10Â objective (see Fig. 1). As with the BF microscopy, it is
essential to adjust the focus planes to obtain the most contrasting UV-images possible. For very fine granular aggregates use
of a 40Â objective might be required. False UV-positive granular aggregates can be observed in crystallization conditions
that include calcium salts.
4. The 2% (w/v) uranyl acetate solution can be used for up to
1 month but should be filtered with a 0.22 μm filter on the day
of grid preparation to obtain best crystal staining results.
5. We routinely use carbon-coated grids with a 400-mesh. These
grids result in less broken areas compared to larger mesh grids
(e.g., 300-mesh, 200-mesh) when blotting by hand from the
back with the P2 filter paper.
6. When preparing the sample to load onto the grid be sure to
visually check that the microcrystals are soaked into the pipette.
In some cases, the sample might stick to the cover slid. In those
circumstances, use the pipette tip or a crystallization tool to
carefully perform slow stirring motions to detach the microcrystals or aggregate from the cover slid. If the reservoir buffer
has a very high viscosity (e.g., a PEG-10000 concentration
above 10% (w/v) or other similar precipitants), it is recommended to use a stabilizing solution containing methyl pentanediol (MPD) instead of the highly viscous reservoir solution
(see also Note 7).
7. The size of glass beads (0.5 mm or 1.0 mm diameter) should be
selected depending on the stability of the protein crystal; smaller beads result in harsher crystal fragmentation (see Fig. 2)
[11]. For fragile crystals (e.g., needles and thin plates), we
recommend using 1.0 mm beads; for sturdier and “chunkier”
crystals, 0.5 mm beads are generally needed. Similarly, the
vortexing time has to be chosen depending on the crystal
sturdiness. In case of temperature-sensitive protein crystals,
the fragmentation process has been carried out at 4
C while
cooling the sample on ice in between vortex intervals to prevent protein denaturation. For scarce material containing fewer
granular aggregates, it is recommended to use stainless steel
beads that can be removed from the tube using a strong magnet. Concentration of the slurry can be achieved by removing
the magnetic beads, followed by low speed centrifugation
(524 rcf) to pellet down crystal fragments followed by removal
of excess stabilizing solution. Crystallization conditions that
have highly viscous solutions (e.g., 25% polyethylene glycol
8000 (peg8K)) are difficult to handle or visualize on EM
grids. For such conditions, it is convenient to find a stabilizing
solution with low viscosity where aggregates do not dissolve. A
solution containing 25% peg8K could be exchanged with a
Detection of Microcrystals for CryoEM
305
essential to adjust the focus planes to obtain the most contrasting UV-images possible. For very fine granular aggregates use
of a 40Â objective might be required. False UV-positive granular aggregates can be observed in crystallization conditions
that include calcium salts.
4. The 2% (w/v) uranyl acetate solution can be used for up to
1 month but should be filtered with a 0.22 μm filter on the day
of grid preparation to obtain best crystal staining results.
5. We routinely use carbon-coated grids with a 400-mesh. These
grids result in less broken areas compared to larger mesh grids
(e.g., 300-mesh, 200-mesh) when blotting by hand from the
back with the P2 filter paper.
6. When preparing the sample to load onto the grid be sure to
visually check that the microcrystals are soaked into the pipette.
In some cases, the sample might stick to the cover slid. In those
circumstances, use the pipette tip or a crystallization tool to
carefully perform slow stirring motions to detach the microcrystals or aggregate from the cover slid. If the reservoir buffer
has a very high viscosity (e.g., a PEG-10000 concentration
above 10% (w/v) or other similar precipitants), it is recommended to use a stabilizing solution containing methyl pentanediol (MPD) instead of the highly viscous reservoir solution
(see also Note 7).
7. The size of glass beads (0.5 mm or 1.0 mm diameter) should be
selected depending on the stability of the protein crystal; smaller beads result in harsher crystal fragmentation (see Fig. 2)
[11]. For fragile crystals (e.g., needles and thin plates), we
recommend using 1.0 mm beads; for sturdier and “chunkier”
crystals, 0.5 mm beads are generally needed. Similarly, the
vortexing time has to be chosen depending on the crystal
sturdiness. In case of temperature-sensitive protein crystals,
the fragmentation process has been carried out at 4
C while
cooling the sample on ice in between vortex intervals to prevent protein denaturation. For scarce material containing fewer
granular aggregates, it is recommended to use stainless steel
beads that can be removed from the tube using a strong magnet. Concentration of the slurry can be achieved by removing
the magnetic beads, followed by low speed centrifugation
(524 rcf) to pellet down crystal fragments followed by removal
of excess stabilizing solution. Crystallization conditions that
have highly viscous solutions (e.g., 25% polyethylene glycol
8000 (peg8K)) are difficult to handle or visualize on EM
grids. For such conditions, it is convenient to find a stabilizing
solution with low viscosity where aggregates do not dissolve. A
solution containing 25% peg8K could be exchanged with a
Detection of Microcrystals for CryoEM
305