Using USC's Cryo-EM Instruments

USC now has two cryo-electron microscopes available for use: Krios with the K3 direct detection camera and Glacios with the Falcon 4 direct detection camera, both manufactured by Thermo Fisher. Both instruments are housed in the Michelson Center for Convergent Bioscience building in the Core Center for Excellence in Nano Imaging (CNI) at USC's University Park campus.

This user guide explains how to get started using the new microscopes, including setting up your cryo-EM project and allocations in the CARC User Portal, reserving the microscopes using CNI's reservation system, configuring the data acquisition process and pre-processing parameters, accessing the microscope data, and analyzing the data.

Creating a cryo-EM project in the CARC User Portal

To use the cryo-EM instruments, you must have a project and associated resource allocations in the CARC User Portal. You can create a new project for your cryo-EM work or add the required resource allocations to an existing project.

The project you use with the cryo-EM instruments will need a cryo-EM subscription allocation, a /project storage allocation, and a Discovery cluster allocation. See the Request New Allocation user guide for detailed instructions for requesting allocations for your project. The allocations should be approved by CARC staff within 24 hours.

You can view all CARC User Portal guides here. These guides explain how to manage your projects and allocations, how to add or remove users for your projects, how to complete your annual project and allocation renewals, and more.

The resources enabled by the cryo-EM subscription allocation are:

• Access to the cryo-EM pre-processing portal, where you can get live feedback during the data acquisition process
• GPU cluster with 10 GPU compute nodes
• Access to a shared storage space (/cryoem1/test) for the datasets collected on the microscopes. Note that datasets will only be stored in this shared space for one month due to the large amount of data generated by the microscopes. You can learn more about transferring data to your personal CARC directories (or elsewhere) in our Data Management user guides.

Reserving a microscope

To reserve time to use the microscopes, you must use CNI's reservation system. If this is your first time using the reservation system, you'll need to request access by signing up as a new user. Your new account should be available shortly.

Once your account is created and you can log in to CLIMS, you must reserve your microscope of choice using the calendar, which can be accessed from the main menu:

The microscopes are typically available between 8:30 AM and 4:00 PM PST. You can select either the Krios or the Glacios microscope using the dropdown at the top of the calendar:

Once you've selected the microscope, you can select a time on the calendar and click "New Reservation" to confirm:

You can delete or edit the details of your reservation by selecting it on the calendar.

Note that use of the microscopes is a paid service and pricing details can be found here.

Using the microscope

When your reservation time begins, log in to the CNI client on the microscope's support PC to power on the microscope you've reserved.

After the microscope has been powered on, use the microscope's control PC to configure the data acquisition process for the microscope you're going to be using.

#Important: Make absolutely sure that you follow the file naming scheme below or your data will not be transferred from the microscope.

The data acquisition protocol is similar for both microscopes:

After setting up the data acquisition process for the microscope, log in to the CARC cryo-EM user portal in a web browser and navigate to the Instrument CheckOut/Return page:

Click the blue CheckOut button for the microscope you reserved and set the following pre-processing parameters:

• Pixel size: Determined during data acquisition setup. Usually between 0.8 and 1.1.
• Dose of electrons per Angstrom square per frame (Dose e-/Å^2): Determined during data acquisition setup. Usually about 60 electrons per 50 frames, so about 1.2.
• keV: 300 for Krios, 200 for Glacios.
• Superresolution: Improve resolution by setting every image pixel to 4 pixels.

These parameters will be used by the Cryo-processor data pre-processing workflow powered by Pegasus, a workflow management system.

Once the microscope has been checked out with the pre-processing parameters set, the transfer and processing of data from the microscope will be automated. If this is your first time using the microscopes, you will be added to a Slack workspace called USC CARC cryo-EM. You will then be added to a private Slack channel dedicated to the microscope session you just created where you will be able to view the pre-processed images from the microscope as they are transferred, with every image labeled with its potential (R)esolution, (A)stigmatism and the average (S)hifts:

Data should begin flowing from the microscope in about ten minutes.

When you are finished using the microscope, you should to "return it" in the CARC user portal using the Instrument CheckOut/Return page. This makes it available for the next user to check out. You can also use the "Switch" option to override another user's session if they forgot to return an instrument you've reserved.

Accessing your data

Data from the microscope will be saved within your project directory in the cryo-EM shared file system (/cryoem1) in a subdirectory with a name of the form <date>_usc_<USC NetID>_<detector>_<sample_name> (e.g., 20210801_usc_ttrojan_k3_mysample). The data in this subdirectory will include the raw data as well as the processed images.

Note: This subdirectory in /cryoem1 is different from your main /project directory in the CARC /project file system.

You can access the cryoem1 file system by logging in to the cryo-EM login node, which requires use of USC's VPN. If you have an active USC account, please go to USC VPN website for further instructions.

Once you can connect to the one of USC VPN endpoints (e.g. connect.usc.edu), you can log in to the cryo-EM login node at knoll.usc.edu using SSH in your terminal:

ssh <username>@knoll.usc.edu

Make sure to substitute your USC NetID as the username. This is the same username for your USC email account (e.g., ttrojan@usc.edu's NetID is ttrojan). After entering the command, you will then be prompted to enter your USC NetID password. This is the same password for your USC email account.

Note: There will be no visual feedback as you enter your password. This is a security feature designed to obscure your password and is expected.

After entering your password, you will then see a Duo two-factor authentication prompt. You can learn more about transferring data from cryoem1 to your personal CARC directories (or elsewhere) in our Data Management user guides.

Analyzing your data

CARC provides access to a managed instance of cryoSPARC processing software, which supports fast and convenient dataset analysis and 3D reconstruction. Moreover, with the use of cryoSPARC Live, you can process your data and observe results in real time. To request access to cryoSPARC, please contact us by submitting a help ticket.

Note: Accessing cryoSPARC also requires use of USC VPN; see the "Accessing your data" section above for details.

• cryoSPARC
• cryoSPARC Live (experimental)

CARC also provides a range of cryo-EM data processing software on the Discovery and Endeavour clusters:

• Relion: A software package that employs an empirical Bayesian approach for cryo-EM structure determination.
• MotionCor2: Correction of electron beam-induced sample motion.
• ctffind4: A fast program for finding the Contrast Transfer Functions of electron micrographs (CPU only version).
• gctf: Accurate and robust real-time GPU accelerated CTF determination.
• pyEM: A selection of Python programs for cryo-EM.
• EMAN2: A broadly based greyscale scientific image processing suite with a primary focus on processing data from transmission electron microscopes.
• EMAN: A suite of scientific image processing tools aimed primarily at the transmission electron microscopy (old, but some tools are still useful).
• IMOD: A set of image processing, modeling, and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections.