Using 3D DNA-origami, we designed a support structure with a defined size and shape that binds specifically to a target protein of interest. The resulting structure is the first artificial scaffold that exerts experimental control over the orientations of individual protein molecules on cryo-EM grids, and protects them from aggregation or harmful interactions with the air-water interface. In addition, the support structure may facilitate the optimization of freezing conditions, and aid in the selection of suitable ice thickness for data acquisition. Although these results are encouraging, this work has not yet led to a ready-to-use solution for high-resolution structure determination of a wide range of different target proteins, but should rather be considered as a proof-of-principle towards achieving this ambitious goal.
By choosing a target protein that naturally binds to dsDNA in a sequence-specific manner, our support structure was designed to act as a goniometer at a molecular level. By using five different positions of the p53 binding sequence on a central dsDNA helix, our structure theoretically provides five different views of a p53 tetramer bound to the support structure. Although the prior information about the five different orientations could indeed be used successfully to calculate an initial tomographic reconstruction of the p53 tetramer bound to DNA, further refinement of the orientations of 2D class averages or individual particle images revealed a distribution in tilt angles that is much broader than one would expect from a rigid dsDNA helix, and somehow seems to disfavor tilt angles around 90°. The intended design of our support structure as a nano-scale goniometer that provides experimental information about the orientations of individual p53 complexes was, therefore, mostly successful.
Our experiments also revealed several challenges that will need to be solved in future studies. The challenges were a low yield in particle numbers, strong signal from the structure affecting the alignment of the target protein. Future designs of larger support structures with a more accessible protein binding site, and with better incorporation of the tilt axis may improve both particle yield and resolution.
Nevertheless, at a resolution of approximately 15 Å, the resulting reconstruction provides useful information about how p53 binds to dsDNA. The observation that our reconstruction shows C2 symmetry with an additional 2-fold translational component along the DNA axis, which is similar to the crystal structure of the tetrameric core of p53 bound to dsDNA, indicates that p53 does not bind to DNA in a previously proposed arrangement with D2 symmetry.
In addition, the design of our support structure contains several useful features that may inspire future experiments. By enclosing the target protein in a hollow structure, it is protected from interactions with copies of itself or with the hydrophobic air-water interface during cryo-EM sample preparation. This may prevent the proteins from aggregation, or from unfolding or adopting preferred orientations against this air-water interface. Also, the observation that the support structures adopt monolayers in an ice layer with the intended thickness is potentially useful. One might for example try to add support structures to standard cryo-EM samples with the idea of using them as 'spacers' to control ice thickness.
Data collection time on a 300 kV electron microscope is still a major limitation in many cryo-EM labs, and in practice such time consuming projects are often not feasible . Therefore, we shifted our focus from DNA origami supports to more classical supports studies. Here, we were able to improve the state-of-the-art preparation techniques of graphene oxide to make it widely applicable for many projects. We published a video on the preparation of graphene oxide grids, which has been well received in the cryoEM community and was fundamental to success in several projects that struggled with grid preparation. Graphene oxide was shown to help with preferred particle orientation (Boland, Martin et al.), protein aggregation (Bokori-Brown, Martin, et al.) and protein unfolding in several studies, some of them I have been deeply involved. But it does not remove the effects of the air water interface completely.
We are currently working on publishing the results of our study on the flexibility of DNA origami and a high-resolution structure of p53 obtained with a graphene oxide support.
