1. I want to do cryoEM-SPR on my protein at Davis. Where do I start?
You came to the right place! The BioEM Core specializes in cryo-electron microscopy for single particle 3-D reconstruction (cryoEM-SPR). In our facility, you can receive training, prepare samples and collect data. Please see our list of services at (https://bioem.ucdavis.edu/services-and-rates).
You can start by reading this page, the cryoEM-SPR Overview page, and Self-Training Links page). If, after reading this material, you are interested in continuing, please contact the BioEM Facility Director, Enoch Baldwin (firstname.lastname@example.org) to schedule a conversation about your project and needs. If cryoEM-SPR is the right tool for you, then you can schedule further discussions with the BioEM Technical Director Fei Guo, Ph.D. (email@example.com).
2. What does cryoEM-SPR tell you?
The results of a successful cryoEM-SPR study are a coulombic (charge) density map of your protein or protein complex, similar to the electron density maps generated by X-ray crystallography. The level of detail (resolution) can vary considerably and is dependent on the quality and properties of the sample. At low resolution (>1nm), the overall molecular envelope and gross features can be discerned. Secondary structure (helices, sheets and some loops) become visible below 9 Å, allowing reasonably accurate placement of pre-existing X-ray crystallographic models. Protein topology such as turns, loops, and beta-strands can be at least partially traced around 3.5 Å to generate de novo atomic models. High resolution (< 3.0 Å) allows reasonably accurate placement of individual atoms and generation of relatively high quality atomic models similar to X-ray crystallography.
3. What sorts of samples are good for cryoEM-SPR?
High resolution structures of larger proteins, membrane proteins, protein complexes, large folded RNAs, and nucleic acid-protein complexes have been achieved using SPR. Unlike X-ray crystallography, crystals are not required, which considerably lowers the bar on getting atomic resolution information. Generally, larger is better (>150 kDa), and irregular, non-spherical shapes are a benefit. In addition, proteins that form helical polymers or with symmetry (e.g. viruses) can be good substrates for SPR. While proteins as small as hemoglobin (64 kDa) have been solved to atomic resolution by SPR, this is not typical. Consult with Fei about how to approach such projects.
4. How typical is it to achieve high resolution?
This is an unknown and depends on numerous variables that are overwhelmingly sample-dependent (see Literature Review Docs on how to achieve the best samples). The main limiting variable is sample compositional and conformational homogeneity on the cryoEM grid. Protein purification and homolog screening play a main role, but grid preparation conditions also contribute. A protein’s intrinsic flexibility will also affect resolution, as will the presence of ligands. Determining sample viability is the primary task of beginning an cyroEM-SPR project.
5. How much sample do I need and at what purity?
For a detailed explanation of sample requirements, please see the Literature Review Docs and Self-Training Links pages, as these will vary from sample to sample. As a rough estimate to begin a project, you will need 0.3-1 mL of 1-5 mg/mL protein (0.3 – 5 mg) that has been purified by size-exclusion chromatography or gradient centrifugation in the final step, appears homogeneous by SDS/PAGE or Native-Blue gels, and is supplied in a minimal buffer with low salt and additives. Inhomogeneous samples generally are not worth pursuing without further clean-up. Initial sample evaluation will be done by negative stain, followed by cryoEM screening using the JEOL 2100. Glacios time will be reserved for promising samples as judged from initial negative-stain and JEOL 2100 data.
6. What does BioEM Facility provide?
We provide initial training and consultation, followed by access to grid preparation equipment and data collection (both on a recharge basis, please see our rates sheet (https://bioem.ucdavis.edu/services-and-rates), raw movie stacks, motion corrected images and, if applicable, 2-D class averages from that data, generated by an automated data analysis pipeline. For very high quality samples, we presently offer access to the BACEM Consortium Krios Titan Microscope at UCSF.
We do NOT generate structures, provide sample-generating materials, licensed software access beyond facility use, generate density maps, build and refine models or provide computer support beyond what is mentioned. These tasks are the user’s responsibility and will require dedicated personnel time (see the User Responsibilities page). There are ample self-training resources for advanced training (Self-Training Links page) as well as the possibility for collaboration with existing facility users.
7. What are the personnel requirements?
The user-researcher is required to provide the main personnel involved in the entirety of cryoEM-SPR projects. Fei Guo will provide them with initial training and resources. These personnel, once trained, will be entirely responsible for project execution, including all sample preparations, initial negative stain characterization, and structural analysis after data have been collected. The amount of time spent will determine the rate of project advancement. Collaborations with existing cryoEM labs is one way to accelerate training and data analysis, but these are formed at the discretion of the lab PIs. Generally, to initiate a feasibility study, 25-50% time for a post-doc or graduate student will be required. If the sample is suitable for SPR analysis, for a project to progress at a reasonable rate, 50% or more of a post-doc or graduate student’s time would be a minimal requirement.
