Core 3: CryoEM Summary Since the CRNA’s inception, cryo-electron microscopy (cryoEM) has been a key structural biology technology provided through the CryoEM Core. During CRNA 1.0 we made many advances in developing cryoEM procedures for studying the structures of small RNAs, initiated CryoEM projects with several CRNA investigators, and held cryoEM workshops to inspire CRNA members and others to use this powerful structure technique. Our most notable achievements are reaching a major technical milestone in determining a subnanometer resolution cryoEM structure of the 30 kDa HIV-1 dimerization initiation site (DIS) RNA dimer and revising the current Rev response element (RRE) structure with rigorous cryoEM structure validation protocols. These studies were done in collaboration with multiple CRNA groups, including Marchant, Summers and Case, to integrate structural information from cryoEM, NMR and molecular dynamics. The DIS structure represents the smallest macromolecule structure determined to date by cryoEM. Moving into CRNA 2.0, we have extended our team to include investigators with complementary expertise from Baylor College of Medicine, the University of Virginia and NIH. We have set three aims: (i) to utilize state-of-the-art cryoEM as a tool for determining the highest possible resolution structures of RNAs, RNA-protein complexes and HIV- related protein complexes, in collaboration with investigators from the CRNA projects and throughout the CRNA; (ii) to develop new experimental and computational protocols in cryoEM for characterizing challenging specimens of RNA and RNA-protein complexes, which are either small, compositionally or conformationally variable, or both; and (iii) to integrate measurements from NMR, X-ray crystallography, cryoEM, molecular dynamics simulation, and biochemistry for partial or intact biological complexes relevant to the CRNA research missions in order to understand their structure and function. In addition, we will continue to offer systematic, hands-on cryoEM training to CRNA students and postdoctoral fellows so that they will become more versatile structural biologists in the coming decades.