Tutorial

Overview

Before run this tutorial, please make sure

• CTFMeasure and its dependencies are correctly installed
• The path has been added into the environment ${PATH}

The tutorial datasets can be downloaded from EMPIAR and the parameter files for running can be downloaded from here.

Tutorial-1 CTF estimation for cryo-ET tilt-series

This section shows how to use CTFMeasure to estimate the CTF parameters of a cryo-ET tilt-series.

• Step 0: Get the tutorial dataset

Download the tilt-series for this tutorial from EMPIAR. This tutorial is based on "mixedCTEM_tomo4.mrc" in EMPIAR-10064.

The parameter files for running can be downloaded from here.

• Step 1: Prepare the input micrograph list file

stack_to_list mixedCTEM_tomo4.st mixedCTEM_tomo4.rawtlt 175.0 mixedCTEM_tomo4.list

• Step 2: Run the program

CTFMeasure --input_micrograph_list mixedCTEM_tomo4.list --pixel_size 2.62 --cs 2.7 --voltage 300 --w 0.07

• Step 3: Analyze the estimated results

The estimated CTF parameters are saved in mixedCTEM_tomo4_ctf.txt. mixedCTEM_tomo4_avg.mrc stores the high-SNR average power spectrum of the tilt-series, and mixedCTEM_tomo4_diag.st stores the power spectrum and Thon ring fitting for every micrograph of the tilt-series. You can use 3dmod from IMOD or other MRC viewers to visualize the power spectrums and check the quality of fitting.

Estimation results for reference:

00@mixedCTEM_tomo4.st 36320.426 33194.453  168.7583 0.000000  175.0000  -60.0000  0.843336  7.7047
01@mixedCTEM_tomo4.st 36344.844 33523.445  165.6700 0.000000  175.0000  -58.0000  0.841390  8.4399
02@mixedCTEM_tomo4.st 35847.801 32724.881  159.3914 0.000000  175.0000  -56.0000  0.831349  7.6251
......
58@mixedCTEM_tomo4.st 38892.008 36136.277  167.8333 0.000000  175.0000   56.0000  0.810796  9.2136

Power spectrums for reference: The right halves are the computed power spectrums. A theoretical CTF and a rotationally-averaged power spectrum are inset in the top left and bottom left of each power spectrum, respectively.

Tutorial-2 CTF estimation for cryo-FIB lamella

This section shows how to use CTFMeasure to estimate the systematic tilt angle offset and the CTF parameters of a cryo-ET tilt-series collected with cryo-FIB milling.

• Step 0: Get the tutorial dataset

Download the tilt-series for this tutorial from EMPIAR. This tutorial is based on "tom1.st" in EMPIAR-10700.

The parameter files for running can be downloaded from here.

• Step 1: Prepare the parameter file

Write the following parameters into the parameter file para_ctf_10700.conf. Or you can just use the downloaded one.

N_zeros=10
resolution_max=10.0
resolution_min=30.0
resolution_adaptive=1
N_avg=1
box_conv=25
skip_offset_estimation=1
skip_offset_refinement=1
pre_offset_estimation=1
it=3
tight_blocks=1
optimize_with_average=0

• Step 2: Prepare the input micrograph list file

stack_to_list tom1.st tom1.rawtlt 85.5 tom1.list

• Step 3: Run the program

CTFMeasure --input_micrograph_list tom1.list --pixel_size 3.418 --cs 2.7 --voltage 300 --w 0.07 --params para_ctf_10700.conf

The above parameter file disables the iterative refinement of the absolute tilt angle offset to run faster. If you need a more accurate estimation of the absolute tilt angle offset, you can set skip_offset_estimation=0 and skip_offset_refinement=0 in the parameter file. However, the program will become much slower when refining the absolute tilt angle offset.

• Step 4: Analyze the estimated results

The estimated CTF parameters are saved in tom1_ctf.txt. tom1_avg.mrc stores the high-SNR tilt-series power spectrum, and tom1_diag.st stores the power spectrum and Thon ring fitting for every micrograph of the tilt-series.

Estimation results for reference:

00@tom1.st 62540.039 61165.461   41.3945 0.000000   85.5000  -61.7215  0.308991 12.3604
01@tom1.st 63178.336 62495.508   23.5812 0.000000   85.5000  -59.7215  0.311229 11.7572
02@tom1.st 62150.324 60971.855   40.9789 0.000000   85.5000  -57.7215  0.327732 12.3309
......
60@tom1.st 61083.574 59616.660   40.2237 0.000000   85.5000   58.2785  0.290625 10.9198

Power spectrums for reference: The angles listed below the power spectrums are the raw stage tilt angles.