Issue Date: March 31, 2014 | Web Date: March 28, 2014
Electron Microscopy Milestone
The first near-atomic-level glimpse of a massive 3-megadalton biological machine has been obtained using only cryogenic electron microscopy (cryoEM), without the need for protein crystallization or extensive purification protocols. The analytical milestone was achieved on the yeast mitochondrial ribosome’s large subunit, a 39-protein complex crucial for making mitochondrial membrane proteins in the energy-producing organelle.
This ribosome is found in eukaryotic mitochondria. It differs from the ribosome found in the cytoplasm of yeast and in cells of other eukaryotes, as well as from the bacterial ribosome, whose three-dimensional X-ray crystal structure garnered Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath the 2009 Nobel Prize in Chemistry. The new 3.2-Å structure was determined by Ramakrishnan, Sjors H. W. Scheres, and their colleagues at the MRC Laboratory of Molecular Biology in Cambridge, England (Science 2014, DOI: 10.1126/science.1249410).
“This paper is one of the most exciting results in structural biology in recent years,” comments Nenad Ban, a structural biologist at the Swiss Federal Institute of Technology, Zurich. “It is a methodological breakthrough to be able to use electron microscopy” to structurally analyze such a large protein complex at atomic-level resolution, Ban says. “This completely changes structural biology” because it eliminates the need for crystallography to solve the structures of massive molecular complexes, he says.
The work provides biomedical researchers with the first near-atomic glimpse of a mitochondrial ribosome. This ribosome is responsible for producing membrane proteins that are essential for the organelle’s energy-making oxidative phosphorylation system.
Dysfunction of the human version of this ribosome is the culprit in several congenital conditions, including forms of deafness and heart-muscle disorders. The human mitochondrial ribosome is also responsible for several side effects of antibiotics that target bacterial cytoplasmic ribosomes. That’s because it is believed to have evolved from bacterial ribosomes about 2 billion years ago, after eukaryotic cells engulfed a bacterium.
The human mitochondrial ribosome is expected to be structurally different from the yeast version analyzed by the Ramakrishnan-Scheres team. Yet the work paves the way for using cryoEM to determine the structure of the human version at atomic resolution, Ramakrishnan says.
The breakthrough was “made possible by a new generation of electron detectors of unprecedented speed and sensitivity,” explains Werner Kühlbrandt of the Max Planck Institute for Biophysics, in Frankfurt, Germany, in an associated commentary (Science 2014, DOI: 10.1126/science.1251652). These paper-thin detectors have extremely high resolution, aren’t destroyed by the high-energy electrons used in cryoEM, and are fast enough to compensate for movements of protein complexes that occur during analysis, Kühlbrandt adds.
Many groups have tried to crystallize mitochondrial ribosomes for X-ray analysis, but low abundance of these complexes and poor sample purity have stymied efforts, Scheres says. To get around these problems, Ramakrishnan’s team skipped the usual biochemical purification protocols and relied instead on newly developed image classification software to select individual mitochondrial ribosomes for analysis.
Ramakrishnan expected there to be structural differences between bacterial and mitochondrial ribosomes, and the study confirmed this. The “biggest surprise” of the study, he says, was that the mitochondrial ribosome’s exit tunnel for newly made proteins is unexpectedly far away, 35 Å, from the bacterial ribosome’s protein outlet.
The team has “achieved something that, less than a year ago, few would have thought possible,” Kühlbrandt notes. “To be able to do this is nothing short of a revolution.”
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