Required inputs#

Tomogram#

Reconstructed tomogram in MRC format.
Allowed extensions are .mrc and .rec. Note that .rec extension does not mean different format of the file, just the extension is different. This naming comes from older versions of IMOD software, where reconstructions stored in MRC format used .rec extension to point out that the data is not stack of images but reconstruction.
IMPORTANT: For the weighting to be correctly computed the tomograms has to have xyz orientation, where z corresponds to the dimension that it perpendicular to the electron beam.

Template in MRC or EM format.
Currently it is safer to use template with a cubic size.
IMPORTANT: The contrast of the template has to be the same as of the tomogram. If the tomogram has features in black (which is typically for cryoET) then the template has to have the same representation. For example, Relion outputs inverted contrast (features are white) and such maps have to be inverted prior running TM.

Mask in MRC or EM format.
Mask is used to mask the template prior and thus should cover the volume of the template that you want to focus on.
It has to have the same size as the template.
It should have smoothened edges. If your mask has sharp edges it is recommended to blur it to avoid artifacts during Fast Fourier Transform (FFT).
The mask can have same shape as the template (so-called shape mask) or can be loose (e.g. a sphere or a cylinder).
It is recommended that the mask cover not only the template in its original orientation but also the template rotated by the smallest angular increment used during template matching. In that way, if a feature in the tomogram has orientation lying below the angular sampling it can still get sufficient correlation with the template.
The mask can be created using cryoCAT cryomask module.

Wedge list file in starfile format.
File containing information about single tilt images used for creating proper weighting during TM.
To fully exploit the power of GAPSTOP™ one should aim for as complete wedge list as possible, i.e. containing information on tilt angles, esimated defocus values for each tilt, electron dose used to correct each tilt during dose-filtering, basic technical information regarding the microscope used for acquisition (Cs, amplitude contrast).
It can be created using cryoCAT wedgeutils module.
To see example of the formatting and values, check the wedge_list.star provided as part of the tutorial.
Note that the wedge list can contain information on multiple tomograms/tilt series. The correct information is found based on the tomo_num value that needs to be correctly set in the parameter file.
The values in the wedge list correspond to the unbinned data - this does not have to be adapted to different binnings. The values are automatically adapted internally based on the binning value specified in the parameter file.

List of rotations used for template rotations.
It is a text file with three values per row, each row specifying one rotation. Rotations are specified by Euler angles following zxz convention. The values denote angles PHI (z1), THETA (x), PSI (z2) and refer to rotation of the reference.
The list can be created manually (if one wants to probe specific rotations), using GAPSTOP™ command line interface, directly on-the-fly (the angle list will be written out), or with cryoCAT.
IMPORTANT: The default convention is to store the angles in zxz order. However, STOPGAP default order is zzx. See the page on differences between GAPSTOP™ and STOPGAP. If you have list generated by STOPGAP you can still use it but you have to set the order parameter to zzx in the parameter file.
Note that the angles can be also specified numerically directly in the parameter file - in such case, the angle list will be created on-the-fly and written out to working directory folder.
The list is important not only during the TM itself but also when fetching the correct angles while creating particle lists.