Getting Started
0. Open Alignframes GUI Open Cygwin64 terminal (on desktop) and go where you copied the IMOD data folder cd /cygdrive/c/Users/YOUR_USERNAME/Documents/IMOD/ Open etomo front page by typing: etomo
IMOD Processing Pipeline
1. Motion Correction 2. Alignment 3. CTF correction 4. Tomogram reconstruction Pre-alignment CTF estimation Tilt/Positioning Post-processing IMOD Processing pipeline
Motion Correction
~100𝑛𝑚 ~100𝑛𝑚 Each frame of a tilt series is composed of “sub-frames” Motion Correction
𝚰 – IMOD processing – Correcting for motion artifacts Two main forces of motion during data collection: stage drift and beam-induced ice deformation Motion during sub-frame acquisition Average of motion
Without motion correction With motion correction 𝚰 – IMOD processing – Correcting for motion artifacts Single “sub-frames”
1. Select the .mdoc Motion Correction
2. Select the .tif images Motion Correction
3. re-select “metadata” box Motion Correction
4. Tick “Gain normalize” box and select the .dm4 gain file Motion Correction
5. Double check the other parameters are similar and go to Alignment tab Motion Correction
6. Select “Advanced” Motion Correction
7. Set Binning to 8 for alignment and 2 for output Motion Correction
8. Check “Do dose weighting” and “Dose weighting from .mdoc file 9. Check all parameters are similar and Run Alignframes Motion Correction
“tomo-g2_af.mrc” has been created, view it in 3dmod with “Open Output Tilt Series” Or through the terminal: 3dmod -I 1 tomo-g2_af.mrc (capital i ) Right click on the image to view the tilt series Motion Correction
Build Tomogram / Setup
Build Tomogram Open etomo front page again by typing in Cygwin terminal: etomo
1. Select the tilt series we motion corrected 3. Scan header and enter “0” for “Fiducial diameter (nm)” 2. Select “Single axis” 5. Check all parameters are similar and “Create Com Scripts” 4. Un-check “Series was..” Setup
Equivalent CLI command: newstack \ -InputFile tomo-g2_af.mrc \ -OutputFile tomo-g2_af_ali.mrc \ -TransformFile tomo-g2_af.xf \ -TaperAtFill 1,0 \ -AdjustOrigin \ -SizeToOutputInXandY 4092,20 \ -OffsetsInXandY 0,0 \ -ImagesAreBinned 0.5 Setup — .com files Command files (C-shell scripts) that drive IMOD processing steps. One .com per step — newst.com, tilt.com, eraser.com, etc. Structure — 3 types of lines # → comment $ → shell command ($setenv, $newstack, $if) Everything else → keyword + value fed to the program via stdin
X-ray Removal
1. Select “Pre- processing” 2 “Find X-rays” and “View X-ray” X-ray removal
1. “Create Fixed Stack” 2. “Use Fixed Stack” 3. “Done” X-ray removal

Interactive: Coarse Alignment

Quick reference for coarse alignment
Coarse Alignment
1. “Calculate Cross- Correlation” 2. Set binning to 4 3. “Generate Coarse Aligned Stack” and inspect tilt series 4. “Done” Alignment model Coarse Alignment

Interactive: Patch Tracking

Quick reference for patch tracking
Alignment Model — Fiducial Model
2. Set “Size of patches” to 300,300 3. Set patch overlap to (0.8,0.8) 4. Check “Use boundary model” and create one 1. Select “Use patch tracking” Alignment model Fiducial model
1. Select “Model” mode 2. Using mouse middle button to draw boundary around the cell 3. Press “s” to save, and close windows Alignment model Fiducial model – Boundary model
“Track Patches” and “Open Tracked Patches” Alignment model Fiducial model
Press “v” to open “Model View” and inspect patch trajectories Inspect patches in movie mode Alignment model Fiducial model
When using too many/small patches Patch residuals vectors Patch trajectories Alignment model Fiducial model
Patch residuals vectors Patch trajectories Without using boundary model Alignment model Fiducial model
“Done” Alignment model Fiducial model

