The 2017 Nobel Prize in Chemistry has been awarded to the team of scientists responsible for developing cryo-electron microscopy (cryo-EM), which allows researchers to view biomolecules with high resolution. The Nobel laureates include Jacques Dubochet of the University of Lausanne, Switzerland; Joachim Frank of Columbia University; and Richard Henderson of the MRC Laboratory of Molecular Biology, Cambridge, UK. Frank is a former member of the American Society for Cell Biology.
This innovative imaging process, still being refined, has become an important research tool for many ASCB members. At the 2016 ASCB Meeting, Grant Jensen, Professor of Biology and Biophysics/HHMI at the California Institute of Technology taught a workshop on the protocol. The technique, which grew out of work pioneered on the vitrification of pure water begun in the early 1980s, is a type of electron microscopy in which samples are held at very cold temperatures. “We use it in biology to study cells and viruses and protein complexes in a nearly-natural state surrounded by frozen water. This gives wonderful information, but there are many technical challenges,” Jensen said.
One of the challenges of using of cryo-EM, said Eva Nogales, Professor of Molecular and Cell Biology/HHMI at University of California Berkeley, has to do with sample preparation. Nogales has been using cryo-EM since graduate school when the technique was in its infancy.
“The purified material has to be vitrified into a thin layer of aqueous solution and that typically involves exposing the sample to a very hydrophobic and disruptive air-water interface that can denature protein and/or disrupt protein-protein interaction and complex integrity,” Nogales said. “Different samples have different responses to this ‘molecular torture’ procedure, and solutions, so far, have been typically obtained by trial and error on a case-by-case basis. This is obviously an area that needs some further development.”
Even so, cryo-EM plays a central role in the Nogales lab. “My lab uses it as our major tool to probe structure-function in our microtubule and kinetochore studies, as well as in our analysis of the molecular machinery involved in gene expression regulation,” Nogales said. “As an example, some of our latest effort to uncover the molecular basis of microtubule dynamic instability and its regulation have used cryo-EM to describe the conformational changes following GTP hydrolysis on microtubule structure, or the effect that anticancer agents like taxol have in the microtubule lattice.”
In the Jensen lab, they use a type of cryo-EM called electron cryotomography that has led to some important recent discoveries.
“One recent finding is the structure of the bacterial type IV pilus machine, which allows bacteria to extend a ‘grappling hook’ out and bind to something in the environment—sometimes a human cell—and then pull itself forward to that target,” Jensen said.
In the future, Jensen said cryo-EM will be used to solve many critical cell biology problems. “It will be used to understand the structure and function of the thousands of protein ‘nanomachines’ that do everything inside cells including holding genetic information, copying it, reading it, expressing proteins, folding proteins, managing the activity of proteins, degrading old proteins, sending signals, digesting food, growing new cells, dividing cells, and so forth.”
Nogales adds that she hopes to see cryo-EM become “more generally applicable, with faster throughput and higher resolution.”
“I hope that we will be able to describe ever more complex conformational and compositional mixtures that carry essential information about the dynamic workings of molecular machines,” Nogales said. “And we all hope that before the present generation of cryo-EMers retires, this kind of structural characterization can be done in the context of the cell or complex cell extracts.”
Jensen offered congratulations to the Nobel laureates and to “their colleagues through the years who pioneered this wonderfully fun field.”
And Nogales added, “The cryo-EM field has been around for many years, and many contributed to its birth and development. Cryo-EM may now have reached adolescence, but by no means maturity. We all look forward to furthering technical development and scientific breakthroughs.”