Cryonegative staining of macromolecular assemblies.
Cryoelectron microscopy (cryo-EM) combined with single-particle reconstruction methods is a powerful technique to study the structure of biological assemblies at molecular resolution (i.e., 3-10 Å). Since electron micrographs of frozen-hydrated biological particles are usually very noisy, improvement of the signal-to-noise ratio (SNR) is necessary and is usually achieved by image processing. We propose an alternative method to improve the contrast at the specimen preparation stage: cryonegative staining. Cryonegative staining aims to increase the SNR while preserving the biological samples in the frozen-hydrated state. Here, we present two alternative procedures to efficiently perform cryonegative staining on macromolecular assemblies. The first is very similar to conventional cryo-EM, the main difference being that the samples are observed in the presence of an additional contrasting agent, ammonium molybdate. The second is based on a carbon-sandwich method and is typically used with uranyl formate or acetate. Compared to air-dried negative staining at room temperature, the advantage of both cryonegative-staining procedures presented here is that the sample is kept hydrated at all steps and observed at liquid nitrogen temperature in the electron microscope. The advantage over conventional cryo-EM is that the SNR is improved by at least a factor of three. For each of these approaches, a few examples of attainable data are given. We cover the technical background to cryonegative staining of macromolecular assemblies, and then expand upon the different possibilities and limitations.
De Carlo
Sacha
S
Department of Chemistry, Institute for Macromolecular Assemblies, City University of New York, City College Campus, New York, USA.
Stark
Holger
H
D000085:Acetates / Q000737:chemistry; D020285:Cryoelectron Microscopy / Q000379:methods*; D046911:Macromolecular Substances / Q000737:chemistry* / Q000648:ultrastructure*; D008982:Molybdenum / Q000737:chemistry; D016624:Negative Staining / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry
D000085:Acetates; D046911:Macromolecular Substances; D009942:Organometallic Compounds; C000472:uranyl formate; D008982:Molybdenum; C022175:ammonium molybdate