Now that our particle poses are mostly correct and we have a good estimate of the reconstructed density, there are several parameters that can be tweaked a posteriori to further refine our result. We will perform these optimisations in
Create Population and Species#
A project in
M is referred to as a
population. To create a new one press the big
+ button in the center of the window, and provide a directory. Here, we put it in
We then need to add a data source. Click on
Manage data sources to add a local source, and select the
*.settings file inside the
frames directory. Make sure to
Include items outside of filter ranges (since we did not use the filters for this project), give it a name such as
HIV and press
CREATE. We also only want to
Use only first 15 frames/tilts in order to refine only based on the high resolution information.
We can then create a Species by pressing the next big ‘+’, selecting
from scratch and filling in the parameters as follows:
Afterwards, select the two last halfmaps generated by the
RELION refinements at 1.6Å/px. For the mask, we will generate a binary mask around the central hexamer (160 Å diameter).
Mask one of the halfmaps to include only the central hexamer in
Dynamo then use your favourite program and determine an appropriate threshold for binarisation. Use this as the
--ini_threshold parameter in the following
relion_mask_create command, replacing
<latest_halfmap> as appropriate:
relion_mask_create --i <latest_halfmap>.mrc --angpix 1.6 --extend_inimask 5 --o mask_1.6Apx.mrc --ini_threshold 0.05
If using Relion3.1+, we need to reformat the final particle positions
.star file using
relion_star_downgrade to the Relion3.0 specification (Warp does not support Relion 3.1+):
relion_star_downgrade -s run_it019_data.star
M will only look for file names with the pattern
*_data.star, so we need to rename the output from
relion_star_downgrade (e.g., from
rln3.0_run_it019_data.star). Now, select this reformatted
.star file containing the final particle positions from Relion as the input for particle poses in M. Click
FINISH to generate the new species.
To start the refinement, click on the
REFINE button and fill in the parameters as follows.
We will do a few iterations of refinements: first some with only spatial parameter optimizations, and then with most parameters. We chose to first optimise a smaller number of parameters to avoid falling into a local minimum, there may be a better way to do this!
First, run four iterations with these parameters:
Afterwards, three iterations as follows:
If everything went well, after the second round of refinements you should be able to get to something like this:
Here’s a closer look to the map compared to the available structures:
Congratulations with your reconstruction!