.. Doppio Tomography Tutorial documentation master file, created by sphinx-quickstart on Tue Feb 18 13:09:38 2025. You can adapt this file completely to your liking, but it should at least contain the root `toctree` directive. Tomo refinement cycle(s) =========================================== Now that we saw the best resolution at the end of high resolution 3D refinement and post processing we reached is 5.08 A. In this section, we describe tomo refinement steps that include the optimisation of per-particle CTF parameters, and the modelling of beam-induced deformations to further improve the resolution. A tomo refinement cycle consists of two steps: *CTF refinement*, and *Bayesian polishing*. These steps go in tandem and their order of execution is irrelavent. Below, we describe one complete tomo refinement cycle, but in practice, one could carry out multiple cycles until no further improvment in resolution as well as the density is obtained. However, care should be taken not to over do this to avoid fitting to noise. **Note on Inputs and Outputs** Tomo refinement should be carried out using the subtomograms extracted at bin 1 level. That means the pixel size should be the original pixel size of the motion corrected images. The reference map and the alignment mask should also be reconstructed at this binning level. As inputs, an ``optimisation_set.star``, reference halfmap ``Reconstruct/jobxxx/half1.mrc``, a file with FSC data ``postporcess.star``, and an alignment mask at bin 1 ``align_mask.mrc`` are required. For the intial cycle, usually, an ``optimisation_set.star`` is not available. In that case, we can input ``tomograms.star`` and ``particles.star`` files, directly. Each step (*CTF refinement*, or *Bayesian polishing*) in one tomo refinement cycle will output an ``optimisation_set.star``. We can use that as input for the next step. We demonstrated how we did this below. Tomo refinement cycle 1 ---------------------------------------------------------------- **CTF Refinement:** We started the tomo cycle by calling CTF Refinement first, and we called it cycle 1. To call CTF Refinement in Doppio, go to :purple:`NEW JOB` tab and type **relion.ctfrefinetomo** in the search bar. Our input parameters for this job is shown in the image below. .. figure:: ../_static/images/STA/ctfrefine-cy1-job-params.png :alt: Example image :width: 100% :align: center At the end of the job the output file ``CtdRefine/job024/optimisation_set.star`` is available. In this file you'll see updated links to ``particles.star`` and ``tomograms.star`` files. Next, we call the second step of this tomo refinement Bayesian polishing below. Before we begin the polishing, we quickly ran a Reconstruct particle job followed by postprocessing to see if the CTF refinement has improved the resolution. Our input parameters are given in tables below: Reconstruct particle (*relion.reconstructparticletomo*) -------------------------------------------------------------- ====================================== ======================================== Input params. Bin 1 ====================================== ======================================== Input optimisation set: ``CtfRefine/job024/optimisation_set.star`` Input tomogram set: Input particle set: Box size (binned pix): 512 Cropped box size (binned pix): 192 Binning factor: 1 Symmetry: ``C6`` **Outputs:** 3D reference density map: ``Reconstruct/job025/merged.mrc`` half1 density map: ``Reconstruct/job025/half1.mrc`` half2 density map: ``Reconstruct/job025/half2.mrc`` ====================================== ======================================== Post process (*relion.postprocess*) -------------------------------------------------------------- ====================================== ======================================== Input params. Bin 1 ====================================== ======================================== One of the 2 unfiltered half-maps ``Reconstruct/job025/half1.mrc`` Solvent mask: ``fsc_mask.mrc`` **Outputs:** Postprocessed starfile: ``Postprocess/job026/postprocess.star`` Postprocessed map: ``Postprocess/job026/postprocess.mrc`` Postprocessed masked-map: ``Postprocess/job026/postprocess_masked.mrc`` ====================================== ======================================== Gold-standard FSC showed the new resolution is **4.63** A. Next, we carried out the second step of the current tomo refinement cycle - Bayesian polishing using outputs from above jobs. **Bayesian polishing:** The input parameters we used are given in the images below. .. figure:: ../_static/images/STA/polish-cy1-job-params1.png :alt: Example image :width: 100% :align: center .. figure:: ../_static/images/STA/polish-cy1-job-params2.png :alt: Example image :width: 100% :align: center At the end of the job, the