that the crystal averages are computed by averaging the boxed unit
cells directly, while the projection maps are computed from the
information in the diffraction spots alone (which is also a form of
averaging). Examples of crystal averages are shown in Supplementary Fig. 3 of [38].
3.4.2 Running
the Pre-Refinement
The script Prepare pre-refinement offers a graphical interface to
generate an mparameters file for frealign-2dx [39]. It will also put
the files required by frealign-2dx into the pre-refine directory, by
default. The default values should be reasonable, but users are
encouraged to fine-tune the parameters to their needs. We draw
the attention here to the Auto-refinement parameters available in
our modified version of frealign-2dx:
– FSC threshold for resolution limit? (thresh_fsc_ref): the resolution limit used in refinement (for preventing overfitting) will
be taken as the resolution where the Fourier shell correlation
(FSC) curve crosses this threshold (default: 0.800).
– FSC threshold for map improvement evaluation? (thresh_fsc_eval): any map improvement between refinement cycles will
be assessed by looking at the resolution where the FSC curve
crosses this threshold (default: 0.143).
– Minimum resolution limit? (res_min): this parameter prevents
the auto-refiner from using a resolution too low, which may
cause the refinement to diverge (default: 40 A ˚ ).
– Stay away from current map resolution? (ref_stay_way): this
parameter prevents the resolution limit from getting too close to
the current map resolution, which would potentially introduce
bias in the refinement (default: 2 A ˚ ).
– Try different combinations of parameter mask? (change_pmask): if enabled, this parameter makes the auto-refiner try
different combinations among the active refinement parameters
(ϕ, θ, ψ, x, y), before declaring convergence, which helps avoiding local minima (default: yes).
– Keep the tilt angle fixed? (no_theta): if active, the tilt angle (θ)
will be kept fixed. Sometimes this is helpful for 2D crystal data
(default: no).
For more information, please see Supplementary Note 2 of
[38]. After preparing the pre-refinement, it can be launched via
the Run pre-refinement script. Alternatively, it can be launched
from the command line by running the frealign_run_refine_auto
program inside the pre-refinement directory. The crystal averages
are only a few hundred for a typical dataset and they have a high
SNR. The pre-refinement should therefore complete in only a few
minutes and noticeably improve the tilt geometry estimation (now
converted to Euler angles), as shown in Fig. 3. Plots of the angular
276
Ricardo Righetto and Henning Stahlberg
cells directly, while the projection maps are computed from the
information in the diffraction spots alone (which is also a form of
averaging). Examples of crystal averages are shown in Supplementary Fig. 3 of [38].
3.4.2 Running
the Pre-Refinement
The script Prepare pre-refinement offers a graphical interface to
generate an mparameters file for frealign-2dx [39]. It will also put
the files required by frealign-2dx into the pre-refine directory, by
default. The default values should be reasonable, but users are
encouraged to fine-tune the parameters to their needs. We draw
the attention here to the Auto-refinement parameters available in
our modified version of frealign-2dx:
– FSC threshold for resolution limit? (thresh_fsc_ref): the resolution limit used in refinement (for preventing overfitting) will
be taken as the resolution where the Fourier shell correlation
(FSC) curve crosses this threshold (default: 0.800).
– FSC threshold for map improvement evaluation? (thresh_fsc_eval): any map improvement between refinement cycles will
be assessed by looking at the resolution where the FSC curve
crosses this threshold (default: 0.143).
– Minimum resolution limit? (res_min): this parameter prevents
the auto-refiner from using a resolution too low, which may
cause the refinement to diverge (default: 40 A ˚ ).
– Stay away from current map resolution? (ref_stay_way): this
parameter prevents the resolution limit from getting too close to
the current map resolution, which would potentially introduce
bias in the refinement (default: 2 A ˚ ).
– Try different combinations of parameter mask? (change_pmask): if enabled, this parameter makes the auto-refiner try
different combinations among the active refinement parameters
(ϕ, θ, ψ, x, y), before declaring convergence, which helps avoiding local minima (default: yes).
– Keep the tilt angle fixed? (no_theta): if active, the tilt angle (θ)
will be kept fixed. Sometimes this is helpful for 2D crystal data
(default: no).
For more information, please see Supplementary Note 2 of
[38]. After preparing the pre-refinement, it can be launched via
the Run pre-refinement script. Alternatively, it can be launched
from the command line by running the frealign_run_refine_auto
program inside the pre-refinement directory. The crystal averages
are only a few hundred for a typical dataset and they have a high
SNR. The pre-refinement should therefore complete in only a few
minutes and noticeably improve the tilt geometry estimation (now
converted to Euler angles), as shown in Fig. 3. Plots of the angular
276
Ricardo Righetto and Henning Stahlberg