Contents

Tomography tutorial (EMPIAR-10164)

Tomography tutorial (EMPIAR-10164)

This tutorial shows how to convert raw tilt-series from EMPIAR-10164 (HIV-1 Gag) into a ~3A resolution structure.

Total running time required to complete this tutorial: ~20 hrs.

We first use the command line to download and decompress a tbz file containing a subset of 5 tilt-series (down-sampled 2x compared to the original data), and an initial model:

# cd to a location in the shared file system and run:

wget https://nextpyp.app/files/data/nextpyp_tomo_tutorial.tbz
tar xvfz nextpyp_tomo_tutorial.tbz

Open your browser and navigate to the url of your nextPYP instance (e.g., https://nextpyp.myorganization.org).

Step 1: Create a new project

Data processing runs are organized into projects. We will create a new project for this tutorial

  • The first time you login into nextPYP, you should see an empty Dashboard:

    Create new project

  • Click on Create new project, give the project a name, and select Create

    Create new project

  • Select the new project from the Dashboard and click Open

    Select new project

  • The newly created project will be empty and a Jobs panel will appear on the right

    Empty project

Step 2: Import raw tilt-series

Import the raw tilt-series downloaded above ( <1 min)

  • Go to Import Data and select Tomography (from Raw Data)

    Import dialog

  • A form to enter parameters will appear:

    File browser

  • Go to the Raw data tab:

    • Set the Location of the raw data by clicking on the icon and browsing to the directory where the you downloaded the raw movie frames

    • Type TS_*.tif in the filter box (lower right) and click on the icon to verify your selection. 205 matches should be displayed

    • Click Choose File Pattern to save your selection

    • Click on the Microscope parameters tab

    File browser

    • Set Pixel size (A) to 1.35

    • Set Acceleration voltage (kV) to 300

    • Set Tilt-axis angle (degrees) to 85.3

    Project dashboard

  • Click Save and the new block will appear on the project page

    Project dashboard

  • The block is in the modified state (indicated by the sign) and is ready to be executed

  • Clicking the button Run will show another dialog where you can select which blocks to run:

    Gain thumbnail

  • Since there is only one block available, simply click on Start Run for 1 block. This will launch a process that reads one tilt image, applies the gain reference (if applicable) and displays the resulting image inside the block

    Gain thumbnail

  • Click inside the block to see a larger version of the image

Step 3: Pre-processing

Movie frame alignment, CTF estimation and particle picking ( 6 min)

  • Click on Tilt-series (output of the Tomography (from Raw Data) block) and select Pre-processing

    File browser

  • Go to the Frame alignment tab:

    • Set Frame pattern to TILTSERIES_SCANORD_ANGLE.tif

    • Click on the CTF determination tab

    • Set Max resolution to 5.0

    • Click on the Tomogram reconstruction tab

    • Set Binning factor for reconstruction to 8

    • Set Thickness of reconstruction (unbinned voxels) to 2048

    • Click on the Virion/spike detection tab

    • Set Virion radius (A) to 500.0

    • Set Virion detection method to auto

    • Click on the Resources tab

    • Set Threads per task to 7

    • Set Memory per task to 14

    • Set other runtime parameters as needed (see Computing resources)

  • Click Save, Run, and Start Run for 1 block. Follow the status of the run in the Jobs panel

    File browser

  • Click inside the Pre-processing block to inspect the results (you don’t need to wait until processing is done to do this). Results will be grouped into tabs:

    Dataset statistics
    Table view
    Gallery view
    Tilt-series (Tilts)
    Tilt-series (Alignment)
    Tilt-series (CTF)
    Tilt-series (Reconstruction)
    Tilt-series (Segmentation)

Tip

While on the Tilt Series tab, use the navigation bar at the top of the page to look at the results for other tilt-series

Step 4 (optional): Virion segmentation

Detection and segmentation of virions (interactive step)

This step is optional, but it showcases tools available in nextPYP to work with virions:

  • Go inside the Pre-processing block, click on the Tilt Series tab, and select Segmentation

    Virion segmentation

  • Select a virion from the table to show its 3D segmentation (8 different thresholds are shown as yellow contours in columns 1-8). The column number highlighted in blue represents the selected threshold value (default is 1, click on a different column to select a better threshold). If none of the columns look reasonable (or if you want to ignore the current virion virion), select the last column (“-“)

  • Repeat this process for all virions in the table and all tilt-series in the dataset

Tip

Click on > Keyboard shortcuts (under the virion image) to reveal instructions on how to speed up the threshold selection process

Step 5: Particle detection

Particle detection from virion surfaces ( 3 min)

  • In the Pre-processing block, click on the menu icon and select the Edit option.

