Cryo-Electron Microscopy
“The beauty of science lies in the intricate complexities of the natural world.” (Dorothy Crowfoot Hodgkin)
Description
The laboratory provides state-of-the-art assistance to the preparation of samples for cryo-electron microscopy (cryo-EM) and the subsequent visualization and data collection on the cryo-microscope. The sample is vitrified on specific supports (grids) using liquid ethane and then manipulated in liquid nitrogen. This technique does not require colouring or fixation and operates directly on the sampe in solution.
The most relevant application of cryo-electron microscopy is the determination of the structure of biological macromolecules (proteins, nucleic acids and their complexes) at near-atomic resolution: by the acquisition of hundreds of thousands of 2D projections of a single particle, it is possible to reconstruct its 3D structure with computational methods (single particle analysis) with a resolution of up to 3 angstrom and it is also possible to identify the presence of different structural conformations in the same sample.
The emergence of cryo-electron microscopy in the latest years has been a revolution in the fields of stuctural biology and drug discovery, rivalling and outcompeting the use of X-ray crystallography and earning its developers J. Dubochet, J. Frank and R. Henderson the Nobel Prize in Chemistry 2017.
This technique can also be used to perform direct imaging of different kinds of nanostructures in suspension at very high magnifications (vesicles, nanoparticles).
Equipment
Software
| Nome | Descrizione | |
|---|---|---|
| TIA (Thermo Fisher Scientific) | for sample visualization on the Ceta detector | |
| EPU (Thermo Fisher Scientific) | for sample visualization, screening and management of data collections on the Falcon detector | link |
Rates
The Centre’s regulations provide for the application of a fee for the use of the equipment:
INTERNAL USERS
For University staff: link
EXTERNAL USERS
| Equipment/Service | Autonomous use (hourly rate) | With technical support (hourly rate) |
|---|---|---|
| Glacios cryo-EM data collection * | not available | € 160,00 |
| Glacios cryo-EM screening | not available | € 186,00 |
| Vitrobot | not available | € 50,00 |
| Consumables Vitrobot for 1 hour (4 grids) | not available | € 80,00 |
| Special treatments of the sample, organization and treatment of data, drafting of reports | not available | € 100,00 |
* For analyses of single samples involving a number of hours greater than 24, specific offers will be drawn up
EXTERNAL USERS WITH AGREEMENTS
| Equipment/Service | Autonomous use (hourly rate) | With technical support (hourly rate) |
|---|---|---|
| Glacios Cryo EM data collection * | not available | € 146,30 |
| Glacios Cryo EM screening | not available | € 170,50 |
| Vitrobot | not available | € 46,20 |
| Consumables Vitrobot for 1 hour (4 grids) | not available | € 80,00 |
| Special treatments of the sample, organization and treatment of data, drafting of reports | not available | € 30,00 |
* For analyses of single samples involving a number of hours greater than 24, specific offers will be drawn up
Publications
FAQ and Protocols
FAQ
| Question | Answer |
|---|---|
| How can a scientist figure out if cryo-EM can be useful for his/her research? | It is recommended to directly contact the technician in charge, Andrea Alfieri (andrea.alfieri@unipv.it; tel. 0382 986062); he will organize a first meeting, in person or remote, to show and discuss the experimental options, expectations, procedures, times and costs. |
| How is the access of users organized? Are you open to external users? | The laboratory is open to users of any affiliation, internal or external, commercial and non-profit. The users interested in the near-atomic determination of macromolecular structures (single particle analysis) will be registered in the management calendar of the Centro Grandi Strumenti (Agendo) and will insert their bookings, to be subsequently validated by the technician in charge; they are called SPA users. Conversely, the users interested in the imaging of nanostructures (imaging users) should contact the technician directly for booking. |
| How long does a typical experiment last? | For all users, the samples are typically prepared in the morning and analyzed on the cryomicroscope in the afternoon. For SPA users, the data collection can either start the following day or be posponed. |
| Can the sample preparation activity be separated from the subsequent visualization? | Yes, because after preparation the grids can be stored in liquid nitrogen in a specific storage of the laboratory until visualization. However the long-term storage of samples in the laboratory is discouraged or limited. |
| Can a user acquire skills and work autonomously? | As far as possible, the technician is available to provide training to interested users. Incremental levels of expertise for autonomous users have been established, but certain operations can be performed exclusively by the technician in charge. |
| Why is the cryomicroscope not always available? | The cryo-electron microscope is a complex machine with several satellite instruments (cooling systems, high-tension generator, liquid nitrogen line, technical gases, storage server); it requires alignments, scheduled cycles at room temperature (cryocycles) and periodic maintenance, besides possible problems. The unavailability is promptly indicated in the calendars for SPA users. |
| How are the delivery and storage of data managed? | For both SPA and imaging users, all the images collected during sample screening can be immediately downloaded through a link provided by the technician. Instead, the data collection images from SPA users (of considerable size, from 1 to several TB) stay on the microscope server to be downloaded as soon as possible via the 10 Gbit/s connection, but the long-term permanence of these data is not allowed, in order to free space for the data that will be subsequently produced. |
| How much does all this cost? | You can refer to the fees of the different services into “Rates” section; please note that discounts are applied to internal users based on the department affiliation and specific offers can be prepared for external users. |
