(d) Repeat above steps 2a through 2c (Subheading 3.4) for
each crystal and determine a tilt range for a set of crystals
to acquire.
3. In the Navigator, set crystal targets to Acquire for a particular
tilt range.
3.5 Data Acquisition
(See Note 3)
1. Adjust camera parameters as necessary for the Record setting.
2. Use the CRmov script [5] to collect data.
3. The script must be edited with parameters appropriate to your
data collection.
4. Acquired datasets, in the form of TIF files, must be converted
to a crystallographic format. Software published by the Gonen
laboratory [9] can read these TIF files generated by SerialEM
and output the corresponding IMG files in SMV format.
4 Notes
1. In step 4 (Subheading 3.1), during the setup of defocuseddiffraction, note that recent versions of SerialEM have the Low
Dose View Mode defocus offset setting disabled for diffraction
mode. In this case, simply set the defocus offset using the focus
knob while in diffraction mode, then use the “Continuous
update” checkbox to save it to the Low Dose View mode.
Additionally, the Low Dose Focus mode can be used for this
purpose (this feature available in SerialEM 3.8 beta); for convenience, this would free the Low Dose View mode to be set up
at the low magnification for the whole-grid atlas, as mentioned
in step 6 (Subheading 3.1).
2. In Subheading 3.1, the microscope must be in imaging mode
in order for the stage to properly move to targets and for the
Eucentricity routine to work.
3. Note that the CRmov script collects each batch of targets at the
same tilt range. Crystals that need to be collected at different
tilt ranges must be run separately.
4. Initially, only microscopes from Thermo Fisher Scientific had
been tested to work with this method because the SerialEM
BackgroundTilt command (used in the CRmov script) was
originally written for that manufacturer’s microscope scripting
interface. However, as of SerialEM version 3.7.5, recent JEOL
TEMs controlled by the TemExt server now have access to
advanced microscope scripting via SerialEM, including the
BackgroundTilt command.
5. Refer to the protocol mentioned previously [5] for more details
on this procedure.
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M. Jason de la Cruz
each crystal and determine a tilt range for a set of crystals
to acquire.
3. In the Navigator, set crystal targets to Acquire for a particular
tilt range.
3.5 Data Acquisition
(See Note 3)
1. Adjust camera parameters as necessary for the Record setting.
2. Use the CRmov script [5] to collect data.
3. The script must be edited with parameters appropriate to your
data collection.
4. Acquired datasets, in the form of TIF files, must be converted
to a crystallographic format. Software published by the Gonen
laboratory [9] can read these TIF files generated by SerialEM
and output the corresponding IMG files in SMV format.
4 Notes
1. In step 4 (Subheading 3.1), during the setup of defocuseddiffraction, note that recent versions of SerialEM have the Low
Dose View Mode defocus offset setting disabled for diffraction
mode. In this case, simply set the defocus offset using the focus
knob while in diffraction mode, then use the “Continuous
update” checkbox to save it to the Low Dose View mode.
Additionally, the Low Dose Focus mode can be used for this
purpose (this feature available in SerialEM 3.8 beta); for convenience, this would free the Low Dose View mode to be set up
at the low magnification for the whole-grid atlas, as mentioned
in step 6 (Subheading 3.1).
2. In Subheading 3.1, the microscope must be in imaging mode
in order for the stage to properly move to targets and for the
Eucentricity routine to work.
3. Note that the CRmov script collects each batch of targets at the
same tilt range. Crystals that need to be collected at different
tilt ranges must be run separately.
4. Initially, only microscopes from Thermo Fisher Scientific had
been tested to work with this method because the SerialEM
BackgroundTilt command (used in the CRmov script) was
originally written for that manufacturer’s microscope scripting
interface. However, as of SerialEM version 3.7.5, recent JEOL
TEMs controlled by the TemExt server now have access to
advanced microscope scripting via SerialEM, including the
BackgroundTilt command.
5. Refer to the protocol mentioned previously [5] for more details
on this procedure.
326
M. Jason de la Cruz