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- EMDB-8538: Structure of the p53/human RNA polymerase II assembly -

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Basic information

Entry
Database: EMDB / ID: EMD-8538
TitleStructure of the p53/human RNA polymerase II assembly
Map dataThe cryo-EM 3D reconstruction of p53-bound human RNA Polymerase II
Sample
  • Complex: Binary complex of p53 with human RNA polymerase II
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.8 Å
AuthorsLiu WL / Singh SK
CitationJournal: Genes Dev / Year: 2016
Title: Structural visualization of the p53/RNA polymerase II assembly.
Authors: Sameer K Singh / Zhen Qiao / Lihua Song / Vijay Jani / William Rice / Edward Eng / Robert A Coleman / Wei-Li Liu /
Abstract: The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have ...The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.
History
DepositionDec 22, 2016-
Header (metadata) releaseFeb 1, 2017-
Map releaseJan 24, 2018-
UpdateJan 24, 2018-
Current statusJan 24, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0075
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0075
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8538.png

Downloads & links

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Map

FileDownload / File: emd_8538.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe cryo-EM 3D reconstruction of p53-bound human RNA Polymerase II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0075 / Movie #1: 0.0075
Minimum - Maximum-0.0074459417 - 0.018558849
Average (Standard dev.)0.00033213312 (±0.0015836135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-179-179-179
NX/NY/NZ359359359
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0070.0190.000

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Supplemental data

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Sample components

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Entire : Binary complex of p53 with human RNA polymerase II

EntireName: Binary complex of p53 with human RNA polymerase II
Components
  • Complex: Binary complex of p53 with human RNA polymerase II

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Supramolecule #1: Binary complex of p53 with human RNA polymerase II

SupramoleculeName: Binary complex of p53 with human RNA polymerase II / type: complex / ID: 1 / Parent: 0
Details: Assembly of native human RNA Polymerase II with human recombinant full-length p53
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa cells
Molecular weightTheoretical: 570 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

Concentration0.026 mg/mL
BufferpH: 7.9
Component:
ConcentrationFormulaName
20.0 mMHepesHepes
0.2 mMEDTAEDTA
2.0 mMMgCl2MgCl2
10.0 %Glycerol
100.0 mMKClPotassium chloride

Details: plus 0.05% NP40, 1 mM DTT and 0.5 mM PMSF
GridModel: Protochips CF-1.2/1.3-4C / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Details: A thin carbon film supported by a 400-mesh carbon-thickened C-flat holey grid with hole diameter 1.2 micron/spacing 1.3 micron (CF-1.2/1.3-4C, Protochips) was freshly glow discharged using a ...Details: A thin carbon film supported by a 400-mesh carbon-thickened C-flat holey grid with hole diameter 1.2 micron/spacing 1.3 micron (CF-1.2/1.3-4C, Protochips) was freshly glow discharged using a high-vacuum evaporator (Denton).
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278.65 K / Instrument: FEI VITROBOT MARK III
Details: The assembled p53/Pol II co-complex was applied directly on the grid for 10 seconds followed by 5.5 seconds of blotting. The sample grid was then washed with 3.5 % Trehalose in 0.1 M KCl/HEM ...Details: The assembled p53/Pol II co-complex was applied directly on the grid for 10 seconds followed by 5.5 seconds of blotting. The sample grid was then washed with 3.5 % Trehalose in 0.1 M KCl/HEM buffer (20 mM HEPES, 0.2 mM EDTA, 2 mM MgCl2, pH 7.9) for 10 seconds, blotted for 5.5 seconds and finally plunged frozen in liquid ethane..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 66.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 22500
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

CTF correctionSoftware - Name: CTFIND3
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 270 / Random conical tilt - Tilt angle: 50 degrees
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 8097
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 1.4)

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