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- EMDB-3816: Structure of an RNA polymerase II-DSIF transcription elongation c... -

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Basic information

Entry
Database: EMDB / ID: EMD-3816
TitleStructure of an RNA polymerase II-DSIF transcription elongation complex, DSIF-EC2
Map dataNone
Sample
  • Complex: RNA polymerase II-DSIF elongation complex
    • Complex: RNA polymerase II
    • Complex: DSIF
    • Complex: DNA-RNA synthetic construct
Biological speciesBos taurus (domestic cattle) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsBernecky C / Plitzko JM / Cramer P
Funding support Germany, 3 items
OrganizationGrant numberCountry
German Research FoundationSFB860, SPP1935 Germany
Volkswagen Foundation Germany
European Research CouncilTRANSREGULON Germany
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Structure of a transcribing RNA polymerase II-DSIF complex reveals a multidentate DNA-RNA clamp.
Authors: Carrie Bernecky / Jürgen M Plitzko / Patrick Cramer /
Abstract: During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA ...During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II-DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.
History
DepositionJul 18, 2017-
Header (metadata) releaseAug 23, 2017-
Map releaseSep 13, 2017-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3816.png

Downloads & links

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Map

FileDownload / File: emd_3816.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å

Surface

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.23098513 - 0.36672285
Average (Standard dev.)-0.000011585955 (±0.010266359)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2310.367-0.000

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Supplemental data

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Mask #1

Fileemd_3816_msk_1.map
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Additional map: None

Fileemd_3816_additional_1.map
AnnotationNone
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Additional map: None

Fileemd_3816_additional_2.map
AnnotationNone
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Additional map: None

Fileemd_3816_additional_3.map
AnnotationNone
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Half map: Halfmap 1

Fileemd_3816_half_map_1.map
AnnotationHalfmap 1
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Half map: Halfmap 2

Fileemd_3816_half_map_2.map
AnnotationHalfmap 2
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Sample components

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Entire : RNA polymerase II-DSIF elongation complex

EntireName: RNA polymerase II-DSIF elongation complex
Components
  • Complex: RNA polymerase II-DSIF elongation complex
    • Complex: RNA polymerase II
    • Complex: DSIF
    • Complex: DNA-RNA synthetic construct

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Supramolecule #1: RNA polymerase II-DSIF elongation complex

SupramoleculeName: RNA polymerase II-DSIF elongation complex / type: complex / ID: 1 / Parent: 0
Molecular weightTheoretical: 690 KDa

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Supramolecule #2: RNA polymerase II

SupramoleculeName: RNA polymerase II / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 520 KDa

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Supramolecule #3: DSIF

SupramoleculeName: DSIF / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 130 KDa

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Supramolecule #4: DNA-RNA synthetic construct

SupramoleculeName: DNA-RNA synthetic construct / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)
Molecular weightTheoretical: 40 KDa

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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

Concentration0.25 mg/mL
BufferpH: 7.25
Component:
ConcentrationNameFormula
5.0 mMHEPES
150.0 mMsodium chlorideNaCl
0.01 mMzinc chlorideZnCl2
1.0 mMdithiothreitol

Details: pH was adjusted at 25 degrees Celsius
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Sample (four microliters) was applied to glow-discharged Quantifoil R 2/1 holey carbon grids, which were then blotted for 8.5s and plunge-frozen in liquid ethane..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-33 / Number grids imaged: 1 / Number real images: 2549 / Average exposure time: 9.9 sec. / Average electron dose: 33.0 e/Å2
Details: Movies were aligned and binned to the physical pixel size of 1.35 angstroms.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.6 µm / Calibrated defocus min: 0.6 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 37000
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: 687928
CTF correctionSoftware - Name: CTFFIND (ver. 4.0.16)
Startup modelType of model: EMDB MAP
EMDB ID:

3218

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 139075
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)

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