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.TH howflared 1 4.6.34 IMOD
.SH NAME
howflared - measure microtubule end flaring and curvature
.SH SYNOPSIS
howflared [options] output_file
.SH DESCRIPTION
Howflared analyzes tracings of the walls of microtubules (MTs) to compute
various measures of the amount of curvature or "flaring" at the MT end. The
IMOD model with MT tracings must meet a number of requirements. The first
step is to extract the MTs into subvolumes so that they are oriented
vertically, with the end facing downward. This can be done with
Mtrotlong(1). The orientations do not need to be perfect. Once subvolumes
have been extracted as a set, they can be loaded into 3dmod together as a
4-dimensional data set. The 4th dimension is referred to as "time".
Alternatively, a central slice can be extracted from each and a stack made
with these central slices.
.P
The program will also work with lines drawn in an arbitrary plane in
the Slicer window, although it cannot analyze pairs of left and right
walls with such data. Each contour is fit to a plane and rotated so
that it lies in an X/Y plane, then X/Y coordinates are analyzed as usual.
.P
The tracings must all be drawn starting near the top of the image in Y and
ending at the MT end. Each tracing defines two regions of an MT wall: a
linear segment before the end, and a potentially curved segment. A line is
fit to a defined part of the linear segment and extrapolated into the region
of the end. The quantitative measures are based on the deviation of the
curved segment from this extrapolated line. Thus, a tracing must include a
linear segment, and there must be some way to indicate where the linear
segment ends and the measurement of the deviation should begin. There are
essentially three different ways to indicate this boundary.
.P
One way is to specify how far down from the top of the tracing the
beginning and the end of the line fit should be. This method is suitable if
all the tracings have similar starting points near the top of the subvolumes
and their curvature starts at similar locations as well. The starting and
ending points can be specified as either absolute units (in pixels) or as a
fraction of the distance from the maximum to the minimum Y value in the
tracing. The second method is to use a horizontal line, consisting of only
two points, entered as a separate contour, to specify the boundary point.
With this method, one might still enter an ending limit, in which case the
maximum of the ending limit and the Y position of the marker line are used
for the end of the linear fit, but the measurement of deviation starts at
the marker position. The third method is to model the linear segment with
just two points. In this case, one would enter 0,0 for starting and ending
limits. The second and third methods can both used in the same model; the
second point will be used as the boundary only if there is no horizontal
marker line.
.P
The program can analyze either two paired walls of a MT, or a single wall.
When two walls are paired, the linear fit finds a single slope for the two
walls, and a width between them. This width is used for normalizing some of
the flaring measurements. If there are also single, unpaired walls, a width
is assumed when normalizing these measurements (see -width option). When a
horizontal marker is used for a pair of walls, it should be drawn from near
the boundary point on the left wall to near the boundary point on the right
wall. A single wall is treated like the wall on the left of a pair; so in
this case a marker line should have its left end near the boundary point on
the wall.
.P
The program needs to be able to tell when two MTs are paired, and when a
marker line belongs with one or two MTs. Nothing special needs to be done
when modeling in only a single plane per MT. If you are modeling in
multiple sub-volumes, the program will segregate the tracing based on their
times; if you are modeling in a stack of single slices from different MTs,
the segregation will be based upon Z value. However, if you want to trace
multiple, unpaired walls in the Slicer, either you need to segregate them by
placing them in either separate objects or separate surfaces, or you need to
use the -nopairs option. Thus, you could have one object for all of the
first tracings in each MT, a second object for the second tracing, etc.
Similarly, you could use the surface number 0 for the first tracing in each
MT, 1 for the second, etc.; or you could simply put each tracing (or tracing
pair) with associated marker into a separate surface. If you are willing to
have no marker lines, then you can use the -nopairs option to indicate that
there are neither pairs nor markers.
.P
The program computes various quantities and stores them in columns of a
data matrix; you specify which columns you want output with -columns. The
items and their units are:
.P
The area between the tracing and the extrapolation of the wall, down to
the minimum Y value of the tracing (square nm).
The square root of this area (nm)
The sum, over all line segments, of the product of the line segment length
and the angle between the segment and the extrapolation of the
wall(nm*radians)
The area divided by the square of the average MT width (square MT widths)
The square of the area divided by the average MT width (MT widths)
The angular sum divided by the average MT width (MT widths * radians)
The total length of the tracing after the boundary (nm)
The final angular deviation of the tracing from the extrapolation of the
wall (degrees)
The angular change in degrees per nm of length
The average radius of curvature in nm.
.P
The first 6 items are placed into columns 1-6 for the left wall of a pair
or for an unpaired wall, and into columns 7-12 for the right wall of a pair.
The sum of the two values for left and right in placed in columns 13-18.
The last 4 items are placed into columns 19-22 for the left wall of a pair
or for an unpaired wall, and into columns 23-26 for the right wall of a
pair.
.P
In the output file, these columns of output are preceded by 3 or 4
numbers. The first number is a model identifier; the second is the object
number. If you used surface numbers to segregate tracings, the third of 4
numbers is the surface number; if you used the -nopairs option, the third of
4 numbers is the contour number. The last number is the time value, or the
Z value.
.P
If the \fB-nopair\fR and \fB-point\fR are both selected, the program
will assume that the start of the flare is at the second point of each
contour, and that different microtubules are modeled with different
times. It will then compute the position of the second point along the
axis of the microtubule for each protofilament and report these
distances in a file with the name "starts." prepended to the name of
the points file. For each protofilament, there will be a line with the
model number, the time, the contour number, and this position, which is
relative to the average starting point of all the contours in the
microtubule. After all the protfilaments for each microtubule, there
will then be a line with the model number and time, "SD", and the
standard deviation of those distances.
.SH OPTIONS
Howflared uses the PIP package for input exclusively (see the manual page
for pip(1)). The following options can be specified either as command line
arguments (with the -) or one per line in a command file or parameter file
(without the -). Options can be abbreviated to unique letters; the
currently valid abbreviations for short names are shown in parentheses.
.P
INSERT OPTION TEXT HERE
.TP
.B -StandardInput
Read parameter entries from standard input.
.SH HISTORY
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Written by David Mastronarde in 1996
Modified for PIP input and incorporation into IMOD, 10/14/06
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.SH BUGS
Email bug reports to mast@colorado.edu.