Single-particle cryo-electron microscopy (cryo-EM) is a powerful technique to determine the structure of macromolecular complexes at high resolution. It circumvents the need to crystalize samples for X-ray crystallography, requires limited quantities (nanogram) of purified material and can provide near atomic resolution information on large and dynamic structures. Over the past three years NIEHS has been collaborating with Duke and UNC Chapel Hill to establish regional capabilities in Cryo-EM . Molecular Microscopy Consortium (MMC) was envisioned as an association between three imaging facilities, one for each institution (Duke, UNC and NIEHS). The operation is unified by a single closely connected core team providing expertise in data acquisition, training and support in specimen preparation and in image processing. The facilities were expected to become operational in the course of two years. NIEHS led the effort by building the Cryo-EM Core in record time. Its FEI Talos Arctica cryo-electron microscope was fully operational in June 2017, nine months after placing the order. During its two years of operation, structural biologists from over two dozen groups were trained in specimen preparation. This fiscal year specimens were examined during over two hundred screening sessions which lead to the identification of conditions suitable for high resolution data collection for several complexes. Initial structures were obtained from data collected at the Talos Arctica. The Duke cryo-EM facility, equipped with a top-of-the-line FEI Titan Krios, was deployed in mid-April of 2018. This greatly accelerated the data collection on specimens that had been previously optimized at the NIEHS facility. In less than four months, data from the Krios lead to the solution of ten additional structures in the range of 3 to 5 (Table). The instrument has been operating in data collection mode uninterrupted 24 hours a day, seven days a week since its deployment. Many other projects are steadily progressing through the optimization process and are expected to yield new structures in the near future. Using their own data, collaborators from different groups were trained in the utilization of software to solve their own structures. In a relatively short period of one year, several laboratories are now capable of independently optimizing specimen preparation and, once high-resolution data is collected, of solving their own structures. This further demonstrates the viability of collaborative training supported by an organization providing high quality data. The UNC facility opened its doors in the Spring of 2019. Equipped with a Talos Arctica cryo-TEM, the new addition dramatically increases the screening capacity. This has allowed both the NIEHS and UNC facilities to dedicate some time to the optimization of data collection in their 200 KeV instruments. These routinely yield structures at 3.2 A resolution. Overall, the operation of the Cryo-EM core has helped establish a vibrant cryo-EM community at the Research Triangle Park.
| Pillon, Monica C; Sobhany, Mack; Borgnia, Mario J et al. (2017) Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage. Proc Natl Acad Sci U S A 114:E5530-E5538 |
