the shutter), change in defocus, image shift in X, image shift in
Y. Units for the last three entries are all in microns. By saving
the array as a persistent variable in SerialEM, this step only
needs to be done once per tilt-scheme.
2. A typical fast-incremental SerialEM script includes the following commands:
(a) “FrameThresholdNextShot 0.1” automatically skips saving blank frames based on a mean value below a threshold
of 0.1. This threshold can be adjusted as desired.
(b) “FrameSeriesFromVar var 31 #d”, where #d is an initial
delay time, followed by the Record command to record
movie frames.
(c) ReportLastFrameFile, to assign the filename to reported
Value1.
(d) “WriteFrameSeriesAngles $reportedValue1.angles” writes
the tilt angles collected into a text file appended with .
angles for post-processing.
3.3 Data Collection
for High-Resolution
(Near-Atomic)
Tomography
Data collection for high-resolution tomography begins with determining the desired resolution, and choosing parameters based on
both the desired resolution and the features of the object of interest. Collection is typically optimized through one or more sessions,
with alignment and subtomogram averaging resolution used to
determine optimal settings.
For higher-resolution collection, samples must be thin (ideally
less than 200 nm) and fiducials must be plentiful (typically 20 or
more beads in the field of view). The data collection is typically
reminiscent of a single-particle averaging collection, with a pixel
size of less than 2 Å, defocus gradient of À1 to À5 μm, and dosefractionated frame collection [28, 42]. If subtomogram averaging is
intended, a phase plate is not recommended. The tilt-scheme
begins at zero degrees and is acquired with symmetric dose alternating between positive and negative angles [28]. Optimal dose is
determined based on the point at which the sample displays visible
damage, but is often approximated at 120 electrons/Å
2 with the
highest accumulated doses at the highest tilt angles. Tilt increment
and dose have frequently been set to À60
to +60
in 3
increments, but smaller tilt increments or a larger range may be beneficial for some samples.
Also, particular attention is paid to minimize the dose used to
find and center the target and for setting the eucentric height and
to minimize drift during exposure. Movies can be collected to
motion-correct any residual drift. In addition, it is recommended
to use off-targets focus spots to maintain focus throughout tiltseries collection. Finally, the beam has to be highly coherent and
parallel to preserve high-resolution signals.
Methods in Cryo-Electron Tomography
101
Y. Units for the last three entries are all in microns. By saving
the array as a persistent variable in SerialEM, this step only
needs to be done once per tilt-scheme.
2. A typical fast-incremental SerialEM script includes the following commands:
(a) “FrameThresholdNextShot 0.1” automatically skips saving blank frames based on a mean value below a threshold
of 0.1. This threshold can be adjusted as desired.
(b) “FrameSeriesFromVar var 31 #d”, where #d is an initial
delay time, followed by the Record command to record
movie frames.
(c) ReportLastFrameFile, to assign the filename to reported
Value1.
(d) “WriteFrameSeriesAngles $reportedValue1.angles” writes
the tilt angles collected into a text file appended with .
angles for post-processing.
3.3 Data Collection
for High-Resolution
(Near-Atomic)
Tomography
Data collection for high-resolution tomography begins with determining the desired resolution, and choosing parameters based on
both the desired resolution and the features of the object of interest. Collection is typically optimized through one or more sessions,
with alignment and subtomogram averaging resolution used to
determine optimal settings.
For higher-resolution collection, samples must be thin (ideally
less than 200 nm) and fiducials must be plentiful (typically 20 or
more beads in the field of view). The data collection is typically
reminiscent of a single-particle averaging collection, with a pixel
size of less than 2 Å, defocus gradient of À1 to À5 μm, and dosefractionated frame collection [28, 42]. If subtomogram averaging is
intended, a phase plate is not recommended. The tilt-scheme
begins at zero degrees and is acquired with symmetric dose alternating between positive and negative angles [28]. Optimal dose is
determined based on the point at which the sample displays visible
damage, but is often approximated at 120 electrons/Å
2 with the
highest accumulated doses at the highest tilt angles. Tilt increment
and dose have frequently been set to À60
to +60
in 3
increments, but smaller tilt increments or a larger range may be beneficial for some samples.
Also, particular attention is paid to minimize the dose used to
find and center the target and for setting the eucentric height and
to minimize drift during exposure. Movies can be collected to
motion-correct any residual drift. In addition, it is recommended
to use off-targets focus spots to maintain focus throughout tiltseries collection. Finally, the beam has to be highly coherent and
parallel to preserve high-resolution signals.
Methods in Cryo-Electron Tomography
101