Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 47, Pages E7456-E7463Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1612720113
Keywords
cryo-EM; DNA-origami; single particle analysis; structural biology; p53
Categories
Funding
- European Molecular Biology Organisation [ALTF-1229-2013, aALTF-778-2015]
- European Commission
- European Research Council [GA 256270]
- Deutsche Forschungsgemeinschaft [Sonderforschungsbereich SFB863]
- Center for Integrated Protein Science Munich
- Nano Initiative Munich
- UK Medical Research Council [MC_UP_A024_1010, MC_UP_A025_1013]
- European Molecular Biology Organisation [ALTF-1229-2013, aALTF-778-2015]
- European Commission
- European Research Council [GA 256270]
- Deutsche Forschungsgemeinschaft [Sonderforschungsbereich SFB863]
- Center for Integrated Protein Science Munich
- Nano Initiative Munich
- UK Medical Research Council [MC_UP_A024_1010, MC_UP_A025_1013]
- Medical Research Council [MC_UP_A024_1010, MC_EX_G0901534, MC_UP_A025_1013] Funding Source: researchfish
- MRC [MC_EX_G0901534, MC_UP_A025_1013, MC_UP_A024_1010] Funding Source: UKRI
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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 similar to 15 angstrom. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.
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