howflared(1) General Commands Manual howflared(1)NAMEhowflared - measure microtubule end flaring and curvatureSYNOPSIShowflared [options] output_fileDESCRIPTIONHowflared 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-nopairand-pointare 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.OPTIONSHowflared 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-OutputFileFilenameOutput file for results. If this option is not entered, the first non-option argument will be used for the filename.-columns(-c)OR-ColumnsToOutputListofintegerrangesList 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-PointOutputFileFilenameOutput 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-nopairsis also entered, there will be an addi- tional output file with positions of flaring start points along the axis, as described above.-pixel(-pi)OR-PixelSizeDefaultFloatingpointDefault 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-WidthDefaultFloatingpointDefault width between walls in nm. This value is used to com- pute normalized areas for unpaired walls. The default is 20.-surface(-s)OR-UseSurfaceNumbersUse 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-NoPairsOrMarkersThere 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-ModelFileFilenameInput model file with tracings of microtubule walls (Successive entries accumulate)-fit(-f)OR-FitTopAndBottomTwofloatsUpper 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-IdentifierIntegerID 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-usagePrint help output-StandardInputRead parameter entries from standard input.HISTORYWritten by David Mastronarde in 1996 Modified for PIP input and incorporation into IMOD, 10/14/06BUGSEmail bug reports to mast at colorado dot edu. IMOD 4.10.15 howflared(1)