Accelerating throughput at Scotland's national Cryo-EM centre - a next generation direct electron detector for SCMI.
Lead Research Organisation:
University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci
Abstract
Structural biologists strive to understand the shape of biological molecules, the 'machinery of life', at the atomic level. In 1953, Watson and Crick published the most famous structure of a biological molecule - that of DNA. In each of our cells, our entire genome, the information that tells our bodies how to make and maintain us, is written in to this elegant double-helix structure, using an alphabet of only four letters. The interpretation of our genome, by our cells, leads to the production of a vast array of protein molecules. These are much more complex structures, and are the machines for which our DNA is the blue-print. To understand biological processes, such as how a virus can enter our bodies and cause disease, requires us to work to understand the shapes of protein molecules. In the case of a virus, such as the coronavirus that caused a pandemic and changed our lives so dramatically these past few years, we wish to understand the structures of the proteins that the virus is made of, and how they interact with our own proteins to cause disease.
To do this, we purify the proteins, freeze them, and image them in a very powerful microscope. This process is called cryogenic electron microscopy, or more commonly 'cryo-EM'. Images from cryo-EM can be processed in computers, to calculate the shapes of the protein molecule of interest at high resolution. This allow us to build a model of the protein in which we can confidently define the position of individual atoms.
In the MRC - University of Glasgow Centre for Virus Research (CVR), we perform cryo-EM experiments using the facilities available at the Scottish Centre for Macromolecular Imaging (SCMI), which is located in our building. This centre was established in 2018 and is one of only a handful of high-performance cryo-EM centres in the United Kingdom, and the only one in Scotland. We wish to build the capacity of the SCMI to solve protein structures faster, and to higher resolution, by the addition of a state-of-the-art camera system. This will dramatically speed up our experiments, allowing researchers to collect enough cryo-EM images to solve their protein structures in four to five hours, rather than the two to three days currently required. Cryo-EM equipment is very expensive to run - one day of microscope time costs more than £1,000. This investment will greatly improve the efficiency of our science and the quality of structures we are able to solve.
To do this, we purify the proteins, freeze them, and image them in a very powerful microscope. This process is called cryogenic electron microscopy, or more commonly 'cryo-EM'. Images from cryo-EM can be processed in computers, to calculate the shapes of the protein molecule of interest at high resolution. This allow us to build a model of the protein in which we can confidently define the position of individual atoms.
In the MRC - University of Glasgow Centre for Virus Research (CVR), we perform cryo-EM experiments using the facilities available at the Scottish Centre for Macromolecular Imaging (SCMI), which is located in our building. This centre was established in 2018 and is one of only a handful of high-performance cryo-EM centres in the United Kingdom, and the only one in Scotland. We wish to build the capacity of the SCMI to solve protein structures faster, and to higher resolution, by the addition of a state-of-the-art camera system. This will dramatically speed up our experiments, allowing researchers to collect enough cryo-EM images to solve their protein structures in four to five hours, rather than the two to three days currently required. Cryo-EM equipment is very expensive to run - one day of microscope time costs more than £1,000. This investment will greatly improve the efficiency of our science and the quality of structures we are able to solve.
Technical Summary
This proposal seeks funding to support the purchase of a next-generation direct-electron detection (DED) camera for the Scottish Centre for Macromolecular Imaging (SCMI). The SCMI was established as Scotland's national centre for structural biology research by cryogenic electron microscopy (cryo-EM) in 2018. The SCMI's front-line automated Cryo-TEM is a JEOL CryoARM 300 that is equipped with a Direct Electron DE64 DED. At the time of purchase the DE64 was considered cutting edge and selected as a versatile imaging tool capable of rapid linear image capture on its large 64 MPixel sensor. Electron counting data collection was comparable in performance to the leading Thermo Fisher DED the Falcon III, but over a four-times larger detection area. Electron counting with 2x binning compared favourably with the Gatan K2 summit. Moreover the DE64 was the only detector for which the vendor supported MicroED data collection.
Since 2018, DED technology has advanced as have data collection strategies. Aberration-free image shift protocols use optics rather than stage movements to collect images over an area spanning more than 10 microns^2. This development has accelerated data collection, such that combined with fast cameras users may acquire sufficient data to determine a near-atomic resolution structure in a few hours (typically ~500 micrographs per hour). Here the performance of the DE64 no longer meets user expectations, it is slow - limiting counted data collections to ~1000 micrographs per 24 hour period.
The Direct Electron Apollo is a revolutionary next-generation DED. By counting electrons on the sensor, rather than reading out whole image frames, Apollo delivers electron counting at equivalent to 2400 frames per second and at flux rates of up to 60 e/A^2. On a CryoARM 300 in Japan, the camera has delivered a <1.5 A structure. We expect that Apollo will deliver better than 10x speed up of data collection, transforming cryo-EM throughput and data quality at the SCMI.
Since 2018, DED technology has advanced as have data collection strategies. Aberration-free image shift protocols use optics rather than stage movements to collect images over an area spanning more than 10 microns^2. This development has accelerated data collection, such that combined with fast cameras users may acquire sufficient data to determine a near-atomic resolution structure in a few hours (typically ~500 micrographs per hour). Here the performance of the DE64 no longer meets user expectations, it is slow - limiting counted data collections to ~1000 micrographs per 24 hour period.
The Direct Electron Apollo is a revolutionary next-generation DED. By counting electrons on the sensor, rather than reading out whole image frames, Apollo delivers electron counting at equivalent to 2400 frames per second and at flux rates of up to 60 e/A^2. On a CryoARM 300 in Japan, the camera has delivered a <1.5 A structure. We expect that Apollo will deliver better than 10x speed up of data collection, transforming cryo-EM throughput and data quality at the SCMI.