  • Go to the Virion/spike detection tab:

    • Set Spike detection method to uniform

    • Set Minimum distance between spikes (voxels) to 8

    • Set Size of equatorial band to restrict spike picking (A) to 800

    • Click on the Particle detection tab

    • Set Detection radius (A) to 50

  • Click Save, Run, and Start Run for 1 block

  • Navigate to the Reconstruction group inside the Tilt-series tab to inspect the virion and spike coordinates:

    Spike coordinates

Step 6: Reference-based refinement

Reference-based particle alignment ( 8 hr)

  • Click on Particles (output of the Pre-processing block) and select Particle refinement

  • Go to the Sample tab:

    • Set Molecular weight (kDa) to 300

    • Set Particle radius (A) to 150

    • Set Symmetry to C6

    • Click on the Extraction tab

    • Set Box size (pixels) to 192

    • Set Image binning to 2

    • Click on the Refinement tab

    • Specify the location of the Initial model by clicking on the icon , navigating to the folder where you downloaded the data for the tutorial, and selecting the file EMPIAR-10164_init_ref.mrc

    • Click in Alignments from sub-volume averaging to select the initial parameters text file tomo-preprocessing-*_original_volumes.txt from Pre-processing

    • Set Max resolution (A) to 8.0

    • Check Use signed correlation

    • Check Skip refinement

    • Click on the Constrained refinement tab

    • Set Last exposure for refinement to 10

    • Set Optimizer - Exhaustive search points to 50000

    • Check Refine particle alignments

    • Set Phi range (degrees) and Theta range (degrees) to 10

    • Set Translation range (voxels) to 50

    • Click on the Reconstruction tab

    • Set Max tilt-angle to 50

    • Set Min tilt-angle to -50

    • Click on the Resources tab

    • Set Walltime per task to 9:00:00

    • Set Threads (merge task) to 6

    • Set Memory (merge task) to 20

  • Save your changes, click Run and Start Run for 1 block

  • One round of refinement and reconstruction will be executed. Click inside the block to see the results

    Iter 2

Step 7. Fully constrained refinement

Tilt-geometry parameters and particle poses are refined in this step ( 1.5 hr)

  • Edit the settings of the existing Particle refinement block and go the Refinement tab:

    • Set Max resolution (A) to 8:10:8:6

    • Set Last iteration to 5

    • Click on the Constrained refinement tab

    • Check Refine tilt-geometry

    • Set Optimizer - Max step length to 100

    • Set Optimizer - Exhaustive search points to 0

    • Set Phi range, Psi range and Theta range to 20.0

    • Click on the Exposure weighting tab

    • Check Dose weighting

    • Set Frame weight fraction to 4

  • Click Save, Run, and Start Run for 1 block to execute three rounds of refinement and reconstruction

  • Click inside the Particle refinement block to inspect the results:

    Iter 5

Tip

Use the navigation bar at the top left of the page to look at the results for different iterations

Step 8. Filter particles

Identify duplicates and particles with low alignment scores ( 4 min)

  • Click on Particles (output of the Particle refinement block) and select Filter particles

  • Go to the Particle filtering tab:

    • Set Score threshold to 2.5

    • Set Min distance between particles (A) to 10

    • Specify the location of Input parameter file by clicking on the icon and selecting the file tomo-coarse-refinement-*_r01_05.par.bz2

    • Set Lowest tilt-angle to -15.0

    • Set Highest tilt-angle to 15.0

    • Check Generate reconstruction after filtering

    • Click on the Refinement tab

    • Specify the location of the Initial model by clicking on the icon and selecting the file tomo-coarse-refinement-*_r01_05.mrc

  • Click Save, Run, and Start Run for 1 block. You can see how many particles were left after filtering by looking at the job logs.

Step 9 (optional): Permanently remove bad particles

Permanently remove bad particles to improve processing efficiency downstream ( 1 min)

  • Edit the settings of the existing Filter particles block

  • Go to the Particle refinement tab:

    • Check Permanently remove particles

    • Uncheck Generate reconstruction after filtering

  • Click Save, Run, and Start Run for 1 block to launch the job

Step 10. Region-based local refinement (before masking)

Constraints of the tilt-geometry are applied over local regions ( 1 hr)

  • Click on Particles (output of Filter particles block) and select Particle refinement

  • Go to the Sample tab:

    • Set Particle radius to 100

    • Click on the Extraction tab

    • Set Box size (pixels) to 384

    • Set Image binning to 1

    • Click on the Refinement tab

    • Specify the location of the Initial model by clicking on the icon and selecting the file tomo-fine-refinement-*_r01_02.mrc

    • Select the location of the Initial parameter file by clicking on the icon and selecting the file tomo-fine-refinement-*_r01_02.par.bz2 (select the file tomo-fine-refinement-*_r01_02_clean.par.bz2 if bad particles were permanently removed in the previous step)

    • Set Max resolution (A) to 6:5

    • Set Last iteration to 3

    • Check Skip refinement

    • Click on the Constrained refinement tab

    • Set Last exposure for refinement to 4

    • Set Number of regions to 8,8,2

    • Set Translation range (voxels) to 20.0

  • Click Save, Run, and Start Run for 1 block to run the job

  • Click inside the Particle refinement block to inspect the results:

