Systemic Upgrade of a Cryo-Electron Microscope

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In this application, we apply for funding to secure the competitive performance of the sole cryo-electron-tomography-suitable (300 kV) electron microscope (Titan Krios G1, Thermo Fisher Scientific, TFS) of the Goethe University Frankfurt (GU) and of the Hessian universities, by an upgrade of the direct-electron detector and the compustage/autoloader electronics of the system. At the GU and the University in Giessen, many groups utilize cryo-electron tomography (cryoET) as well as single-particle cryo-electron microscopy (cryoEM) for high-end structural and cellular biology. Demands on both the resolution and throughput of the system is increasing, driven by the proven enhanced applicability of cryoEM/ET in both structural biology and structural (in-situ) cell biology. The Krios was commissioned in 2011 and was collaboratively operated with the Max Planck Institute of Biophysics until 2021 when the 10-year cooperation agreement expired. It is currently operated within an infrastructure center of the GU (Frankfurt Center for Electron Microscopy, FCEM). The focus of the research papers over the last decade has been on membrane dynamics and membrane-attached complexes. Previous successful projects include the structure of multi-domain signaling molecules, the structure and dynamics of RNA-Polymerase I, deep insights into mitochondrial function, structure and dynamics of cellular interfaces, mechanisms of antibiotic resistance and others. In the meanwhile, the electronics of the microscope, in particular the compustage and autoloader, have become antiquated and are no longer manufactured. Thus, service and replacement parts from TFS are not guaranteed. To allow for further operation of the Krios, a complete electronics replacement/upgrade is therefore necessary. The electronics replacement must be accompanied by an operating system upgrade, as the newer electronic parts require a different operating system. In addition, the mechanics of the compustage shows massive wear, resulting in extremely prolonged data acquisition times for cryoEM and importantly, prohibits any fast-tilting scheme for cryoET which is currently the standard. In addition to the electronics upgrade, we also apply for an upgrade of the direct electron detector of the system. The current direct electron detector lacks both performance and throughput compared to the current models available in the market. Of critical importance is that the service of the direct electron detector terminates in 2023, and thereafter an exchange of parts or even the detection chip will no longer be supported by the vendor (Gatan). Overall, the systemic upgrade of our Krios, as proposed here, is the best value for money to conduct current and future projects in a competitive manner for the next decade.