Cryo-EM Developers Win Nobel Prize in Chemistry
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It may sound like science fiction, but inventors of a technique in which mixtures of biomolecules are frozen in time, blasted with an electron beam, and resolved into three-dimensional structures at atomic resolution just won this year’s Nobel Prize in chemistry. Still a work in progress, cryo-electron microscopy dramatically improved through decades of dedicated tinkering by Jacques Dubochet of the University of Lausanne in Switzerland, Joachim Frank of Columbia University in New York, and Richard Henderson of MRC Laboratory of Molecular Biology, Cambridge, England. The imaging feat has delivered a windfall of structures for biologists including neurodegenerative disease researchers, who in recent years got to lay eyes upon γ-secretase, Aβ fibrils, and tau fibrils at atomic resolution.
Inklings of cryo-EM arose in the 1970s, when Henderson and colleagues were attempting to discern the structure of bacteriorhodopsin. The protein refused to form crystals amenable to X-ray diffraction, so the researchers turned to electron microscopy instead. They blasted a frozen solution of the protein with a beam of electrons, which passed through the sample and were focused by a lens onto photographic film. That led to a three-dimensional structure of the protein in 1975, though its blob-like appearance was a far cry from the exquisite detail of today’s structures.
Covered Horseshoe. The four subunits of γ-secretase make a lopsided horseshoe with a nicastrin lid (green). A previous study assigned each of the 20 transmembrane domains to a specific subunit, and the new atomic-level structure confirmed those assignments. [Courtesy of Bai et al., Nature 2015.]
Over the following decades, Henderson, Frank, Dubochet, and a cadre of other researchers have improved every aspect of the technique. Dubochet developed a rapid freezing method using liquid ethane, which prevented biomolecules from drying out and distorting under the ensuing bombardment of electrons. Henderson and others created far more sensitive electron detectors, and Frank was instrumental in advancing computational algorithms that yielded a crisp, authoritative three-dimensional structure by merging millions of images.
Pretty Pair.
As seen by cryo-EM, stacks of symmetrically paired C-shaped protofilaments generate the tau paired helical filaments found in an Alzheimer’s brain. [Courtesy of Fitzpatrick et al., Nature 2017.]
All of these improvements have paid off. Rather than scratching their heads at the sight of the amorphous splotches of the early days, researchers now use cryo-EM to scrutinize relationships between individual amino acids within devilishly complex protein machines, such as the ribosome and spliceosome.
An atomic level structure of γ-secretase—the conglomerate that churns out Aβ—was a boon to Alzheimer’s researchers. An initial, near-atomic level structure revealed how each of the complex’s 20 transmembrane regions laid in the cell membrane, and assigned each to one of the four protein subunits (May 2015 news). A few months later, an atomic-level structure unveiled interactions between specific amino acids, offering up clues about how mutations in the enzyme might lead to the overproduction of Aβ42 (Aug 2015 news). Yigong Shi of Tsinghua University in Beijing led both studies.
Stacking Up.
A cross section of Aβ42 fiber backbone showing parallel packing of Aβ monomers resolved by cryo-EM. [Courtesy of Gremer et al., Science/AAAS.]
Shi earned his cryo-EM chops with the help of Sjors Scheres—a cryo-EM expert Henderson recruited to the MRC. Scheres also developed an image-processing algorithm called RELION that helped take Shi’s initial γ-secretase structure to the atomic level. Scheres has put his stamp on other AD-related structures as well. This year, he collaborated with Michel Goedert, also at the MRC, to dazzle the field with atomic-level structures of tau fibrils (Jul 2017 news).
Scheres told Alzforum that amyloid proteins present a unique problem for cryo-electron microscopists. Compared with a protein complex like the ribosome, with its distinctive bumps and curves, amyloid fibrils are composed of meticulously stacked β-sheets, and thus are extremely smooth and difficult to get a handle on. “You have to start at the resolution where two β-strands can be separated,” he told Alzforum, and cryo-EM has finally advanced to the level where this is possible. Case in point, researchers led by Dieter Willbold and Gunnar Schröder at Heinrich-Heine-Universität, Düsseldorf, and the Forschungszentrum Jülich in Germany published an atomic-level structure of Aβ fibrils just last month using cryo-EM (Sep 2017 news).
