- Time requirement: 40 min
- Goal: Get a quick start into how CryoCloud works and determine a 3.5 Angstrom structure of Apoferritin.
This tutorial will walk you through the analysis of the EMPIAR dataset 10146 containing Apoferritin. When you sign up for CryoCloud, the dataset EMPIAR-10146 will have been automatically linked for you.
Creating a Project
To start, you need to create a new project. Navigate to the Project view by clicking on the Project tab in the left sidebar. To Create a new Project, click on the top right button "+Add new project". Enter a name and specify the type (SPA for this tutorial).
Once you confirm, the project grid will open automatically.
Linking a dataset
To run any type of analysis in a CryoCloud project, you need to link a dataset to your project.
Click on the "Dataset" tab on the top left, and select Empiar-10146.
To set-up a motion correction, click on the "+" tile in the Pre-processing column. The job type (Motion Correction) and the linked dataset will already be pre-selected in the dropdowns, so you just need to confirm the selection.
Once confirmed, the Job setup form will open. The movies.star will be already pre-selected as it was created for the linked Empiar dataset during upload - there is no need to run a separate Import Job on CryoCloud, as the .star files will be automatically created during dataset upload.
You can keep all parameters to default except for the patches. For this dataset, we want to use patches of 3x3.
This dataset also does not contain a gain reference, so you don't need to specify one. But keep in mind to specify one for your other datasets (if you linked a gain reference to your dataset during upload, the field for the gain reference in the Motion Correction job will already be correctly pre-filled).
After that, confirm the setup and submit the job by clicking "Save & Run" on the bottom right. The job status will change to submitted, and analysis will start in 3- 5min, during which an instance is spun up for you. The Motion COrrection itself should finish in 8 seconds.
You can queue a job to a running job or a job draft. Simply click on the "+" sign on the top right of a job tile. After setting up and submitting the job, it's status will be shown as queued and it will automatically start when the previous job has finished.
After submitting the MotionCor job, you can go back to the overview and setup the next CTF job - there is no need to wait for the job to be finished, as you can queue the CTF job to the running MotionCor job.
Simply click on the "+" in the top right corner of the MotionCor Job, select CTF job with the Motion job as parent, and specify all parameters in the job view. For this job, you can keep all default parameters - so if you like simply jump to the bottom and click "Save & Run".
The Job should finish in about 3 seconds. After it completed, you can inspect the results by clicking on the micrographs tab and then opening one of the micrographs in the table. It should looks as follows:
The Micrograph Table should also show that most Micrographs have a maximum resolution of 4- 4.5 Angstrom. If there were outliers with considerable worse resolution you could run a Selection job and set a cutoff for CTF Max Resolution. However, this it not required for this dataset so we can proceed to particle picking.
Setup a particle picking job, either as before by clicking on the "+" of the CTF job, or by hovering over the pre-processing column, clicking the "+" sign and specifying the CTF job as input. For this dataset, you can use the default values with the LoG picker. You just need to specfiy the minimum and maximum diameter as 100 nm 160 nm respectively.
Just like for the CTF job you can inspect the results of your picking job once its finished in the results section.
To analyze your picked particles you need to extract them first. Setup an extract job via the "+" sign on the Picking job tile, and specify the input micrographs from your Motion Correction job. As box size, specify 180 pixels.
You can keep the default values for the other settings and submit the job.
Setup a class2D job, specify the particles.star from your previous Extraction job as input, and set the numner of classes to 20.
For this job, we will use Relions VDAM algorithm with 200 iterations. This is the default setting in both Relion4 and CryoCloud, so you can keep it like that. Specify the mask diamter as 160 Angnstrom, and submit the job.
The 2D classification job should finish in about 5 minutes.
2D Class Selection
Once the Class 2D job is finished, setup a Selection job from the class2D job tile.
In the job form, select the last iteration from the Class 2D parent job, and click "Load selector" to load the 2D class averages. You can then interactively select the correct classes (shown below).
If you used all the settings from this tutorial, the selection should result in roughly 2,300 particles.
Once you selected all classes, click the Run bottom to submit the job.
Uploading a reference map
Before we go to the last job of 3D refinement, you need to upload a reference map to your project.
In CryoCloud, all references, massks and other auxiliary files can be uploaded to the Project Archive located next to Datasets on the top left. You can use these files as input for jobs that require external data (masks, references, high resolution maps for model building).
Simply click on the Archive button. This will open a Side Panel that allows you to upload new files and inspect all current files in the Archive. Click on upload file and select a reference.
You can download the following reference, and upload it to your project archive:
It was generated from the map EMD-24797 and rescaled to 1.5 Angstrom and a box size of 180 pixels. So it will have the correct dimensions for this tutorial, and you can use it as a reference for the next 3D refinement job.
Use the selected particles from the Class 2D job and the uploaded reference in the archive as input for the Refine 3D job. You need to specify the following parameters:
- Initial low-pass filter: 8
- Symmetry: O (the letter 'oh', not zero!)
- Mask Diameter: 160 A
You can keep all other settings as default, and submit the job.
The 3D refinement should finish in about 6 minutes and results in a 3.5 Angstrom resolution map.
Feel free to download the resulting map from the Refine3D job, create a mask using the Mask Create job and run a post-processing job.
You can even run polishing and CTF refinement jobs with the available data, even though the resolution of this dataset will be limited by the movie pixel size of 1.5 Angstrom.
If you have any input or questions during the tutorial, let us know via the in-app messenger or an email to firstname.lastname@example.org.
If you have read through the tutorial but not signed up for a free 30 day CryoCloud trial, you can do that here.