Schematic diagram illustrating monolayer recovery and freezing. The diagram is based on binding of chicken smooth muscle α-actinin to a peptide corresponding in sequence to the β1-integrin cytoplasmic domain, which in turn is binding to a Ni chelating lipid (Kelly and Taylor, 2005). (A) To recover monolayer samples for cryoEM, we placed the grid bar side down onto the monolayer surface. After ~ 30 seconds, the grid sinks through the monolayer thereby facilitating contact between the lipid layer and the reticulated carbon film. The grid can then be lifted for freezing. A magnified representation of a single grid square is also indicated. (B) The orientation of the monolayer in the grid square of a Quantifoil grid allows blotting from the “back” side of the grid, as opposed to the “front” side on which the reticulated carbon film is located. (Recovery using the front side would normally be used for negative staining.) This highly magnified side view of the grid (with grid bars represented as copper colored surfaces) demonstrates how the grid bars help to shield the crystals from being disrupted by the filter paper during blotting. (C) After adequate blotting from the back side of the grid, the specimen is plunged into liquefied ethane (Dubochet et al., 1988) after which specimen manipulations are the same as for any other frozen specimen.

Four examples of holes with variable ice thickness. In each image a white line to the left is used to indicate differences in the ice thickness at the edges. Qualitatively, the variations in the appearance of holes can be described as: (A) clear-flat hole – ordered diffraction is not obtained from images with this appearance; (B) clear with a ring - typically, these holes yield diffraction spots about 35% of the time; (C) thick with a ring - holes with this appearance show crystalline diffraction 45% of the time; (D) a hole with ice that is too thick for useful imaging although ordered arrays are commonly found in these holes about 38% of the time.

(A) Low magnification survey view of the frozen-hydrated specimen recovered using a Quantifoil grid. Magnification of 170x. In this example, about 52% of the 220 grid squares would be potentially useful. The others have broken films or excessively thick ice. (B) Medium magnification (3500x) view of a grid square shown in (A). Here the reproducible character of the holes within a single grid square can be readily seen. Note that although the crystallization solution is topologically on the side of the grids with the grid bars, the ice is thinnest around the grid bars. This is counter to expectations, but is what occurs normally with this method. (C) High magnification view of one hole in the area shown in (B). The frozen hydrated holes are readily identified by the relatively thicker ring of ice near the edges of the holes. (D) High magnification (29,000x) of one hole with a crystal and its Fourier Transform in the lower right hand corner.

Radial density plots obtained from the four hole-types shown in Figure 3. (A) Raw data. The large fluctuations near a radius of zero are due to the relatively few pixels in this region. Note that the clear-flat profile, which lacks the water ring around the lip of the hole, is noticeably different than the other three hole-types, which contain the ring. (B) The same data plotted but this time normalized to constant density for the carbon matrix. This would be correct only if the carbon matrix contained no ice film across it. Normalizing to the carbon matrix makes all four hole-types appear as if they had constant ice thickness, but this is probably not true since Fourier transforms of clear-flat holes never show crystalline diffraction whereas the other hole-types do.