Interactive: Fine Alignment

Quick reference for fine alignment (tiltalign)
Fine Alignment
1. Check “robust fitting” 2. Go to “Global Variables” tab Alignment model Fine Alignment
Set Rotation, Magnification, Tilt Angle, and Distortion solution types Alignment model Fine Alignment
1. Run cross-validation 2. Compute Alignment Alignment model Fine Alignment
“View 3D model” “View residual vectors” Alignment model Fine Alignment
Tomogram Positioning
Tomogram positioning & Volume trimming X Y X Z Y=1/4 Y=2/4 Y=3/4 Prompts user to delineate tomogram volume Y=1/4 Y=2/4 Y=3/4
1. Set tomogram thickness to 3000 2. Check “Use whole tomogram” and use “Binning 8” 3. Run “Create Whole Tomogram” and “Create Boundary Model Tomogram positioning & Volume trimming
1. Rotate the tilt series to view the Z/X view 2. Set Z value to roughly 1/3rd the thickness 3. (in “Model” mode) use middle-click to set 2 pairs of points on either side of the biological sample Tomogram positioning & Volume trimming 4. Repeat for Z ≈ 2/4 and Z ≈ 3/4 5. Press “s” to save and close
Tomogram positioning & Volume trimming Opening XYZ view can be helpful
1. Run “Compute Z shift” and “Create Final Alignment” 2. “Done” Tomogram positioning & Volume trimming
CTF Estimation
CTF estimation “Thon” rings visible in Power spectrum (Fourier transform) Defocus and microscope abherations introduce spatial frequency (phase) distorsions. Contrast Transfer Function (CTF) needs to be approximated to recover true object

Interactive: CTF & Phase Contrast

Explore defocus, phase contrast, and the contrast transfer function
1. Set Binning to 4 and run “Create Full Aligned Stack” CTF estimation
1. Click “View Full Aligned Stack” 2. In 3dmod press Shift+F to view tilt series in Fourier space CTF estimation
1. Click “Correct CTF” tab CTF estimation
1. Click “View Full Aligned Stack” CTF estimation
4. “Scan for Defocus then Autotune” Set “lower end of defocus range” to 2 and “higher end” to 8” 3. Check “Vary exponent of CTF function” Check “Do auto weighing and truncation” 6. “Save to File” 5. “Autofit All Views” 1. Set “Autofit” from -60 to 60 2. Check “Fit each view seperately” CTF estimation
1. Click “Correct CTF”, “View CTF Correction” and “Use CTF Correction” CTF estimation Before CTF correction After CTF correction
Tomogram Reconstruction
Tomogram Reconstruction “Generate Tomogram”
Tomogram Reconstruction 1. Check “Rotate around X axis 2. “Trim Volume” We can reorient the tomogram if it’s in the wrong orientation
Tomogram Reconstruction
CryoCARE Denoising
~100𝑛𝑚 “Sub-frames” are separated into “even” and “odd” stacks CryoCARE denoising Preparing “Even/Odd” stacks ~100𝑛𝑚
“Even” motion corrected frame “Odd” motion corrected frame CryoCARE denoising Preparing “Even/Odd” stacks alignframes -mdoc s1.st.mdoc -output tomo-g2_af.mrc -adjust -bin 8,2 -gain SuperRef_tomo-g2_00000_-11.0.dm4 -pixel 1.283 -evenodd 1 Creates “tomo-g2_af_even.mrc” and “tomo-g2_af_odd.mrc”
Use “combined” transformation files Use “combined” transformation files We apply the same transformation files to “Even” and “Odd” frames to generate tomograms that are identical in structure but differ only in the noise. Train & denoise ~2 hour ~10 mn ~10 mn Odd Tomogram Even Tomogram CryoCARE denoising Preparing “Even/Odd” stacks Denoised Tomogram
CryoCARE denoising Reconstructing “Even/Odd” tomograms mkdir even mkdir odd move tomo-g2_af_even.mrc even move tomo-g2_af_odd.mrc odd copy tomo-g2_af.xf even copy tomo-g2_af.xf odd copy tomo-g2_af.tlt even copy tomo-g2_af.tlt odd 1. Create folders and move even/odd stacks 2. Copy alignment files into both folders cd even newstack -input tomo-g2_af_even.mrc -output tomo-g2_af_even_ali.mrc -xform tomo-g2_af.xf -taper 1,0 -origin -size 1024,1440 -bin 4 3. Align even stack (newstack) 4. Reconstruct even tomogram (tilt) tilt -input tomo-g2_af_even_ali.mrc -output tomo-g2_af_even_rec.mrc -IMAGEBINNED 4 -TILTFILE tomo-g2_af.tlt -THICKNESS 1800 -RADIAL 0.35,0.035 -FalloffIsTrueSigma 1 -LOG 0.0 -SCALE 0.0,250.0 -MODE 1 - AdjustOrigin 6. Do the same in odd folder (replace the _even_ by _odd_ in the commands) clip rotx tomo-g2_af_even_rec.mrc tomo-g2_af_even_rotx.mrc 5. Rotate even tomogram (clip)
train_data_config.json train_config.json predict_config.json CryoCARE training and prediction is controlled through 3 configuration files and 3 command lines CryoCARE denoising Runnig CryoCARE
~30 mn training 20 epochs / 75iterations ~6 hours training 200 epochs / 100 iterations Original CryoCARE denoising

Interactive: CryoCARE Explained

Understand how CryoCARE denoising works