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- EMDB-8885: RNA Pol II complex arrested by a CPD lesion -

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Basic information

Entry
Database: EMDB / ID: EMD-8885
TitleRNA Pol II complex arrested by a CPD lesion
Map dataRNA Pol II elongation complex with a CPD lesion containing scaffold
Sample
  • Complex: Binary complex of RNA Pol II and transcription scaffold
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsLahiri I / Leschziner AE
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM27681 United States
CitationJournal: Nature / Year: 2017
Title: Structural basis for the initiation of eukaryotic transcription-coupled DNA repair.
Authors: Jun Xu / Indrajit Lahiri / Wei Wang / Adam Wier / Michael A Cianfrocco / Jenny Chong / Alissa A Hare / Peter B Dervan / Frank DiMaio / Andres E Leschziner / Dong Wang /
Abstract: Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block ...Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II). Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II-CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.
History
DepositionAug 11, 2017-
Header (metadata) releaseSep 6, 2017-
Map releaseDec 6, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.99
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.99
  • 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_8885.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA Pol II elongation complex with a CPD lesion containing scaffold
Voxel sizeX=Y=Z: 3.6 Å
Density
Contour LevelBy AUTHOR: 0.99 / Movie #1: 0.99
Minimum - Maximum-0.9382218 - 2.7755256
Average (Standard dev.)-0.0071814493 (±0.13135828)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 460.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.9382.776-0.007

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Supplemental data

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Sample components

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Entire : Binary complex of RNA Pol II and transcription scaffold

EntireName: Binary complex of RNA Pol II and transcription scaffold
Components
  • Complex: Binary complex of RNA Pol II and transcription scaffold

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Supramolecule #1: Binary complex of RNA Pol II and transcription scaffold

SupramoleculeName: Binary complex of RNA Pol II and transcription scaffold
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c

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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: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 7.7 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC
Details: The particles contributing to the structure adopted a strong preferred orientation.
Number images used: 51119

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