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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Curr Opin Colloid Interface Sci. 2017 Dec 22;34:1–8. doi: 10.1016/j.cocis.2017.12.009

Table 1.

Approaches that have been identified as possible ways to improve the quality of cryo-grids for specimens that have proven to be “difficult”. In each case, examples of ways to implement a given approach are provided, and comments are made about caveats and known weak points of each.

APPROACHES EXAMPLES COMMENTS
Stabilize the structure by optimizing the buffer conditions

  • Stabilizing cosolutes such as glycerol, trehalose, or ammonium sulfate

  • Optimized pH, ionic strength, or ionic composition

  • Glycerol may cause excessive bubbling and beam-induced motion

  • Salt concentrations above 0.15 M may be problematic
Stabilize macromolecular complexes by chemical crosslinking

  • The Grafix method of crosslinking with glutaraldehyde

  • Crosslinking with BS3 (bis(sulfosuccinimidyl)suberate)

  • Surface charge is changed since all lysine residues are modified, whether crosslinked or not

  • Rare or off-pathway conformational states can be trapped by cross-linking
Minimize interaction with the air-water interface by adding a pre-emptive, structure-friendly surfactant

  • Detergents often used during purification (e.g. NP-40) may help

  • Other detergents or surfactants (Tween 20, fluorinated Fos-choline- 8, amphipol; nanodisks; LMNG (lauryl maltose-neopentyl glycol) or phospholipids) may be effective

  • Surfactants may not have sufficiently high surface pressure to completely block access of proteins to the air- water interface
Minimize interaction by ultrafast thinning and quenching

  • The Spotiton strategy combined with self-blotting grids

  • Outrunning all interaction with the air-water interface may result in specimens with too few particles per unit area
Adsorption to carbon (or other) films to prevent diffusion to the air-water interface

  • Glow-discharge treated, evaporated-carbon films

  • Chemically functionalized carbon films

  • Graphene oxide

  • The chemical makeup and homogeneity is not well characterized

  • Preferential orientation is still a possibility
Immobilization onto structure- friendly affinity grids

  • Ni-NTA functionalized lipid monolayers

  • Antibodies bound to evaporated-carbon films

  • Streptavidin monolayer crystals

  • Additional strategies may be needed to avoid preferential orientation

  • Continuous support films necessarily generate unwanted structural noise in the image