Answering Real Biological Questions by Combining Cryo-TEM, XRD and NMR

A new frontier exists in unraveling interactive biological and biochemical processes and pathways at the macromolecular level. Of critical importance is the three-dimensional visualization of protein complexes and molecular machines in their native functional state. Three techniques play a major role in orchestrating this.Nuclear magnetic resonance (NMR) has the capability to study specific protein domains or fragments and their role in protein folding and dynamics and in ligand binding. X-Ray crystallography (XRD) provides high-resolution but more static 3D structures of apo and liganded proteins, mainly in a monomeric or dimeric state after crystallization. To unravel more physiologically relevant situations, it is essential to visualize multimeric complexes in their tertiary and quaternary state and their interaction with other complexes. By performing cryo-transmission electron microscopy (cryo-TEM) applications like single particle analysis or tomography, this can be achieved. Cryo-TEM provides complementary information to NMR and XRD that can be crucial for understanding the structure of protein complexes that when combined can help answer real biological as well as medically relevant questions.Recent developments in cryo-TEM have brought the three major structural biology technologies closer together. When combined with molecular dynamics simulations, a continuum has been reached on all important aspects with regards to resolution and macromolecular scales which allows for full deployment of the combination of these technologies.Here, we will illustrate the historical context of these technologies with respect to one another and show how recent developments have reached the critical requirements needed to fully unleash the power of structural biology for not just answering fundamental questions, but actually contributing to curing diseases and improving health. We will discuss the future of structural biology based on results from recent publications and expected developments over the next several years.