@article{c77781ced02a42f58a6e9f9713c3cfe6,
title = "Design of a molecular support for cryo-EM structure determination",
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 {\AA}. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.",
keywords = "Cryo-EM, DNA-origami, P53, Single particle analysis, Structural biology",
author = "Martin, {Thomas G.} and Bharat, {Tanmay A.M.} and Joerger, {Andreas C.} and Bai, {Xiao Chen} and Florian Praetorius and Fersht, {Alan R.} and Hendrik Dietz and Scheres, {Sjors H.W.}",
note = "Funding Information: We thank Christos Savva, Shaoxia Chen, Toby Darling, and Jake Grimmett for technical support, and Miriana Petrovich for protein purification. This work was supported by the European Molecular Biology Organisation through a long-term postdoctoral fellowship (ALTF-1229-2013 to T.G.M.) and an advanced fellowship (aALTF-778-2015 to T.A.M.B.), and by European Commission Marie Sk{\l}odowska-Curie postdoctoral fellowships (to T.G.M. and X.-c.B.). The project was further supported by European Research Council Starting Grant GA 256270 (to H.D.); by the Deutsche Forschungsgemeinschaft through grants provided within the Sonderforschungsbereich SFB863, the Center for Integrated Protein Science Munich, and the Nano Initiative Munich; and UK Medical Research Council Grants MC-UP-A024-1010 (to A.R.F.) and MC-UP-A025-1013 (to S.H.W.S.). Funding Information: We thank Christos Savva, Shaoxia Chen, Toby Darling, and Jake Grimmett for technical support, and Miriana Petrovich for protein purification. This work was supported by the European Molecular Biology Organisation through a long-term postdoctoral fellowship (ALTF-1229-2013 to T.G.M.) and an advanced fellowship (aALTF-778-2015 to T.A.M.B.), and by European Commission Marie Sk?odowska-Curie postdoctoral fellowships (to T.G.M. and X.-c.B.). The project was further supported by European Research Council Starting Grant GA 256270 (to H.D.); by the Deutsche Forschungsgemeinschaft through grants provided within the Sonderforschungsbereich SFB863, the Center for Integrated Protein Science Munich, and the Nano Initiative Munich; and UK Medical Research Council Grants MC-UP-A024-1010 (to A.R.F.) and MC-UP-A025-1013 (to S.H.W.S.).",
year = "2016",
month = nov,
day = "22",
doi = "10.1073/pnas.1612720113",
language = "English (US)",
volume = "113",
pages = "E7456--E7463",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "47",
}