    Iter 3

Step 11: Create shape mask

Use most recent reconstruction to create a shape mask ( <1 min)

  • Click on Particles (output of Particle refinement block) and select Masking

  • Go to the Masking tab:

    • Select the Input map by click on the icon and selecting the file tomo-coarse-refinement-*_r01_03.mrc

    • Set Threshold for binarization to 0.45

    • Check Use normalized threshold

  • Click Save, Run, and Start Run for 1 block to run the job

  • Click on the menu icon of the Masking block, select the Show Filesystem Location option, and Copy the location of the block in the filesystem (we will use this in the next step))

  • Click inside the Masking block to inspect the results of masking.

Note

You may need to adjust the binarization threshold to obtain a mask that includes the protein density and excludes the background (a pre-calculated mask is provided with the raw data if you rather use that).

Step 12: Region-based constrained refinement

Constraints of the tilt-geometry are applied over local regions ( 2 hr)

  • Edit the settings of the existing Particle refinement block and go to the Refinement tab:

    • Set Max resolution (A) to 6:5:5:4:3.5

    • Uncheck Resume refinement

    • Set First iteration to 4

    • Set Last iteration to 6

    • Specify the location of the Shape mask produced in Step 11 by clicking on the icon , navigating to the location of the Masking block by copying the path we saved above, and selecting the file frealign/maps/mask.mrc

  • Click Save, Run, and Start Run for 1 block to run the job

  • Click inside the Particle refinement block to inspect the results:

    Iter 6

Step 13: Particle-based CTF refinement

Per-particle CTF refinement using most recent reconstruction ( 3 hr)

  • Click on the menu icon from the Particle refinement block and choose the Edit option

  • Go to the Refinement tab:

    • Set Max resolution (A) to 3.1

    • Set Last iteration to 7

    • Click on the Constrained refinement tab

    • Set Last exposure for refinement to 10

    • Uncheck Refine tilt-geometry

    • Uncheck Refine particle alignments

    • Check Refine CTF per-particle

  • Click Save, Run, and Start Run for 1 block

  • Click inside the Particle refinement block to inspect the results

    Iter 7

Step 14: Movie frame refinement

Particle-based movie-frame alignment and data-driven exposure weighting ( 3 hr)

  • Click Particles (output of Particle refinement block) and select Movie refinement

  • Go to the Sample tab:

    • Set Particle radius to 80

    • Click on the Refinement tab

    • Specify the Initial model by clicking on the icon and selecting the file tomo-coarse-refinement-*_r01_07.mrc

    • Specify the Input parameter file by clicking on the icon and selecting the file tomo-coarse-refinement-*_r01_07.par.bz2

    • Set Max resolution (A) to 3.2

    • Click on the Constrained refinement tab

    • Set Last exposure for refinement to 4

    • Uncheck Refine CTF per-particle

    • Check Movie frame refinement

    • Check Regularize translations

    • Set Spatial sigma to 200.0

  • Click Save, Run, and Start Run for 1 block

  • Click inside the Particle refinement block to inspect the results:

    Iter 2

Step 15: Refinement after movie frame refinement

Additional refinement using new frame alignment parameters ( 1 hr)

  • Click on the menu icon from the Movie refinement block and choose the Edit option.

  • Go to the Refinement tab:

    • Set Max resolution (A) to 3.3

    • Click on the Constrained refinement tab

    • Set Min number of projections for refinement to 2

    • Check Refine tilt-geometry

    • Set Translation range (pixels) to 10.0

    • Set Tilt-angle range (degrees) and Tilt-axis range (degrees) to 1.0

    • Check Refine particle alignments

    • Set Phi range (degrees) to 1.0

    • Set Psi range (degrees) to 1.0

    • Set Theta range (degrees) to 1.0

    • Set Translation range (voxels) to 10.0

    • Uncheck Movie frame refinement

  • Click Save, Run, and Start Run for 1 block

  • Click inside the Particle refinement block to inspect the results:

    Iter 3

Step 16: Map sharpening

Apply B-factor weighting in frequency space ( <1 min)

  • Click Movies (output of Movie refinement block) and select Post-processing

  • Go to the Post-processing tab:

    • Specify the First half map by clicking on the icon and selecting the file tomo-flexible-refinement-*_r01_half1.mrc (output of the Movie refinement block)

    • Set Automask threshold to 0.4

  • Click Save, Run, and Start Run for 1 block

  • You can inspect the result by clicking inside the Map sharpening block:

    Final map

Note

Running times were measured running all tilt-series in parallel on nodes with 124 vCPUs, 720GB RAM, and 3TB of local SSDs

See also

previous

Single-particle tutorial (EMPIAR-10025)

next

Classification tutorial (EMPIAR-10304)