As with most Nobel prizes, significant contributions in the field came from far more people than just the awardees. However, Scheres told Alzforum that he and others in the field largely agree that Henderson, Dubochet, and Frank made seminal advances deserving of the prize.—Jessica Shugart
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Max Planck Institute for Biophysical Chemistry
The Nobel Prize for cryo-electron microscopy honors the development of and recent breakthroughs that have been achieved by this extremely powerful structural biology technique. Currently there is hardly a week in which an exciting cryo-EM structure of a challenging protein complex is not published in one of the top scientific journals. A key advantage of cryo-electron microscopy is that no crystallization of the protein is required. This allows structural characterization of a large number of critical biological interactions, which have been out of reach for many years. In addition, because rather small sample volumes and no specific modification of the protein are required, protein complexes of interest can be purified from natural sources, overcoming the problems of recombinant protein production and assembly of protein complexes from individual components. The ability to obtain high-resolution structural information without protein crystallization furthermore allows access to a larger amount of functional and pathological protein states.
High-resolution cryo-electron microscopy has already started to provide the first critical contributions to our structural understanding of key processes in neurodegenerative disorders. This includes the determination of an atomic structure of human γ-secretase (Bai et al., 2015) and elucidation of the architecture of the human mTOR complex 1 (Aylett et al., 2016). A scientific highlight is clearly also the characterization of the structure of paired and straight filaments of the tau protein purified from the brain of a patient with Alzheimer’s disease (Fitzpatrick et al., 2017). Indeed, the highly regular structure of amyloid fibrils suggests that it should be possible to determine the three-dimensional structure of amyloid fibrils purified from the brains of patients who suffer from a variety of neurodegenerative disorders (Vázquez-Fernández et al., 2016), as well as those fibrillar polymorphs prepared in vitro (Schmidt et al., 2009; Gremer et al., 2017).
Another structural biology technique, which does not require crystallization of the protein, is nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy is particularly powerful for the analysis of the structure and dynamics of flexible and heterogeneous protein states. In addition, transient protein interactions with lower affinity are difficult to capture by cryo-electron microscopy and X-ray crystallography. NMR spectroscopy is therefore the only method that can currently provide high-resolution insight into the interaction of flexible proteins such as tau and α-synuclein with a variety of molecular partners, including microtubules and molecular chaperones. In addition, NMR spectroscopy can look into the interior of even heterogeneous protein oligomers, which are believed to be the primary toxic species in neurodegeneration (Lashuel et al., 2013), as well as liquid droplets, which have been linked to neurodegenerative disorders (Aguzzi and Altmeyer, 2016; Banani et al., 2017; Ambadipudi et al., 2017; Zhang et al., 2017).
Without doubt we will witness in the next few years many additional breakthroughs in the structural understanding of neurodegeneration using cryo-electron microscopy. This will likely not be limited to amyloid fibrils, but include transporters such as the dopamine transporter, and might potentially extend to structural studies in cells using cryo-electron tomography (Baker et al., 2017).
References:
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Schmidt M, Sachse C, Richter W, Xu C, Fändrich M, Grigorieff N. Comparison of Alzheimer Abeta(1-40) and Abeta(1-42) amyloid fibrils reveals similar protofilament structures. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):19813-8. PubMed.
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Aguzzi A, Altmeyer M. Phase Separation: Linking Cellular Compartmentalization to Disease. Trends Cell Biol. 2016 Jul;26(7):547-58. Epub 2016 Apr 1 PubMed.
Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. 2017 May;18(5):285-298. Epub 2017 Feb 22 PubMed.
Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M. Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat Commun. 2017 Aug 17;8(1):275. PubMed.
Zhang X, Lin Y, Eschmann NA, Zhou H, Rauch JN, Hernandez I, Guzman E, Kosik KS, Han S. RNA stores tau reversibly in complex coacervates. PLoS Biol. 2017 Jul;15(7):e2002183. Epub 2017 Jul 6 PubMed.
Baker LA, Grange M, Grünewald K. Electron cryo-tomography captures macromolecular complexes in native environments. Curr Opin Struct Biol. 2017 Sep 12;46:149-156. PubMed.
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