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When picking filaments, it is important to identify each filament individually. This allows specific spacing of the boxes (i.e., the helical rise) to maximize the number of particles. CrYOLO supports this method of picking filaments.
Filament mode on actin:
Filament mode on MAVS (EMPIAR-10031) :
The first step is to create the training data for your model. Right now, you have to use the e2helixboxer.py for this:
e2helixboxer.py --gui train_image/*.mrc
After tracing your training data in e2helixboxer, export them using File → Save. Unfortunately you have to do that with each image separately.
Make sure that you uncheck the boxes “Write Helices” and “Particle Images” and check the box “Particle Coordinates”, as this the only format supported right now (see screenshot). Also remove the “_helix_ptcl_coords” suffix in the path field. The coordinate files have to have the same name as the micrographs.
In the following example, it is expected that you exported into a folder called “train_annot”.
For projects with roughly 20 filaments per image we successfully trained on 40 images (⇒ 800 filaments).
If you followed the installation instructions, you now have to activate the crYOLO virtual environment with
source activate cryolo
You can use crYOLO either by command line or by using the GUI. The GUI should be easier for most users. You can start it with:
The crYOLO GUI is essentially a visualization of the command line interface. On left side, you find all possible “Actions”:
Each action has several parameters which are organized in tabs. Once you have chosen your settings you can press [Start] (just as example, don't press it now ), the command will be applied and crYOLO shows you the output:
It will tell you if something went wrong. Moreover, it will tell you all parameters used. Pressing [Back] brings you back to your settings, where you can either edit the settings (in case something went wrong) or go to the next action.
You now have to create a configuration file for your picking project. It contains all important constants and paths and helps you to reproduce your results later on.
You can either use the command line to create the configuration file or the GUI. For most users, the GUI should be easier. Select the config action and fill in the general fields:
At this point you could already press the [Start] button to generate the config file but you might want to take these options into account:
Since crYOLO 1.4 you can also use neural network denoising with JANNI. The easiest way is to use the JANNI's general model (Download here) but you can also train JANNI for your data. crYOLO directly uses an interface to JANNI to filter your data, you just have to change the filter argument in the Denoising tab from LOWPASS to JANNI and specify the path to your JANNI model: I recommend to use denoising with JANNI only together with a GPU as it is rather slow (~ 1-2 seconds per micrograph on the GPU and 10 seconds per micrograph on the CPU)
You can also modify all options and parameters directly in the config.json file. It can be opened by any text editor. Please note the wiki entry about the crYOLO configuration file if you want to know more details.
Now you are ready to train the model. In case you have multiple GPUs, you should first select a free GPU. The following command will show the status of all GPUs:
For this tutorial, we assume that you have either a single GPU or want to use GPU 0.
In the “Optional arguments” tab you can change the GPU that should be used by crYOLO. If you have multiple GPUs (e.g. nvidia-smi lists GPU 0 and GPU 1) you can also use both by setting the GPU argument to '0 1'.
The default number of warmup epochs3) is fine as long as you don't want to refine an existing model. During the warmup training epochs it will not try to estimate the size of your particle, which helps crYOLO to converge.
When you start the training, it will stop when the “loss” metric on the validation data does not improve 10 times in a row. This is typically enough. In case you want to give the training more time to find the best model can increase the “not changed in a row” parameter to a higher value by setting the early argument in the “Optional arguments” to, for example, 15.
The final model will be written to disk as specified in saved_weights_name in your configuration file.
Now select the “Filament options” tab and check “Activate filament mode”, specifiy the filament width (e.g. 100) and define the box distance (e.g. 20 for 90% overlap when using a box size if 200):
output_boxes will be created and all results are saved there. The format is the eman2 helix format with particle coordinates.
You can find a detailed description how to import crYOLO filament coordinates into Relion here.
To visualize your results you can use the boxmanager:
As image_dir you select the
full_data directory. As box_dir you select the
CBOX folder (or
EMAN_HELIX_SEGMENTED in case of filaments).
CBOX files contain besides the particle coordinates more information like the confidence and the estimated size of each particle. When importing .cbox files into the box manager, it enables more filtering options in the GUI. You can plot size- and confidence distributions. Moreover, you can change the confidence threshold, minimum and maximum size and see the results in a live preview. If you are done with the filtering, you can then write the new box selection into new box files. The video below shows an example.