Instructions

1. Starting

Create Online Account and Submit EICN Application

Once the project is approved, the PI will be notified of via an email with instructions on creating an online Core Lab Management System (CLMS) account (for scheduling) (www.clms.cnsi.ucla.edu) and completing a separate EICN application (for user management). The user will be given a voucher ID which will be entered as the PO number on the CLMS form during account creation.

Upon receiving all required forms, the UCLA Finance Office will review and approve the CLMS account. At the end of every month, UCLA Finance Office will deduct any accumulated charges from the voucher account.

2. Preparation

(i) Submit Project Description for Committee Review

To start a new project, the principal investigator (PI) of a consortium laboratory will need to submit a one-page project description to the consortium user coordinator. This project description serves two purposes. First, a supervisory committee will evaluate this description for suitability and need for the high-end Titan Krios instrument. Upon approval, each laboratory will be awarded a voucher in the amount of $10,000/year for instrument access. UCLA approved pricing information for instrument access and staff assistance is listed here. Second, UCLA’s annual report of the consortium grant to NIH will be based on descriptions of approved projects. For this reason, a new project description must be submitted for each new project (project description example is available here). The total amount for all projects in each laboratory is $10,000/year (see rules at the bottom).

(ii) Get Training (If Needed)

Training is required in order to use EICN equipment. Thereafter, users are permitted to use the facility 24 hours a day, 7 days a week. Please email eicnhelp@cnsi.ucla.edu to set an appointment for training. All training sessions are billed using the hourly rates associated with each instrument. Training is not available without a completed EICN Application form.

Users are permitted to use the EICN facility after creating a CLMS account, presenting a signed EICN User Agreement and completing training. Using instruments without a reservation on CLMS is not permitted.

Users must comply with general CNSI and internal EICN policies and be aware of charges related to using EICN.

Users have equal access to the EICN facility on a first come, first serve basis during normal working hours via CLMS.

3. Scheduling and Sample Shipping

(i) Log In to EICN Scheduler

Consortium laboratories may click here to log in to your schedule. When competition for time exists, EICN will coordinate scheduling via email to utilize the facility and resources and discuss project proposals to optimally utilize the capabilities of the facility. If necessary, telephone conferences will be held with all participants to mediate any issues that arise.

The online reservation system allows users to reserve slots at their convenience. Each day is divided into three 8-hour slots so the facility is operated on a 24-hour, 7- day schedule.

(ii) Contact Coordinator and Ship Samples

Cost of sample shipping to EICN will be covered. The sample label below provides the information needed for shipping to EICN, including the information for the “Payment” section on the label:

(iii) Coordinate Sample Loading and Imaging Set up
Sample Criterion: A consortium user from each regional institute will submit a project description and request for access to the consortium user coordinator with supporting images obtained from their local low-end cryo electron microscopes. With those images, the participant must demonstrate that the concentrated samples are isolated, purified, and meet the minimal sample criterion for reaching near-atomic resolution as demonstrated in Figure 1.

Figure 1. Sample criterion of the UCLA Titan Krios instrument.
An example of minimal sample quality to qualify for Titan Krios
(Jiang et al., 2015). (a) Negative stain EM micrograph of anthrax PA pore
particles. Some representative top-view and sideview particles are selected
with circles and squares, respectively. (b) CryoEM micrograph of PA pore
particles showing well-separated particles with different views. Some
representative side-view particles of PA pore are indicated by arrows.
(c), Representative cryoEM 2D class averages of particles demonstrating
different orientations.

Specimen samples should be prepared and optimized by the users, and cryoEM grids can be prepared in the user’s laboratory or EICN. After setting up Leginon or EPU automation software, cryo-grids are loaded into the Titan Krios microscope. A users’ responsibility is to select desired/good areas for imaging. Data is recorded on either the K2 (counting mode) or Falcon3 camera at a magnification of ~48,000x or ~140kx with a pixel size of ~1.0?/pixel on the specimen. To obtain the best performance of the DED cameras, the optimal dose rates of ~8 or ~50e-/pixs will be used for the K2 (counting mode) and falcon3 cameras, respectively. The image stacks could be recorded at 4 frames/sec on the K2 camera for ~10 seconds, or 17 frames/sec on the Falcon3 for ~2 seconds. After drift correction, the first frames of each image stack are merged to generate a final image with a total dose of ~30e-/?2. After the session, all data will be stored on external disks and mailed to the users.

On-site staff will set up the TeamViewer/VNC software for consortium users to remotely access and control the Titan Krios TEM computer from anywhere. Therefore, PIs/key personnel will simply need to send their samples to UCLA, and our experienced staff will load the samples into the TEM and set up the initial data collection for PIs/key personnel. All data will be saved on external hard drives and be sent back to the respective PI/key personnel by our staff.

Figure 2. Use of Leginon to control instruments for data collection.

(a) Atlas of a cryo-grid; (b) An enlarged square image selected from (a);

(c) A magnified hole image for choosing final targets; (d) Final image

chosen in (c).

4. Imaging Data

Trained users do not have to travel onsite to record images with the Titan Krios instrument. The users reservations can be made by individual user groups through the online reservation system and execution of reserved sessions will be facilitated through user coordinator, in consultation with staff who will provide assisted data acquisition service.

After our staff loaded the frozen grids delivered to UCLA, consortium users would be able to operate the UCLA Titan Krios continually from their computer, “counting” electrons passing through sample areas of interest on the grid to record “movies”.

As the host institution, UCLA will provide to participating institutions access to the Titan Krios cryo-electron microscope with existing pre- and post-GIF direct electron detectors, and on-site staff assistance to collect data.

To increase data collection efficiency and ensure image quality, the K2 and Falcon II DEDs have been operated with automation software packages Leginon and EPU, respectively, working continuously 24/7 at rate of ~1 image per minute, producing more than 1000 images per day. With both automation software packages, users take the atlas of a grid (Figure 2), on which squares with thin ice are chosen for magnified images for picking good regions. Targets with thin ice and good particle distribution are selected for final imaging. Users can operate instruments remotely from different cities using a graphical desktop sharing system such as VNC.

To ensure image quality, the instruments will be checked and carefully tuned before each data collection session. Specifically, EICN staff will align the microscope and minimize the beam tilt by aligning the beam as precisely as possible to minimize beam-tilt artifacts. We deliberately tilt the beam left and right along the X axis, measure defocus in the Fourier transformations of the two images, and compute the X-tilt that equalizes left and righted ocus. We then do the same up and down along the Y axis. This procedure is commonly called “coma-free alignment.” We also use a parallel beam to avoid the type of beam tilt introduced by divergence of the beam and to insure constant magnification for images at different defocus levels. The performance of the microscope will be regularly monitored by checking Thon rings in the power spectrum of carbon images (Figure 2 and 1a). Tore cover the bad pixels generated by electrons, the camera sensor of the K2 DED will be conditioned by raising the temperature to 50 C overnight every week.

All data collected will be saved onto two external hard drives, one as backup, and be sent back to the respective PI/key personnel by EICN staff. Upon confirmation of receipt of the data by the user, EICN will delete their backup copy. We encourage the receiving party to make another copy of the data for their records. Users can get data on line. If necessary data stored on external disks can be mailed to the users.

Rules: This $10,000 amount is automatically renewed in August. Any unused balance on the voucher at the time of renewal will be forfeited. Vouchers are laboratory specific and non-transferable to other laboratories. A consortium laboratory may submit a purchase order to UCLA if it needs additional instrument time exceeding that allotted by the awarded voucher amount and if such additional time is available based on communication with the user coordinator.

To make reservation, please go to CLMS Titan Krios reservation page