stabilizing solution containing 30–40% methyl pentanediol
(MPD) during several rounds of concentrating, removing
excess liquid and slowly adding the new stabilizing solution.
8. Hold the grid at a perpendicular angle relative to the filter
paper and slowly move it along the filter paper with only its
edge touching the filter paper. Make sure to visually check the
filter paper during to blotting process for signs of the blotted
liquid (see Fig. 3). When instead blotting from the front, the
crystals might be inadvertently removed from the grid as well,
while blotting from the back might not be possible with a
non-holey grid due to the high buffer viscosity. Generally, the
more viscous the reservoir solution, the slower the blotting
process will be and it will take about 2–10 s to fully blot away
the excess liquid.
9. Within a couple of seconds after blotting, place the blotted grid
sample side down on top of the first drop of uranyl acetate
solution in order to keep the sample from completely drying
out before the staining. It is important to fully blot away excess
liquid but not to let the crystals crack due to dehydration when
trying to optimize staining results, therefore a thorough blotting and fast transfer are essential.
10. For the TEM screening, we have predominantly used an FEI
TECNAI T12 electron microscope operating at 120 kV with a
single-tilt specimen holder. Images are acquired with a
2 k  2 k Gatan UltraScan 1000 CCD camera, typically at
magnifications between 11,000 and 52,000Â. Fast Fourier
Transform (FFT) calculations of the lattices are used to determine the crystal quality. The experimenter should be prepared
to spend up to 2 h looking for crystal lattices on each grid.
When looking for crystals, it is important to focus on objects
with sharp symmetric edges, which show a dark fringe and a
brighter color in its central parts. Some crystals are radiation
sensitive and the lattice might “melt” during the focus adjustment. In these cases, it might be necessary to use the “Low
Dose” mode of the microscope for imaging purposes.
Acknowledgments
This work was supported by NIH grant R01GM112686 (G.C.),
P50GM082251 (G.C. and S.W.), and BioXFEL-STC1231306 (S.
W). S.V. acknowledges support from grant R01GM097082. The
contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of
NIGMB or NIH.
306
Simon Weiss et al.
(MPD) during several rounds of concentrating, removing
excess liquid and slowly adding the new stabilizing solution.
8. Hold the grid at a perpendicular angle relative to the filter
paper and slowly move it along the filter paper with only its
edge touching the filter paper. Make sure to visually check the
filter paper during to blotting process for signs of the blotted
liquid (see Fig. 3). When instead blotting from the front, the
crystals might be inadvertently removed from the grid as well,
while blotting from the back might not be possible with a
non-holey grid due to the high buffer viscosity. Generally, the
more viscous the reservoir solution, the slower the blotting
process will be and it will take about 2–10 s to fully blot away
the excess liquid.
9. Within a couple of seconds after blotting, place the blotted grid
sample side down on top of the first drop of uranyl acetate
solution in order to keep the sample from completely drying
out before the staining. It is important to fully blot away excess
liquid but not to let the crystals crack due to dehydration when
trying to optimize staining results, therefore a thorough blotting and fast transfer are essential.
10. For the TEM screening, we have predominantly used an FEI
TECNAI T12 electron microscope operating at 120 kV with a
single-tilt specimen holder. Images are acquired with a
2 k  2 k Gatan UltraScan 1000 CCD camera, typically at
magnifications between 11,000 and 52,000Â. Fast Fourier
Transform (FFT) calculations of the lattices are used to determine the crystal quality. The experimenter should be prepared
to spend up to 2 h looking for crystal lattices on each grid.
When looking for crystals, it is important to focus on objects
with sharp symmetric edges, which show a dark fringe and a
brighter color in its central parts. Some crystals are radiation
sensitive and the lattice might “melt” during the focus adjustment. In these cases, it might be necessary to use the “Low
Dose” mode of the microscope for imaging purposes.
Acknowledgments
This work was supported by NIH grant R01GM112686 (G.C.),
P50GM082251 (G.C. and S.W.), and BioXFEL-STC1231306 (S.
W). S.V. acknowledges support from grant R01GM097082. The
contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of
NIGMB or NIH.
306
Simon Weiss et al.