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Controlled cryo-EM sample preparation through DNA-Origami pillars

Periodic Reporting for period 1 - EM-Pillars (Controlled cryo-EM sample preparation through DNA-Origami pillars)

Reporting period: 2015-08-01 to 2017-07-31

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful technique for high-resolution structure determination of protein complexes. While cryo-EM has been successfully used for structure determination of many protein complexes, others have been proven difficult. One of the biggest challenges lies in the sample preparation. In order to make samples suitable for cryo-EM, proteins are placed in a very thin film of water on top of a supporting metallic grid that is then rapidly frozen. Just before freezing, the proteins are exposed subjected to a large hydrophobic air-water interface. Interactions with this interface can cause the protein structures to unfold, or can induce specific orientations of the proteins against this interface, which may prevent structure determination.
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.
During our research, we found that a simpler strategy for preparing cryo-EM samples is the use of graphene oxide monolayers as supporting material. We were able to improve the state of the art preparation techniques of graphene oxide to make it widely applicable for many projects. We have published a video on the preparation of graphene oxide grids, which has been well received by the community and was key in several projects for optimal grid preparation. We observed that graphene oxide is especially useful to deal with preferred orientations and aggregation of protein complexes, as well as projects in which particle absorption to the grid needed to be improved. While it is not as harsh as the air-water interface itself, the graphene oxide layer is also a surface that interacts with the protein and it does not remove the effects of the air-water interface completely.
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
With our work, we established tools for cryo-EM sample preparation that are helping other researchers to discover the structures of protein complexes. In the long run, the resulting structures will further our knowledge of how the biology in a cell works which will help pharmaceutical companies to think of new strategies for drug development.
DNA Origami support for small DNA binding proteins