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TitleDesign of a molecular support for cryo-EM structure determination.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 113, Issue 47, Page E7456-E7463, Year 2016
Publish dateNov 22, 2016
AuthorsThomas G Martin / Tanmay A M Bharat / Andreas C Joerger / Xiao-Chen Bai / Florian Praetorius / Alan R Fersht / Hendrik Dietz / Sjors H W Scheres /
PubMed AbstractDespite 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.
External linksProc Natl Acad Sci U S A / PubMed:27821763 / PubMed Central
MethodsEM (single particle)
Resolution15.0 Å
Structure data

EMDB-3453:
Cryo-EM structure of human p53 bound to a molecular support structure made of DNA origami.
Method: EM (single particle) / Resolution: 15.0 Å

Source
  • Homo sapiens (human)

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