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- EMDB-2923: Electron cryo-microscopy of a transcribing Pol IIp complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2923
TitleElectron cryo-microscopy of a transcribing Pol IIp complex
Map dataReconstruction of a transcribing Pol IIp complex
Sample
  • Sample: transcribing Pol IIp complex from yeast
  • Protein or peptide: DNA-directed RNA polymerase II
  • Other: DNA-RNA Scaffold
KeywordsTranscription / pre-mRNA capping
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsMartinez-Rucobo FW / Kohler R / van de Waterbeemd M / Heck AJR / Hemann M / Herzog F / Stark H / Cramer P
CitationJournal: Mol Cell / Year: 2015
Title: Molecular Basis of Transcription-Coupled Pre-mRNA Capping.
Authors: Fuensanta W Martinez-Rucobo / Rebecca Kohler / Michiel van de Waterbeemd / Albert J R Heck / Matthias Hemann / Franz Herzog / Holger Stark / Patrick Cramer /
Abstract: Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by ...Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5'-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5' end. Thus, the RNA 5' end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5'-exonucleases during early transcription.
History
DepositionFeb 27, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseSep 23, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing 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
  • Download
  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2923_squ...

Downloads & links

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Map

FileDownload / File: emd_2923.map.gz / Format: CCP4 / Size: 12.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a transcribing Pol IIp complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 148 pix.
= 296. Å		2 Å/pix.
x 148 pix.
= 296. Å		2 Å/pix.
x 148 pix.
= 296. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.31250411 - 0.41651475
Average (Standard dev.)0.00077728 (±0.01971558)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions148148148
Spacing148148148
CellA=B=C: 296.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z148148148
origin x/y/z0.0000.0000.000
length x/y/z296.000296.000296.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS148148148
D min/max/mean-0.3130.4170.001

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Supplemental data

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Sample components

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Entire : transcribing Pol IIp complex from yeast

EntireName: transcribing Pol IIp complex from yeast
Components
  • Sample: transcribing Pol IIp complex from yeast
  • Protein or peptide: DNA-directed RNA polymerase II
  • Other: DNA-RNA Scaffold

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Supramolecule #1000: transcribing Pol IIp complex from yeast

SupramoleculeName: transcribing Pol IIp complex from yeast / type: sample / ID: 1000
Oligomeric state: One monomer of Pol IIp contains Pol IIp contains 1 DNA/RNA scaffold of 2 DNA and 1 RNA molecules
Number unique components: 2
Molecular weightExperimental: 540 KDa / Theoretical: 540 KDa / Method: native mass spectroscopy

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Macromolecule #1: DNA-directed RNA polymerase II

MacromoleculeName: DNA-directed RNA polymerase II / type: protein_or_peptide / ID: 1 / Name.synonym: RNA polymerase II / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 520 KDa / Theoretical: 520 KDa

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Macromolecule #2: DNA-RNA Scaffold

MacromoleculeName: DNA-RNA Scaffold / type: other / ID: 2 / Name.synonym: template DNA, non-template DNA, mRNA / Classification: DNA/RNA / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 20 KDa / Theoretical: 20 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 / Details: 5mM HEPES, 100mM KCl, 3mM DTT
GridDetails: 200 mesh grid with thin carbon support
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Cs0
DateJan 7, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 74000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Cs: mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 66214

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