collected from the liquid nitrogen. Furthermore, ice crystals
loosely attached to the grid may evaporate and re-condense
onto the cold grid surface, thus forming a continuous or speckled ice layer sometimes difficult to detect. This continuous
contamination accumulates over time due to water molecules
present in the vacuum of the microscope. Working in a dry
environment, changing liquid nitrogen frequently to avoid ice
crystal accumulation, handling the grids in liquid nitrogen or
very close to the surface, and heating/emptying the containers
between two freezing sessions will reduce ice contaminations.
If contamination builds up rapidly in the microscope, check the
anticontamination devices of your microscope.
6. Damaged sample. During cryo-EM sample preparation, protein complexes may be damaged when placed in a thin layer of
suspension due to its interactions with the air–water interface.
This may result to partial denaturation, dissociation of subunits, and aggregation. Sample cross-linking with glutaraldehyde or BS3 as described in Subheading 3.1.2 generally helps
stabilizing the molecular assemblies. Alternatively, the air–
water interface may be screened with amphiphilic molecules
such as detergents. In this case, the particle concentration may
need to be increased since the air–water interface does not
concentrate the biomolecules any more. The possibility to
adsorb the proteins on a continuous carbon film is worth
exploring to avoid the air–water interface.
7. Lack of particles. It is difficult to predict the protein concentration needed to produce projection image with densely packed
particles. Recent experiments showed that in most cases as
much as 90% of the particles accumulate at the air–water interface [6]. The number of particles adsorbed on the grid depends
on their propensity to accumulate at the interface. In the
absence of surface accumulation, an extremely rare situation,
particle concentrations of 100 mg/mL (10% of the volume)
may be needed to produce densely populated images. Absence
of particles may therefore also arise from competition for the
surface and high concentrations of detergents or of hydrophobic peptides that may shield the surface and prevent surface
accumulation of the sample. In extreme cases, most of the
particles are denatured and form a grainy background. A
cross-linking experiment generally helps sorting out this
problem.
8. The quality of the frozen hydrated grid depends critically on
sample homogeneity and stability. It is essential to give the
highest priority to protein purification to increase the chances
to produce an exploitable grid.
Specimen Optimization for Single Particle cryo-EM
255
loosely attached to the grid may evaporate and re-condense
onto the cold grid surface, thus forming a continuous or speckled ice layer sometimes difficult to detect. This continuous
contamination accumulates over time due to water molecules
present in the vacuum of the microscope. Working in a dry
environment, changing liquid nitrogen frequently to avoid ice
crystal accumulation, handling the grids in liquid nitrogen or
very close to the surface, and heating/emptying the containers
between two freezing sessions will reduce ice contaminations.
If contamination builds up rapidly in the microscope, check the
anticontamination devices of your microscope.
6. Damaged sample. During cryo-EM sample preparation, protein complexes may be damaged when placed in a thin layer of
suspension due to its interactions with the air–water interface.
This may result to partial denaturation, dissociation of subunits, and aggregation. Sample cross-linking with glutaraldehyde or BS3 as described in Subheading 3.1.2 generally helps
stabilizing the molecular assemblies. Alternatively, the air–
water interface may be screened with amphiphilic molecules
such as detergents. In this case, the particle concentration may
need to be increased since the air–water interface does not
concentrate the biomolecules any more. The possibility to
adsorb the proteins on a continuous carbon film is worth
exploring to avoid the air–water interface.
7. Lack of particles. It is difficult to predict the protein concentration needed to produce projection image with densely packed
particles. Recent experiments showed that in most cases as
much as 90% of the particles accumulate at the air–water interface [6]. The number of particles adsorbed on the grid depends
on their propensity to accumulate at the interface. In the
absence of surface accumulation, an extremely rare situation,
particle concentrations of 100 mg/mL (10% of the volume)
may be needed to produce densely populated images. Absence
of particles may therefore also arise from competition for the
surface and high concentrations of detergents or of hydrophobic peptides that may shield the surface and prevent surface
accumulation of the sample. In extreme cases, most of the
particles are denatured and form a grainy background. A
cross-linking experiment generally helps sorting out this
problem.
8. The quality of the frozen hydrated grid depends critically on
sample homogeneity and stability. It is essential to give the
highest priority to protein purification to increase the chances
to produce an exploitable grid.
Specimen Optimization for Single Particle cryo-EM
255