Project Summary Project Title: A high precision piezo driven replacement goniometer for cryoelectron microscopy Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Joseph Stevick Summary: Biomolecular structures resolved by cryoelectron microscopy (Cryo-EM) have significantly advanced our understanding of life processes, specifically in the areas of drug design, vaccines, and other microbiological health solutions. The technique has been so successful and transformative that it was awarded the Nobel Prize for Chemistry in 2017, and more recently was used to solve the structure of the RNA-dependent RNA polymerase from the COVID-19 virus. The biochemical models produced by Cryo-EM studies like this one are vital to researching anti-viral drugs and other microbiological solutions in addition to helping shape our basic understanding of molecular machinery. However, there is a significant technology gap in Cryo-EM hardware that has been overlooked. Transmission electron microscope (TEM) side-entry goniometers (sample positioning systems) were designed before the Cryo-EM technique was developed, and cryogenic side-entry holders are unstable and inconvenient. As a result, closed-loop automated of Cryo-EM workflows becomes inefficient and limited to what the positioning system can achieve rather than what is scientifically important. Time consuming image tracking and error correction methods are currently used to compensate for the poor mechanical positioning capabilities of goniometers and side-entry holders. Our solution is to design a modern sample motion control stage and a corresponding sample holder as a purpose-built combination that is optimized for repeatability, accuracy, and stability at cryogen temperatures. The proposed design will improve state-of-the- art Cryo-EM automation by more than an order of magnitude in precision and increase Dewar life by a factor of 5. This improvement is made possible by a unique ground-up design that optimizes the stage, holder, and cryo-system simultaneously. In Aim 1, we will optimize the stage and holder, utilizing an unconventional design that limits coupled motion, and employs modern mechatronic technologies and sensing methods that will dramatically improve the closed-loop precision of our stage in comparison with current goniometers. In Aim 2, we will optimize the cryo-system design for stable -170°C sample temperatures and ease of use in concert with the stage and holder. As a commercial product, this device would act as a simple retrofit replacement to goniometers on thousands of TEMs that are already dedicated to biological research. Our long-term objective is to empower more scientists with the instruments they need to achieve the best possible Cryo-EM results without having to struggle with basic hardware issues.