Cryogenic electron microscopy (cryo-EM) enables structure determination of macromolecular objects and their assemblies. Although the techniques have been developing for nearly four decades, they have gained widespread attention in recent years due to technical advances on numerous fronts, enabling traditional microscopists to break into the world of molecular structural biology. Many samples can now be routinely analyzed at near-atomic resolution using standard imaging and image analysis techniques. However, numerous challenges to conventional workflows remain, and continued technical advances open entirely novel opportunities for discovery and exploration. Here, I will review some of the main methods surrounding cryo-EM with an emphasis specifically on single-particle analysis, and I will highlight challenges, open questions, and opportunities for methodology development.

High-resolution insights into the intasome: An essential step in the life cycle of lentiviruses such as HIV-1 is when viral DNA integrates into the host genome, establishing a permanent infection of the host cell. The viral integrase enzyme catalyzes this process and is a major drug target. During viral integration, integrase binds the ends of viral DNA, forming a higher-order structure called the intasome. Passos et al. and Ballandras-Colas et al. used cryo—electron microscopy to solve the structures of the intasomes from HIV-1 and maedi-visna virus (ovine lentivirus), respectively. These structures reveal how integrase self-associates to form a functional intasome and help resolve previous conflicting models of intasome assembly. Science , this issue p. 89 , p. 93

Strengths and weaknesses of an HIV drug: Retroviruses replicate by inserting a copy of their RNA, which has been reverse transcribed into DNA, into the host genome. This process involves the intasome, a nucleoprotein complex comprising copies of the viral integrase bound at the ends of the viral DNA. HIV integrase strand-transfer inhibitors (INSTIs) stop HIV from replicating by blocking the viral integrase and are widely used in HIV treatment. Cook et al. describe structures of second-generation inhibitors bound to the simian immunodeficiency virus (SIV) intasome and to an intasome with integrase mutations known to cause drug resistance. Passos et al. describe the structures of the HIV intasome bound to a second-generation inhibitor and to developmental compounds that are promising drug leads. These structures show how mutations can cause subtle changes in the active site that affect drug binding, show the basis for the higher activity of later-generation inhibitors, and may guide development of better drugs. Science , this issue p. 806 , p. 810