We are proposing to establish a national resource at UCLA for the development, training and dissemination of advanced electron diffraction technologies. Cryo-electron microscopy (cryoEM) methods promise new life to high-throughput macromolecular structure determination. CryoEM overcomes the fundamental barrier to X-ray diffraction determination of macromolecular complexes: growing X-ray grade crystals. Fortunately, the emergence of microcrystal electron diffraction (MicroED) facilitates the determination of new protein structures at atomic resolution from vanishingly small crystals. MicroED exploits the strong interaction between electrons and nano-scale three-dimensional crystals and takes advantage of emerging cryoEM instrumentation coupled to established crystallographic methods. We pioneered MicroED to achieve milestone discoveries, namely we determined previously unknown protein structures at atomic resolution from crystals smaller than the wavelength of visible light. These technological advances, coupled with the greater availability of advanced cryoEM instruments, present an opportunity for further improvement of high-throughput structure determination. The development of new and more efficient approaches to structure determination by MicroED opens new avenues for comprehensive exploration of complex macromolecular structures that remain out of reach for standard methods. These systems include macromolecular complexes that grow small, fragile, or imperfect crystals. Our proposed innovations to technology development in MicroED are in four categories: (1) improved sample screening and preparation, (2) novel refinement and phasing methods, (2) demonstrating the applicability of MicroED to natural products and small molecules, and (4) engineering new hardware for investigating structure dynamics in real time. The biomedical problems associated with these types of assemblies are broad and impact biomedicine, both through the basic understanding of disease and through enabling new therapeutic platforms. Our group of key personnel brings together expertise from all modalities of cryoEM, crystallography, biochemistry, molecular biology, and computation to help develop the next generation of MicroED tools. This effort will be informed by projects led by expert groups around the country working on projects that pose significant structural challenges. Together, we propose a resource that can make nanocrystallography and MicroED routine methodologies for non-experts. Extensive user training and community engagement will further disseminate the MicroED technology and bring new structures to light.
By developing and applying new, advanced tools and methods in electron diffraction, we aim to facilitate the determination of new structures from macromolecular assemblies. To achieve this, we have assembled a team of experts in cryoEM, crystallography, biochemistry and computation that will establish MicroED as an accessible tool for experts and novices alike. With a focus on method development tested on driving biological projects, community engagement through workshops, symposiums, collaborations and one on one training, we forecast that our MicroED resource would have a long and lasting impact on biomedical research.