A well established usage case for cryo-TEM is three-dimensional reconstruction of isolated macromolecules, virus particles, or filaments. On one hand, these approaches are based on averaging of repetitive structures – either due to numerous identical molecules, repetitive patterns on a filament, or symmetries, to reduce the noise inherent to cryo-TEM. On the other hand, different views of these complexes required for three-dimensional reconstruction can be either obtained from (ideally) random orientation of the particles in the ice or from the helicality of filaments.
Mohit Kumar from Dieter Blaas' Group at the Max F. Perutz Laboratories (Vienna) was interested in the interaction of rhinovirus particles with lipid membranes and the events leading to endocytosis of the particles . They decided to employ single particle reconstruction methods to obtain three-dimensional models of the particles docking to artificial membranes (liposomes), using numerous individual virion/membrane complexes selected from many micrographs.
For collection of many micrographs, as required by this type of experiment, or for freezing of numerous grids of similar specimens, reproducibility of results – in this case ice thickness and minimized contamination – is a major factor. The Leica EM GP addresses problems with blotting intensity arising from moist, flexing filter paper or bent grids with a unique sensor: this sensor detects the moment of contact between the filter paper and the sample droplet, establishing a reference point. By moving the filter paper forward by a defined distance from this reference point, the pressure applied varies very little from one grid to the next, yielding highly reproducible results if the same specimen, volume and timing are being used.
Using this blotting sensor, results could be obtained very efficiently from 4 µl samples applied onto glow discharged 400 mesh Cu grids with a R1.2/1.3 Quantifoil film that were allowed to spread for 25 seconds in the environmental chamber with 99 % humidity and 30 °C. Subsequently, they were blotted for 0.8 seconds and plunged into liquid ethane. Particles binding to liposomes of similar diameter were picked from the micrographs of these frozen and unstained specimens acquired on a 300 kV cryo-TEM (Figure 1). From these selected particles, the authors calculated reconstructions of the virus/membrane assemblies in absence of cellular receptors, mimicking the intimate contact of lipid membranes over a twofold axis of icosahedral symmetry, illustrating the binding mechanism of the tight virus-membrane complex (Figure 2).