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Mask #1

Fileemd_10486_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_10486_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_10486_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_10486_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Source (natural)Organism: Homo sapiens (human)
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:
ConcentrationName
100.0 mMNaClSodium chloride
20.0 mMNa-HEPES
1.0 mMDTT
20.0 mMTRIS-HClTris
GridModel: UltrAuFoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / 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 grids imaged: 3 / Number real images: 13679 / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 611983
CTF correctionSoftware - Name: Warp
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 39445
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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