|Tutorial - Tomographic Reconstructions in Priism (1/2)|
|This tutorial will guide you through the reconstruction process. There is a cryo dataset available as an example. Start by downloading the dataset and taking it through the entire process. Many of the intermediate files are also available for download to compare your results.
From the microscope: UCSF_TOMO outputs an ordered stack of data starting from the most negative tilt through to the most positive tilt (Movie 1). Contained within the header of this file are the tilt angles and the tilt axis and shifts used to collect the data. Note, the values of the tilt axis and shifts, while quite good, may need to be further refined as described below. If your data is coming from some other source, you can use the programs CopyRegion and insert_tilts to reorder and put the proper tilt angles in for the rest of the process. Merge_reorder can be used in place of CopyRegion.
|Movie 1. Drosophila centrosome imaged at 300kV, +/- 60 degrees every 2 degrees with a 20eV slit. Total electron dose =200 e-/A2. Note at one point the slit becomes visible.|
|1. Bin data (DataViews Resolution ReduceRes) = file_b4.mrc, file_tb2.mrc We usually begin working on a 500 x 500 file. Working on a smaller file greatly speeds up the process and also increases the signal to noise during cross correlation. (We bin the data initially on the chip, producing a 2k by 2k image. Then binning 4x give us the starting file and 2x the intermediate resolution file.)|
|2. Open EMTAR (Processing EM Processing Alignment + Reconstruction) There is excellent documentation for this process at EMTAR. NOTE: Much of this process is computationally expensive. See notes on parallel processing if on a cluster: likely EC3 or EC4
3. Set TiltSeries = file_b4.mrc (starting with binned 4 data)
4. Select Rough alignment defaults are correct (use header) Note: If a box has been selected, the box will appear darker.
B. Uncheck Rough Alignment, leave EMTAR open.
This outputs fileb4.bprmMn that contains the shifts used by UCSF_TOMO to track the sample during data collection. However, since these shifts are based upon the unbinned file, it is necessary to adjust them to match the 4x binned file (below). Note, if your data did not come from UCSF_TOMO, uncheck the use header box under Rough Alignment and a cross-correlation based alignment will be performed. If you know the tilt axis, even roughly, input it under EMROUGH: special parameters. (Skip step 5).
5. Set .bprmMn file to correct binning (Processing EM Processing Alignment File Manipulator)
A. Input file = file_b4.bprmMn
B. Output file = file_b4.bprmMn (file will be written in place)
C. Sampling ratio = 1/binning function in step 2, i.e. binning 4 = sampling ratio 0.25
D. Close Alignment File Manipulator
This next section, from Mass normalization to EWBP can all be done in one shot, especially if you are doing multiple data sets and have determined the parameters that work best at each point. I would recommend going through step by step at least the first time and examining your data at each point to make sure the data make sense and that you understand what these programs are doing. To do them all at once, simply check all of the boxes of the programs you want to run.
A. Quantity to fit = Average intensity (see documentation to understand average versus background)
B. Axis orientation = enter this if you know it better than the header
C. Special parameters Change Save to Show and Save
D. DoIt => file_b4.bprmMn and graph of fits will pop up (below).
E. Uncheck Mass normalization
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