期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：47
页码：E7456-E7463
DOI：10.1073/pnas.1612720113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceAs the scope of macromolecular structure determination by cryo-electron microscopy (cryo-EM) is expanding rapidly, it is becoming increasingly clear that many biological complexes are too fragile to withstand the harsh conditions involved in making cryo-EM samples. We describe an original approach to protect proteins from harmful forces during cryo-EM sample preparation by enclosing them inside a three-dimensional support structure that we designed using DNA origami techniques. By binding the transcription cofactor p53 to a specific DNA sequence, and by modifying the position of this sequence in our support structure, we also sought to control the relative orientation of individual p53:DNA complexes. 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 [IMG]f1.gif" ALT="A" BORDER="0">. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.
