script presents a number of parameters to the user, the most important of which will be explained below.
3.3.1 Particle Picking
The classical unbending algorithm [26] generates a crosscorrelation (CC) profile (Fig. 1). The peaks in this CC profile
indicate the position of the unit cells, available after running the
Unbend II script during the 2D crystal processing in Subheading
3.2 (Fig. 1a, b). This information, stored in the file
_profile.dat for each micrograph, will be used to
pick the particles from the 2D crystals (Fig. 1c).
Optionally, a Phase Origin Shift can be applied to the CC
peaks. This is a translational shift, here presented in degrees in
relation to the unit cell dimensions. A phase origin shift of 180
in one direction means shifting by half the length of the unit cell.
Such a shift may be required when the center of the protein does
not coincide with the center of the crystal unit cell, as shown in
Fig. 2. The shift is calculated accordingly for non-tilted crystals,
based on the current estimate of the tilt geometry.
The Box size parameter defines the side length of the (squared)
box into which the particles will be windowed, in pixels. The
particles, in this context, are patches of the 2D crystals, and this
parameter defines how large such patches should be.
Differently from conventional SPA, it is not possible here to
have a single protein unit isolated in the box. Also, in the tilted
views the projection of the neighboring proteins overlaps with that
of the central protein, which has to be taken into account during
the particle alignments (see Fig. 4). For this reason, the box size
should be large enough to contain the protein of interest plus at
Fig. 1 Particle picking from unbending profile. (a) The unbending algorithm compares the observed and
predicted unit cell positions. Displacement vectors are 10Â exaggerated for visualization, with colors
indicating the displacement direction. (b) CC peaks indicating unit cell positions after unbending. Darker
dots indicate stronger peaks. (c) Particle picking based on the information from (b). Green circles are picked
particles (i.e., above user-selected threshold) and magenta circles are ignored particles. The blue squares
indicate the size (416 A ˚ in this case) of the overlapping patches to be extracted, with the crosses indicating the
box centers. Scale bar: 500 A ˚
272
Ricardo Righetto and Henning Stahlberg
3.3.1 Particle Picking
The classical unbending algorithm [26] generates a crosscorrelation (CC) profile (Fig. 1). The peaks in this CC profile
indicate the position of the unit cells, available after running the
Unbend II script during the 2D crystal processing in Subheading
3.2 (Fig. 1a, b). This information, stored in the file
pick the particles from the 2D crystals (Fig. 1c).
Optionally, a Phase Origin Shift can be applied to the CC
peaks. This is a translational shift, here presented in degrees in
relation to the unit cell dimensions. A phase origin shift of 180
in one direction means shifting by half the length of the unit cell.
Such a shift may be required when the center of the protein does
not coincide with the center of the crystal unit cell, as shown in
Fig. 2. The shift is calculated accordingly for non-tilted crystals,
based on the current estimate of the tilt geometry.
The Box size parameter defines the side length of the (squared)
box into which the particles will be windowed, in pixels. The
particles, in this context, are patches of the 2D crystals, and this
parameter defines how large such patches should be.
Differently from conventional SPA, it is not possible here to
have a single protein unit isolated in the box. Also, in the tilted
views the projection of the neighboring proteins overlaps with that
of the central protein, which has to be taken into account during
the particle alignments (see Fig. 4). For this reason, the box size
should be large enough to contain the protein of interest plus at
Fig. 1 Particle picking from unbending profile. (a) The unbending algorithm compares the observed and
predicted unit cell positions. Displacement vectors are 10Â exaggerated for visualization, with colors
indicating the displacement direction. (b) CC peaks indicating unit cell positions after unbending. Darker
dots indicate stronger peaks. (c) Particle picking based on the information from (b). Green circles are picked
particles (i.e., above user-selected threshold) and magenta circles are ignored particles. The blue squares
indicate the size (416 A ˚ in this case) of the overlapping patches to be extracted, with the crosses indicating the
box centers. Scale bar: 500 A ˚
272
Ricardo Righetto and Henning Stahlberg