output files ``optimisation_set.star``, ``tomograms.star``, ``particles.star`` and ``motion.star`` are written to ``Polish/job028`` folder. The ``motion.star`` file contains motion trajectories of individual particles, ``particles.star`` contains the new particle positions, and ``tomograms.star`` file contains the updated tilt series alignment. As before, we assessed the results by reconstructing a particle followed by postprocessing. Reconstruct particle (*relion.reconstructparticletomo*) -------------------------------------------------------------- ====================================== ======================================== Input params. Bin 1 ====================================== ======================================== Input optimisation set: ``Polish/job028/optimisation_set.star`` Input tomogram set: Input particle set: Box size (binned pix): 512 Cropped box size (binned pix): 192 Binning factor: 1 Symmetry: ``C6`` **Outputs:** 3D reference density map: ``Reconstruct/job029/merged.mrc`` half1 density map: ``Reconstruct/job029/half1.mrc`` half2 density map: ``Reconstruct/job029/half2.mrc`` ====================================== ======================================== Post process (*relion.postprocess*) -------------------------------------------------------------- ====================================== ======================================== Input params. Bin 1 ====================================== ======================================== One of the 2 unfiltered half-maps ``Reconstruct/job029/half1.mrc`` Solvent mask: ``fsc_mask.mrc`` **Outputs:** Postprocessed starfile: ``Postprocess/job030/postprocess.star`` Postprocessed map: ``Postprocess/job030/postprocess.mrc`` Postprocessed masked-map: ``Postprocess/job030/postprocess_masked.mrc`` ====================================== ======================================== We observed a clear improvement in resolution from 4.63 A to **4.25** A. Once a tomo refinement cycle is complete, it is best practice to run a 3D refinement using the improved alignment (tomograms) and particles. For that, we extracted bin1 subtomograms using the ``Polish/job028/optimisation_set.star``. Extraction of pseudosubtomograms (*relion.pseudosubtomo*) ---------------------------------------------------------------- ====================================== ======================================== Input params. Bin 1 ====================================== ======================================== Input optimisation set: ``Polish/job028/optimisation_set.star`` Box size (binned pix): 512 Cropped box size (binned pix): 192 Binning factor: 1 **Outputs:** Pseudosubtomograms star file: ``Extract/job031/optimisation_set.star`` ====================================== ======================================== 3D refinement (relion.refine3d.tomo) ------------------------------------ ====================================== ================================================== Input params. Bin 1 ====================================== ================================================== Input optimisation set: ``Extract/job031/optimisation_set.star`` Reference map: ``Reconstruct/job029/half1.mrc`` Reference mask (optional): ``align_mask.mrc`` Ref. map is on absolute greyscale? Yes Resize reference if needed? Yes Initial low-pass filter (A): 4.5 Symmetry: ``C6`` Do CTF-correction? Yes Ignore CTFs until first peak? No Mask diameter (A): 230 Mask individual particles with zeros? Yes Use solvent-flattened FSCs? Yes Use Blush regularisation? No Initial angular sampling: 1.8 Initial offset range (pix): 5 Initial offset step (pix): 1 Local searches from auto-sampling: 1.8 Relax symmetry: Use finer angular sampling faster? No Prior width on tilt angle (deg): 10 Use parallel disc I/O? Yes Number of pooled particles: 30 Skip padding? No Skip gridding? Yes Pre-read all particles into RAM? No Copy particles to scratch directory: Combine iterations through disc? No Use GPU acceleration? Yes Number of MPI procs: 5 Number of threads: 8 **Outputs:** Achieved Resolution (A) 4.39 Refined particles ``Refine3D/job032/run_data.star`` Optimisation set ``Refine3D/job032/run_optimisation_set.star`` Refined averaged density map: ``Refine3D/job032/run_class001.mrc`` half1 density map: ``Refine3D/job032/run_half1_class001_unfil.mrc`` half2 density map: ``Refine3D/job032/run_half2_class001_unfil.mrc`` ====================================== ================================================== At the end of the 3D refinement, we again ran **Reconstruct particle** followed by **Postprocess** to reach **4.25** A resolution map. .. figure:: ../_static/images/STA/final-fsc.png :alt: Example image :width: 100% :align: center Final FSC curves We refer you to the Relion documentation for more details. `Relion Tomo refinement documentation `_.