What Could Go Wrong? A Practical Guide to Single-Particle Cryo-EM: From Biochemistry to Atomic Models

Cryo-electron microscopy (cryo-EM) has enjoyed explosive recent growth due to revolutionary advances in hardware and software, resulting in a steady stream of long-awaited, high-resolution structures with unprecedented atomic detail. With this comes an increased number of microscopes, cryo-EM facilities, and scientists eager to leverage the ability to determine protein structures without crystallization. However, numerous pitfalls and considerations beset the path toward high-resolution structures and are not necessarily obvious from literature surveys. Here, we detail the most common misconceptions when initiating a cryo-EM project and common technical hurdles, as well as their solutions, and we conclude with a vision for the future of this exciting field.