8. What are the computational requirements?
Please see a detailed explanation of computational requirements for a full structure solution (Computational Hardware and Software page). Our facility provides data pre-processing (motion/CTF corrected frames), short-term data storage, initial particle picking, and 2-D class averages. However, these services are provided via an automated pipeline without parameter optimization. While some initial processing can be done in the facility, the actual image processing will require user-provided computing resources, including long term data storage of very large data sets (1 GB/movie, 2-4 TB for each data set).
9. What is the typical BioEM cost and timeline for solving a structure?
This depends entirely on the trajectory of the project and how long it takes to generate a high-quality sample. Examples of some step-wise time estimates are
Negative-stain evaluation (1 week – 1 month)
Preliminary cryo grid evaluation (1 week – 1 year)
Glacios screening evaluation (1 week – 1 month)
Glacios high quality data collection (1 day – 1 month)
Image processing/3-D reconstruction (1 week – 6 months)
Please see our rate sheet for unit costs.
10. How do I learn more about cryoEM-SPR?
Community is one of the best teachers. To subscribe our cryoEM user mailing list, please visit https://lists.ucdavis.edu/sympa/info/cryoem for UC Davis users, or send a request to Fei Guo (firstname.lastname@example.org) for non-UC Davis users. To assist in building a strong UC Davis BioEM Community, we will also hold bi-monthly User meetings. These meetings will deal both with week-to-week BioEM operations but also discussions of cryoEM-SPR topics and research at UCD. In the future, BioEM faculty will be designing and implementation courses concerning cryoEM-SPR theory and practice. We will also hold periodic workshops on basic cryoEM-SPR tasks and implementation. A schedule of these events can be found at (https://bioem.ucdavis.edu/events).
In addition, there are numerous on-line resources and publications addressing every step of the process. We have collected some links to publications and videos we find useful (Self-Training Links page).
11. How is data collection scheduled and prioritized?
Scheduling should be discussed with Fei Guo and depends on your sample needs.
Scheduling Calendars can be found at (https://bioem.ucdavis.edu/schedules)
-Requires approved training and dedicated personnel
-Requires dedicated personnel and consultation with Fei Guo for each use
Glacios (screening and data collection)
-Requires consultation with Fei Guo for each use
-Requires dedicated personnel
-Requires consultation with Fei Guo for each use
-Requires Data Collection Request form (Download Krios Data Request Form)
12. I want to make cyroEM-SPR part of my NIH Grant. What do I do?
Consult with Fei Guo. NIH Grant reviewers will typically want to see evidence that a sample or set of samples are cryoEM-friendly. Depending on what stage the project is at, you will at least need to provide preliminary images and class averages, that is do a Feasibility Study. With enough lead time, some limited funding for these can be obtained through the Office of Research CRCF Pilot and Feasibility Program (https://research.ucdavis.edu/research/core-facilities-services/core-resources/). If you decide to incorporate cryoEM-SPR into your proposal, Fei can write you a letter of support (email@example.com) and facilities information can be downloaded from our website (Facilities Statement for Proposals).
13. What are the specifications of the individual microscopes and computers in the BioEM core?
Please go to the equipment page for links to machine specifications:
- JEOL 1230 (Negative stain) 100kV, LaB6 filament, TVIPS 2K CCD camera
- JEOL 2100 (cryo grid screening) 200kV, FEG, DirectElectron DE-20 camera
- Glacios (cryo grid screening and data collection) 200kV X-FEG, Gatan K3 camera
- Titan Krios (UCSF) 300kV X-FEG, GIF, phase plate, Gatan K3 camera,
- Gatan K3 computer: on-the-fly data collection
- Maestro: on-the-fly image processing (motion correction/particle picking/2-D classification), x1 CPU: Intel i7-6900K@3.2GHz, x4 GPU: Nvidia GTX 1080 Ti, RAM: 128 GB, Storage: 1TB ssd, 4TB RAID drive
- Gordo (short term storage): 84TB for short term data storage
- Primo (short term data processing) : x2 CPU: Intel E5-2640 v4 @ 2.4GHz, x2 GPU: Nvidia GTX 1080, RAM: 128 GB, Storage: 1T SSD, 26TB RAID drive