The implementation of advanced algorithms with user-friendly software packages has made the cryo-EM method straightforward in practice. Despite these exciting advances, major challenges still remain, such as higher throughput, mapping continuous deformations, reconstruction of small macromolecules, and in sample preparation. For pleomorphic structures, cryo-electron tomography (cryo-ET) is the method of choice, but it is typically limited to lower resolution compared to cryo-EM, and it remains a daunting challenge to determine macromolecular structures in situ at high resolution. Overcoming these major challenges in cryo-EM and cryo-ET requires significant advances in sample preparation, hardware development and computational algorithms. The goal of this workshop is to bring together leading experts in cryo-EM, biologists, applied mathematicians and physicists to discuss and debate the current challenges and future perspectives of this very exciting cross-disciplinary field.