9. Initial Model Generation
An initial model or 3D reference structure is required for the subtomogram averaging workflow in RELION. There are multiple ways to obtain a reference structure. If your data is good enough, you can generate an initial model from your data as explained below, and is the best way to avoid the reference bias. There are two ways to do this in RELION:
9.1. Particle Reconstruct
If you have picked your particles by using sphere annotation or filement annotation in Napari-picker, the particles also contain
their oreintations relative to context. In the tutorial data, we have used sphere annotation for picking, thus, each particle
has its ortientation information coded in rlnTomoSubtomogramRot, rlnTomoSubtomogramTilt and rlnTomoSubtomogramPsi columns of the particles.star file.
Using this information together with coordinates, RELION can generate an initial model by reconstructing the particles directly.
To do this in Doppio, go to NEW JOB tab in Doppio GUI and search for Reconstruct 3D particle job.
In the previous step of the workflow, we extracted pseudosubtomograms at bin 6 level. It is important to use the same binning level here and the same box size and cropped box size. In the next step (during 3D refinement), we will use pseudosubtomograms and the reference generated here. RELION can resize the reference to match the the of the pseudosubtomogram size automatically, but it is good to make sure you control the parameters properly to avoid possible confusion later.
Our input parameters are shown in the image below:
Once the job is finished, the output initial model is Reconstruct/job007/merged.mrc.
Note
Reconstruct 3D particle job will always try to reconstruct half1.mrc and half2.mrc files for half-set validation.
However, this requires the random assignement of particles to half-sets have done in the particle file.
But the particles.star file from the picking job does not have this information. Thus, the job will only generate
merged.mrc file.
Once the job is finished, the merged.mrc will be displayed in the RESULTS tab automatically.
This figure shows the orthogonal slices of the initial model generated from the picked particles. You can immediately guess that the initial model is a pan-like structure, which represents a part of the sphere.
To get the better view, open the 3D view of the Reconstructed map in the Doppio image viewer.
We will use this initial model for the next 3D refinement step.
9.2. de novo Initial Model Generation
If you do not have orientation information for your particles, you can still generate an initial model de novo using RELION’s initial model generation tool. To do this in Doppio, go to NEW JOB tab in Doppio GUI and search for RELION initial model(Tomography) job. Please refer to the RELION documentation below for job parameters.
9.3. Reference model from other sources
If you have a reference model from other sources (e.g., from PDB or EMDB), you can use that as an initial model as well. However, make sure to low-pass filter and rescale the model to match the data before using it as an initial model.