EM-Pillars · Controlled cryo-EM sample preparation through DNA-Origami pillars

Controlled cryo-EM sample preparation through DNA-Origami pillars

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.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз