Negatively stained GroEL protein.

GroEL is a bacterial chaperone protein and is required for the correct folding of many proteins. It consists out of two barrel shaped heptamer rings, the subunits are 58-kDa and identical. Cryo-TEM has been used to investigate the mechanism by which it works. This image was acquired on the Talos at 120kV.

Mitochondria in cell

Mitochondria generate much of the chemical energy used by a cell. They are cell organelles with a double membrane, an outer membrane and a distinctively folded inner membrane which increases the surface area on which chemical reaction can occur. This image has been acquired on the Talos at 80kV.

Talos L120C TEM system:

The Talos transmission electron microscope offers the ability to screen samples with high contrast and high sensitivity. Specifications include: 0.204nm Line resolution and <0.37nm point resolution. This system is housed in MSB G056 and will be used for negative stain imaging and for cryo-EM sample screening. It is also available for imaging of traditional TEM samples. 

The Talos is equipped with a Gatan Elsa (698) side-entry cryo-holder for imaging cryo-EM samples.

Mark IV Vitrobot:

This system allows for reproducible vitrification of biological samples for cryo-EM analysis. Vitrification results from a rapid-freezing method that forms transparent, amorphous ice without damaging the biological samples. The Vitrobot interface offers control over temperature, humidity and other grid blotting settings allowing for reproducible sample freezing conditiions.

Glacios 200 kV cyogenic TEM:

Glacios 200 kV cryogenic TEM equipped with both a Ceta-16M camera and a TFS Falcon 4 direct electron detector, in addition to a Selectris Energy Filter. This system will has the potential for solving atomic resolution structures for Single Particle Analysis projects.

ThermoFisher

negative staining workbench

A Negative Stain bench provides all the materials needed for the safe handling, preparation, and disposal of uranyl-salt solutions and other materials for negative staining. 

Time for using it can get reserved on the Stratocore website for the core. 

How to get started and what proteins can get imaged?

How do I get started on a project?
Information for new users.
Stratocore:
PPMS is a core facility management software provided by Stratocore. The CASB facility uses it for scheduling, billing and project management. If you or your PI do not yet have a Stratocore account you will need to establish one. Navigate to the entry portal: https://med.uc.edu/research/core for directions for requesting an account.
Please note that Cincinnati Children's also uses Stratocore software, but that account information is not shared between Children’s and UC. You will need an account at UC.
Starting New Projects:
Please initiate all new projects by contacting the staff to ask for a project consultation. The consultation request can be made by anyone but should always be done with the approval of the PI(s) involved in the proposed work. After contacting the facility for a consultation, you will be asked to fill out and return a short questionnaire detailing sample information and your imaging goals. A consultation meeting will be scheduled after the completed questionnaire has been received and reviewed. In most cases, the staff will simply arrange a face-to-face meeting with the person who made the request. This meeting should involve all lab personnel who will be involved in the project. The purpose of such a meeting is to establish details (including the potential costs, the state of the sample(s) to be examined, expectations for the project's time-to-completion (including possible benchmarks along the way) and involvement of the staff versus lab personnel (for example, specific roles of the staff about providing advice, training, imaging and/or data analysis). There is never a fee for staff time spent in such meetings.
Before work on the project can begin, a PI will need to authorize the facility to use a specific account usually through Statocore (Cincinnati Children’s researchers need to provide a contact person who can provide a PO for services rendered.)
_______________________________________________________

What Proteins are suitable targets for cryo electron microscopy?
While X-ray crystallography and NMR continue to be valuable techniques for solving atomic structures, cryo-EM is rapidly becoming the method of choice for structural biologists who seek to study complex molecular machines or challenging targets such as membrane proteins.
Single particle analysis (SPA) is a popular method for characterizing the molecular organization of biological samples. SPA is fundamentally an averaging technique, where thousands of low-signal/high-noise images from similar views/orientations are sorted into class averages to improve signal to noise ratio. Anything that decreases similarity like different conformations of the protein increases the number of particles needed for imaging and analysis.

Factors which affect the difficulty of the task:
Protein size: While proteins as small as ~50kDa have been determined by cryo-EM, these represent extremely challenging targets. Proteins above 200kDa are considered more promising targets. To obtain a structure, images of the particles must be aligned to each other accurately. This can be difficult to do with small proteins. Larger targets with more features are easier to identify and easier to align during reconstruction. Distinct features of a protein will make the analysis easier.
Flexibility: While proteins with different conformations can be analyzed, this will require a minimum number of particles to be analyzed for each conformation.
Purity: proteins need to be >99% pure for analysis and their transient or permanent modifications should be minimal. Several microliters of a prote

Sharma, M; Sheth, M; Poling, HM; Kuhnell, D; Langevin, SM; Esfandiari, L. Rapid purification and multiparametric characterization of circulating small extracellular vesicles utilizing a label-free lab-on-a-chip device. (2023) SCIENTIFIC REPORTS. 13(1):18293 PMC10600140

Chutipongtanate S, Cetinkaya H, Zhang X, Kuhnell D, Benefield D, Haffey W, Wyder M, Patel R, Conrey SC, Burrell AR, Langevin S, Nommsen-Rivers L, Newburg DS, Greis KD, Staat MA, Morrow AL. Prenatal SARS-CoV-2 infection alters postpartum human milk-derived extracellular vesicles. (2023) bioRxiv. PMC10312504

Chimote, AA; Lehn, MA; Bhati, J; Mascia, AE; Sertorio, M; Lamba, MA; Ionascu, D; Tang, AL; Langevin, SM; Khodoun, MV; Wise-Draper, TM; Conforti, L. Proton Treatment Suppresses Exosome Production in Head and Neck Squamous Cell Carcinoma. (2024). CANCERS(Basal) 16(5):1008 PMC10931005