ctfplotter(1) ctfplotter(1) NAME ctfplotter - estimate defocus values of a tilt series SYNOPSIS ctfplotter options DESCRIPTION This GUI program will plot the logarithm of a rotationally averaged power spectrum of an input tilt series after subtracting the noise floor. The method is based on periodogram averaging; namely, averaging of spectra from small, overlapping areas of the images, referred to as tiles. The user can interactively choose which projection views are included in the averaging. The noise floor is based on actual measurements of noise in an image taken with no specimen in the beam. Thus, in order to use this pro- gram, you need to collect a set of such blank images, at a series of beam intensities, and place each image in a separate file. These noise files are specific not only to the microscope but also to the CCD cam- era used, the operating voltage, and the binning. The mean counts in these files should span the range of the mean counts in the data being analyzed. The interval between intensities can be a factor of 1.5; interpolation is used to estimate the noise for an image whose mean counts do not match one of the noise files. For example, for a camera that has a gain of 10 counts per electron, one might collect images at mean counts near 100, 150, 225, etc., up to 2210. With a 1 nm pixel size, these noise files could be used with images where the dose recorded at the camera ranged from 0.1 to 2.2 electrons per square Angstrom. If you need files for analyzing both unbinned and binned data, you can take one set of unbinned noise images and then bin them with a Newstack command like this: newstack -bin 2 -mult 4,0 input_file output_file The multiplication by 4 (the square of the binning factor) is necessary to make the counts be the same as they would be for binned images from the microscope with the same exposure. If there are significant numbers of X-rays in the noise images, you should clean them with Ccderaser, such as with: ccderaser -find -peak 8 -diff 6 input_file output_file The noise files must have positive means; i.e., their values should be proportion to the recorded electrons. If they are acquired with soft- ware that subtracts 32768 before storing as signed integers, then you need to add this offset back to the data in order to use them as noise files. When you start the program, it will first load and analyze all of the noise files. Then it will read in the images within the specified ini- tial angular range and compute power spectra from the central tiles. This initial computation of tile power spectra is the most time-consum- ing step, whereas recomputing a summed power spectrum from the spectra of individual tiles is quick. The program thus stores the power spec- trum for each tile in a cache when it is computed. You will have to wait several seconds whenever you add non-central tiles from the cur- rent angular range, or when you change the angular range to include views that have not been analyzed before. However, once all of the tiles on all views have been analyzed, the program will respond quickly when you change angular ranges or defocus values. To read a general description of the plots and tool buttons, click the handbook icon to access the HTML help page. Found defocus values can be saved in a text file that can be used as input to the CTF correction program ctfphaseflip. A sample command file for running the program at the command line can be found in the IMOD/com directory. Options Ctfplotter uses the PIP package for input (see the manual page for pip). Options can be specified either as command line arguments (with the -) or one per line in a command file (without the -): -input OR -InputStack File name Input stack whose defocus will be estimated. -angleFn OR -AngleFile File name File containing tilt angles for the input stack. Each line of this file is the tilt angle for a view of the input stack. The angles are listed in order starting from view 1. -invert OR -InvertTiltAngles Invert the sign of the tilt angles to compensate for a left- handed coordinate system in the microscope. When the sign of the tilt angles and the value of the tilt axis rotation angle are such that reconstructions are generated with inverted hand- edness, then this option is needed to keep power spectra for off-center tiles from being shifted in the wrong direction. -offset OR -OffsetToAdd Floating point The program must analyze data values where 0 corresponds to no electrons recorded. Use this option to specify a value to add to your input stack that will make values positive and propor- tional to recorded electrons. The value will not be added to noise files; they are required to be positive. The program automatically sets this value to 32768 if it recognizes a file from FEI data acquisition software with a minimum below 0, or if the minimum and mean of a non-FEI file are sufficiently nega- tive. -config OR -ConfigFile File name The configure file specifies the noise files used to estimate the noise floor, one file per line. The files can be specified with either absolute paths or with paths relative to the loca- tion of the configure file itself. -defFn OR -DefocusFile File name File to store found defocuses. Each entry in the file will con- sist of the starting and ending view number of the range of views being fit to (numbered from 1), the starting and ending angle of the tilt angle range being fit to, and the defocus value in nanometers (underfocus positive). If this file already exists when the program starts, it will be read in and the results displayed in a table in the Angle Range and Tile Selec- tion dialog. The previous version will become a backup file (with ~ added to its name) when new results are saved to this file. -aAngle OR -AxisAngle Floating point Specifies how much the tilt axis deviates from vertical (Y axis). This angle is in degrees. It follows the right hand rule and counter-clockwise is positive. -psRes OR -PSResolution Integer The number of points over which CTF will be computed. The Nyquist frequency is divided into equal intervals delineated by these points. -tileSize OR -TileSize Integer The tile size each strip will be tessellated into. The size is in pixels and the tiles are square. Each view is first divided into strips that are considered to have constant defocus. -volt OR -Voltage Integer Microscope voltage in kV. -cache OR -MaxCacheSize Integer To speed up computation, ctfplotter uses a cache to hold the rotationally averaged power spectra of individual tiles at 20 times the final power spectrum resolution. This option controls the cache size. The default value is 1000 megabytes, which should be enough for all slices in a 4Kx4K tilt series with the default tile size. -cs OR -SphericalAberration Floating point Microscope spherical aberration in millimeters -defTol OR -DefocusTol Integer Defocus tolerance in nanometers defining the center strips. The center strips are taken from the central region of a view that has defocus difference less than this tolerance. These kind of center strips from all views within AngleRange are considered to have a constant defocus and are used to compute the initial CTF after being further tessellated into tiles. -pixelSize OR -PixelSize Floating point Image pixel size in nanometers. -ampContrast OR -AmplitudeContrast Floating point The fraction of amplitude contrast. For Cryo-EM, it should be between 0.07 and 0.14. -expDef OR -ExpectedDefocus Floating point Expected defocus at the tilt axis in nanometers, with a positive valie for underfocus. The frequency of the first zero of the CTF curve is first computed based on this expected defocus. The segments of the CTF curve of the input stack around that fre- quency are selected to be fitted. -leftTol OR -LeftDefTol Floating point Defocus tolerance in nanometers for strips to the left of the center strip. When non-center strips are included in the aver- age, strips to the left of center are included if their defocus difference is less than the given value. -rightTol OR -RightDefTol Floating point Defocus tolerance in nanometers for strips to the right of the center strip. When non-center strips are included in the aver- age, strips to the right of center are included if their defocus difference is less than the given value. -range OR -AngleRange Two floats Starting and ending tilt angles for initial analysis. Views with a tilt angle within this range are used to compute the CTF curve. -debug OR -DebugLevel Integer Debug level, 0-3. 0: quiet. 1: user messages. 2: cache, tile iteration messages. 3: additional fitting messages. the default is 1. -param OR -Parameter Parameter file Read parameter entries from this file. -StandardInput Read parameter entries from standard input AUTHORS Quanren Xiong David Mastronarde SEE ALSO ctfphaseflip BUGS Prior to IMOD 4.0.29, Ctfplotter had a bug in which the view numbers written to the defocus file were numbered from 0, not 1. When Ctfplot- ter reads in an existing defocus file, it will do its best to detect this situation and adjust all the view numbers up by 1. If it does detect an inconsistency between view numbers and angular ranges, it will issue a warning. Email bug reports to mast at colorado dot edu. BL3DEMC 4.3.7 ctfplotter(1)