howflared(1) General Commands Manual howflared(1)
NAME
howflared - measure microtubule end flaring and curvature
SYNOPSIS
howflared [options] output_file
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 require-
ments. 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. 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.
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.
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 seg-
ment. 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.
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 suit-
able 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 hor-
izontal line, consisting of only two points, entered as a separate con-
tour, 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 lin-
ear fit, but the measurement of deviation starts at the marker posi-
tion. 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 lim-
its. 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 horizon-
tal marker line.
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 sin-
gle, unpaired walls, a width is assumed when normalizing these measure-
ments (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.
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 trac-
ing 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.
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:
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.
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.
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.
If the -nopair and -point 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.
OPTIONS
Howflared uses the PIP package for input exclusively (see the manual
page for pip). 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.
-output (-o) OR -OutputFile File name
Output file for results. If this option is not entered, the
first non-option argument will be used for the filename.
-columns (-c) OR -ColumnsToOutput List of integer ranges
List of columns to output (values ranging from 1 to 26). For
paired MT walls, columns 1-6 and 19-22 are for the left walls,
7-12 and 23-26 are for the right walls, and 13-18 are the sum of
values from 1-6 and 7-12. Unpaired walls are treated as left
walls, and their values will appear in both columns 1-6 and
13-18. See description of computed values above.
-point (-po) OR -PointOutputFile File name
Output file for points in each wall, starting at the point where
the flare measurement begins. The points are rotated so that
the wall above that point would be vertical, and they are
expressed in nanometers relative to the starting point. For
each MT wall, there is a line starting with the number of
points, then containing the 3 or 4 numbers described above as
preceding the columns of output in the standard output file, and
ending with 1 for a left or unpaired wall, or 2 for a right
wall. If the -nopairs is also entered, there will be an addi-
tional output file with positions of flaring start points along
the axis, as described above.
-zout (-z) OR -OutputRotatedZ
Output Z values after rotation into a plane along with X and Y
in the point output file.
-pixel (-pi) OR -PixelSizeDefault Floating point
Default pixel size in nm, which will be used for any models that
do not have a pixel size defined. The default is 1, namely
results will be in pixels.
-width (-w) OR -WidthDefault Floating point
Default width between walls in nm. This value is used to com-
pute normalized areas for unpaired walls. The default is 20.
-surface (-s) OR -UseSurfaceNumbers
Use surface numbers to determine whether two wall tracings are
paired and whether a horizontal marker contour matches a wall
tracing. This is not the default because surface numbers might
have been introduced by accident.
-nopairs (-n) OR -NoPairsOrMarkers
There are no paired tracings and no horizontal marker lines.
With this option you do not need to segregate tracings in sepa-
rate objects or surfaces.
-model (-m) OR -ModelFile File name
Input model file with tracings of microtubule walls (Successive
entries accumulate)
-fit (-f) OR -FitTopAndBottom Two floats
Upper and lower limits for the line fit. The limits are entered
as either an absolute distance from the maximum Y for a MT or a
fraction of the distance from the maximum to the minimum Y. If
the upper limit is 0, the maximum Y is used. If the lower limit
is 0, the limit used depends on whether there is a horizontal
marker line or not. If there is a line, it is used as the
limit; if not, the second point of the contour is used as the
lower limit. Enter this option once to use the same limit for
all models, or once per model. If it is not entered, the
default limits are 0,0. (Successive entries accumulate)
-id (-i) OR -Identifier Integer
ID number for output. Enter this option either once to specify
a starting ID number, or once per model. If it is not entered,
the ID numbers start at 1. (Successive entries accumulate)
-help (-h) OR -usage
Print help output
-StandardInput
Read parameter entries from standard input.
HISTORY
Written by David Mastronarde in 1996
Modified for PIP input and incorporation into IMOD, 10/14/06
BUGS
Email bug reports to mast at colorado dot edu.
IMOD 5.2.0 howflared(1)