X-ray Crystallography, Transmission-EM and Cryo-EM share a common uncertainty.

Considering Transmission Electron Microscopy (TEM) first, to create an image of a protein quaternary structure the sample is allowed to adsorb to a surface, is dried, fixed and stained. TEM practitioners know that the method can be useful for identifying viruses or proteins but is so vulnerable to artefacts that the resulting images cannot be relied upon to represent real structural features. For a brief critique, read the Introduction to this 1984 work about Cryo-EM. For any particular preparation of any particular protein, it is not known which of the sample treatments create artefacts - whether adsorption, drying, fixing or staining. I suggest that there is a reasonable possibility that for some, possibly most proteins, being at or near a solid interface influences the quaternary structure. The reason would be that the interaction with the interface rivals the interaction between protein subunits, resulting in a modified quaternary structure. If we accept that this is one of the sources of uncertainty in TEM images, it is reasonable to suppose it is a source of uncertainty for X-ray Crystallography as well. Before incorporation into a crystal each protein complex must first encounter the crystal surface. As a result of thermal motion, some proteins will have been near or at the surface for a long time, some for a shorter time. The uncertainties then are; is the protein one whose intersubunit interactions are rivalled by interactions with the crystal surface, do any of the protein molecules spend enough time at the surface to rearrange themselves, and are rearranged quaternary structures more or less likely to be incorporated into the crystal? We must conclude that, for any particular protein, there is uncertainty about whether its solution quaternary structure will be represented in the crystal. The uncertainty hanging over Cryo-EM is not so straightforward to understand. It is all bound up in the complexities of solidification of water. Dubochet and colleagues mistakenly believed they had found a way to supercool water below the well-known limit of between -30 deg C and -40 deg C and thus achieve what they called vitrification without a defined phase change. It seems that instead they had stumbled upon a metastable, partly disordered form of ice that initially forms whenever supercooled water solidifies. (Malkin et al. 2015). This post is long enough. I will detail the Cryo-EM story in the next post.