Deformed grids for single-particle cryo-electron microscopy of specimens exhibiting a preferred orientation

Abstract

For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (>30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-particle cryo-EM of samples showing a limited range of orientations.

Figure 1. Set-up for producing deformed EM grid

(A). A mold is made from a tweezers tip, and a holey EM grid with a thin layer of carbon support film is placed on the surface with ridges. A pencil eraser is used as a stamping tool. A deformed grid is produced by applying pressure on the EM grid. (B) Picture of a deformed EM grid taken on an Olympus dissecting microscope showing a wrinkled surface.

Figure 2. Deformed EM grids have locally tilted carbon support films

A CCD image of a deformed Quantifoil grid captured on a FEI Tecnai F20 electron microscope, showing that some of the circular holes appear elliptical due to localized tilting of the carbon film. Numbers next to the holes are the local tilt angles determined from the ratio of the transverse and conjugate diameters of the ellipses. Scale bar = 5 μm.

Figure 3. Deformed EM grids increased frequency of particles displaying high Euler angles

Plots of the Euler angle distribution of RyR2 particle orientations on regular holey EM grids (A) and on deformed EM grids (B), particles (3,756 particles from regular grids, and 3,573 from deformed grids) are from images that were collected without tilting the specimen grids in the microscope. Since the RyR2 is a homo-tetramer, 4-fold symmetry is usually applied during image processing, and so the Euler angle distribution only covers one quarter of the 3D sphere. The diameters of the circles indicate the relative numbers of particles. More particles are distributed toward high Euler angles (≥ 30°) in the deformed grids than in the regular EM grids.