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- EMDB-3453: Cryo-EM structure of human p53 bound to a molecular support struc... -

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Basic information

Entry
Database: EMDB / ID: EMD-3453
TitleCryo-EM structure of human p53 bound to a molecular support structure made of DNA origami.
Map dataNone
Sample
  • Complex: Tetramer of truncated human p53 (residues 1-360) bound to dsDNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsMartin TG / Bharat TAM / Joerger AC / Bai X / Praetorius F / Fersht AR / Dietz H / Scheres SHW
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Design of a molecular support for cryo-EM structure determination.
Authors: Thomas G Martin / Tanmay A M Bharat / Andreas C Joerger / Xiao-Chen Bai / Florian Praetorius / Alan R Fersht / Hendrik Dietz / Sjors H W Scheres /
Abstract: Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or ...Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.
History
DepositionNov 1, 2016-
Header (metadata) releaseNov 16, 2016-
Map releaseNov 16, 2016-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3453.png

Downloads & links

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Map

FileDownload / File: emd_3453.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.76 Å
Density
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.005
Minimum - Maximum-0.023299422 - 0.029008184
Average (Standard dev.)-0.00009371434 (±0.0035132633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 158.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.761.761.76
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z158.400158.400158.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-0.0230.029-0.000

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Supplemental data

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Sample components

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Entire : Tetramer of truncated human p53 (residues 1-360) bound to dsDNA

EntireName: Tetramer of truncated human p53 (residues 1-360) bound to dsDNA
Components
  • Complex: Tetramer of truncated human p53 (residues 1-360) bound to dsDNA

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Supramolecule #1: Tetramer of truncated human p53 (residues 1-360) bound to dsDNA

SupramoleculeName: Tetramer of truncated human p53 (residues 1-360) bound to dsDNA
type: complex / ID: 1 / Parent: 0
Details: The dsDNA to which p53 was bound is part of a large DNA origami support structure that was used in the structure determination process.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: Modified pET24a expression vecto
Molecular weightTheoretical: 160 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

BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
DetailsP53 tetramers were bound to large DNA origami support structures.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF / Energy filter - Lower energy threshold: -20 eV / Energy filter - Upper energy threshold: +20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-20 / Number grids imaged: 7 / Number real images: 2562 / Average exposure time: 16.0 sec. / Average electron dose: 38.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 272914
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: Tomographic reconstruction from angles experimentally determined by DNA origami support alignment.
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 1.4)
Details: Tomographic angles experimentally determined by DNA origami support alignment.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: RELION (ver. 2) / Software - details: Beta
Details: FSC=0.143 indicates a resolution around 10A.However, visual inspection of the map indicates this may be a bit too high. As some developmental image processing procedures were used to take ...Details: FSC=0.143 indicates a resolution around 10A.However, visual inspection of the map indicates this may be a bit too high. As some developmental image processing procedures were used to take into account the experimental information provided by the DNA origami support structures, we chose to stick to a more conservative 15A resolution estimate.
Number images used: 9271

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