Design of a molecular support for cryo-EM structure determination

Thomas G. Martin, Tanmay A.M. Bharat, Andreas C. Joerger, Xiao Chen Bai, Florian Praetorius, Alan R. Fersht, Hendrik Dietz, Sjors H.W. Scheres

Research output: Contribution to journalArticle

36 Scopus citations

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 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 filmof 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.

Original languageEnglish (US)
Pages (from-to)E7456-E7463
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number47
DOIs
StatePublished - Nov 22 2016

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Keywords

  • Cryo-EM
  • DNA-origami
  • P53
  • Single particle analysis
  • Structural biology

ASJC Scopus subject areas

  • General

Cite this

Martin, T. G., Bharat, T. A. M., Joerger, A. C., Bai, X. C., Praetorius, F., Fersht, A. R., Dietz, H., & Scheres, S. H. W. (2016). Design of a molecular support for cryo-EM structure determination. Proceedings of the National Academy of Sciences of the United States of America, 113(47), E7456-E7463. https://doi.org/10.1073/pnas.1612720113