Single-particle tutorial (EMPIAR-10025)

This tutorial shows how to convert raw movies from EMPIAR-10025 (T20S proteasome) into a ~3A resolution structure.

We first use the command line to download and decompress a tbz file containing a subset of 20 movies, the gain reference, and an initial model:

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

wget https://nextpyp.app/files/data/nextpyp_spr_tutorial.tbz
tar xvfz nextpyp_spr_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 movies

Import the raw movies downloaded above ( <1 min)

  • Click Import Data and select Single Particle (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 clicking on the icon and browsing to the directory where the you downloaded the raw data

    • Type 14*.tif in the filter box (lower right) and click on the icon to verify your selection. 20 matches should be displayed

    • Click Choose File Pattern

    • Click on the Gain reference tab

    File browser

    • Set the Location of the gain reference by clicking the icon and navigating to the directory where you downloaded the data for the tutorial. Select the file Gain.mrc and click Choose File

    • Check Flip vertically

    • Click on the Microscope parameters tab

    File browser

    • Set Pixel size (A) to 0.66

    • Set Acceleration voltage (kV) to 300

    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, top bar) and is ready to be executed

  • Clicking the button Run will show another dialog where you can select which blocks to run. Since there is only block available, simply click on Start Run for 1 block. This will launch a process that reads the first movie, applies the gain reference and displays a thumbnail inside the Single Particle (from Raw Data) block

    Gain thumbnail

    Tip

    Click inside the Single Particle (from Raw Data) block to see a larger version of the image

Step 3: Pre-processing

Movie frame alignment, CTF estimation and particle picking ( 2 min)

  • Click on Movies (output of Single Particle (from Raw Data) block) and select Pre-processing

    File browser

  • Go to the Particle detection tab:

    • Set Particle radius (A) to 65

    • Set Detection method to all

    • Set Min distance (pixels) to 40

    • Click on the Resources tab

  • Click Save, Run, and Start Run for 1 block. You can monitor the status of the run using 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

    Data processing details (particle picking, drift trajectory, CTF profile, power spectrum)

    Micrograph view

    Tip

    While on the Micrographs tab, use the navigation bar at the top of the page to look at the results for other micrographs

Step 4: Reference-based refinement

Reference-based particle alignment ( 3 min)

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

    File browser

  • Go to the Sample tab:

    • Set Molecular weight (kDa) to 700

    • Set Particle radius (A) to 80

    • Set Symmetry to D7

    • Click on the Extraction tab

    File browser

    • Set Box size (pixels) to 128

    • Set Image binning to 4

    • Click on the Refinement tab

    File browser

    • Set the location of the Initial model by clicking on the icon , navigating to the folder where you downloaded the data for the tutorial, selecting the file EMPIAR-10025_init_ref.mrc, and clicking Choose File

    • Set Max resolution (A) to 8:7:6

    • Check Use signed correlation

    • Set Last iteration to 5

    • Uncheck Skip refinement

    • Check Use alignment priors

    • Click on the Reconstruction tab

    File browser

    • Set Fraction of particles to 0

    File browser

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

    The new block will appear on the Dashboard and a thumbnail will be displayed inside after the run is finished

    File browser

    This process executes four rounds of global orientation search (iterations 2-5). The fraction of good particles at each iteration will be determined automatically (Fraction of particles = 0) and used for reconstruction

  • Click inside the Pre-processing block to inspect the results:

    Iteration 5

Step 5: Filter bad particles

Identify particles with low alignment scores ( 1 min)

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

    File browser

  • Go to the Particle filtering tab:

    • Check Automatic score threshold

    • Set Min distance between particles (A) to 20

    • Select the Input parameter file by clicking on the icon and selecting the file sp-coarse-refinement-*_r01_05.par.bz2

    • Check Generate reconstruction after filtering

    • Check Permanently remove particles

    • Click on the Refinement tab

    • Select the Initial model by clicking on the icon and selecting the file sp-coarse-refinement-*_r01_05.mrc

  • Click Save, Run, and Start Run for 1 block to execute particle cleaning and produce a reconstruction with only the clean particles

  • Click inside the Particle filtering block to look at the reconstruction after cleaning:

    Iteration 2

Step 6: Particle refinement

Reconstruction and additional refinement using 2x binned particles ( 9 min)

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

    File browser

  • Go to the Extraction tab:

    • Set Box size (pixels) to 256

    • Set Image binning to 2

    • Click on the Refinement tab

    • Select the Initial model by clicking on the icon and selecting the file sp-fine-refinement-*_r01_02.mrc

    • Select the Input parameter file by clicking on the icon and selecting the file sp-fine-refinement-*_r01_02_clean.par.bz2

    • Set Max resolution (A) to 6:4:3

    • Check Use signed correlation

    • Set Last iteration to 6

    • Set Search mode to local

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

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

    Iteration 2

    Tip

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

Step 7: Create shape mask

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

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

  • Enter parameter values for the Masking tab:

    • Select the Input map by clicking on the icon and selecting the file sp-coarse-refinement-*_r01_06.mrc

    • Set Threshold for binarization to 0.3

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

  • Click on the 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

Step 8: Local refinement

Additional refinement iterations using 2x binned data ( 2 min)

  • Go one block upstream to the Particle refinement block, click on the icon and select the Edit option from the menu

  • Go to the Refinement tab:

    • Set Last iteration to 7

    • Select the Shape mask by clicking on the icon , navigating to the path of the Masking block copied above, and selecting the file frealign/maps/mask.mrc

  • Click Save, then Run. We now need to uncheck the box for the Masking block (since we don’t want to re-run this block), then click Start Run for 1 block

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

    Iteration 7

Step 9: Particle-based CTF refinement

Per-particle CTF refinement using most recent reconstruction ( 9 min)

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

  • Go to the Refinement tab:

  • Set Last iteration to 8

  • Click on the Constrained refinement tab

  • Set Number of regions to 8,8

  • Check Refine CTF per-particle

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

  • Click inside the Particle refinement block to inspect the results

Step 10: Movie frame refinement

Particle-based movie-frame alignment and data-driven exposure weighting ( 8 min)

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

  • Go to the Refinement tab:

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

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

    • Set Max resolution (A) to 3

    • Set Last iteration to 3

    • Check Skip refinement

    • Go to the Constrained refinement tab

    • Set Last exposure for refinement to 60

    • Check Movie frame refinement

    • Check Regularize translations

    • Set Spatial sigma to 15

    • Go to the Exposure weighting tab

    • Check Dose weighting

  • Click Save, then Run to launch Movie refinement. Uncheck the box for the Masking block and click Start Run for 1 block

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

    Iteration 3

Step 11: Refinement after movie frame refinement

Additional refinement using new frame alignment parameters ( 8 min)

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

  • Go to the Refinement tab:

    • Set Last iteration to 4

    • Uncheck Skip refinement

    • Click on the Constrained refinement tab

    • Uncheck Movie frame refinement

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

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

    Final map

Step 12: Map sharpening

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

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

  • Go to the Post-processing tab:

    • Select the First half map by clicking on the icon and selecting the file sp-flexible-refinement-*_r01_half1.mrc

    • Set Automask threshold to 0.5

    • Set Adhoc B-factor (A^2) to -50

  • Click Save, then Run. Uncheck the box for the Masking block and click Start Run for 1 block

  • Click inside the Map sharpening block to inspect the results:

    Post processing

Info

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