Scientific Understanding of Consciousness
Consciousness as an Emergent Property of Thalamocortical Activity

Imaging Large Molecular Complexes via cryo-EM and Computer Processing


Science 28 March 2014:  Vol. 343  no. 6178  pp. 1443-1444

Biochemistry: The Resolution Revolution

Werner Kühlbrandt

Department of Structural Biology, Max Planck Institute of Biophysics, 60538 Frankfurt, Germany.


Precise knowledge of the structure of macromolecules in the cell is essential for understanding how they function. Structures of large macromolecules can now be obtained at near-atomic resolution by averaging thousands of electron microscope images recorded before radiation damage accumulates. This journal reports the structure of the large subunit of the mitochondrial ribosome at 3.2 Å resolution by electron cryo-microscopy (cryo-EM). Together with other recent high-resolution cryo-EM structures, this achievement heralds the beginning of a new era in molecular biology, where structures at near-atomic resolution are no longer the prerogative of x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy.

Ribosomes are ancient, massive protein-RNA complexes that translate the linear genetic code into three-dimensional proteins. Mitochondriasemi-autonomous organelles that supply the cell with energy—have their own ribosomes, which closely resemble those of their bacterial ancestors. Many antibiotics, such as erythromycin, inhibit growth of bacteria by blocking the translation machinery of bacterial ribosomes. When designing new antibiotics, it is essential that they do not also block the mitochondrial ribosomes. For this it is of great value to know the detailed structures of both. The structures of other ribosomes have been determined by x-ray crystallography. Researchers have now determined the high-resolution structure of the mitochondrial ribosome by cryo-EM.

To be able to do this without crystals is nothing short of a revolution, made possible by a new generation of electron detectors of unprecedented speed and sensitivity. The new sensors detect electrons directly, rather than first converting them into photons that are then reconverted into photoelectrons. The latter is what the widely used CCD (charge-coupled device) cameras do, but they do not perform well at high resolution. Photographic film works in principle much better for high-resolution imaging, but is incompatible with rapid electronic readout and high data throughput, which are increasingly essential.

Researchers have designed a sensor that detects electrons directly and that combines the advantages of CCD cameras and film. They have developed detectors that use essentially the same active pixel sensor technology as the camera chips in most cell phones. However, cell phone chips cannot be used in the electron microscope because the intense electron beam would destroy them instantly. The sensors therefore had to be made radiation-hard. Second, the pixels needed to be much larger to prevent the energy-rich electrons from exciting more than one pixel at a time. Third, the camera chip, complete with readout electronics in each of its 1.6 million pixels, had to be very thin, otherwise electron scattering would blur the image and compromise resolution. Current sensors are about half as thick as a sheet of paper.

Cryo-EM requires only small amounts of material. Samples that cannot be isolated in large enough quantities for x-ray crystallography can now yield high-resolution structures. The same holds for heterogeneous samples or flexible complexes that do not crystallize readily, because cryo-EM images of different particles or conformations are easily separated at the image processing stage.

The new detectors offer another decisive advantage: Their fast readout makes it possible to compensate small movements that inevitably happen when the electron beam strikes the thin, unsupported cryo-sample. Before the new cameras were developed, blurring by beam-induced movement was an insidious, seemingly insurmountable problem. Now, dozens of images of one area are taken in rapid succession, and beam-induced movements are detected and reversed in the computer. The impact of this deblurring is dramatic.

The new cameras also promise a major breakthrough in electron cryo-tomography, which images three-dimensional volumes of whole cells, cell slices, or cellular compartments, such as mitochondria. Averaging of recognizable molecular features in tomographic volumes is already revealing subnanometer detail even with standard CCD cameras. The new detectors are bound to make an enormous difference in this area.

Concurrently with the new cameras, powerful maximum likelihood image processing routines define reliable and objective criteria for averaging tens or hundreds of thousands of single-particle images, as is necessary to achieve high resolution. This combination of advanced detectors and software now produces cryo-EM structures that look, in terms of clarity and map definition, considerably better than x-ray structures at the same nominal resolution, owing to the high quality of the phase information contained in cryo-EM images.

Does the resolution revolution in cryo-EM mean that the era of x-ray protein crystallography is coming to an end? Definitely not. For the foreseeable future, small proteins—in cryo-EM, anything below 100 kD counts as small—and resolutions of 2 Å or better will remain the domain of x-rays. But for large, fragile, or flexible structures (such as membrane protein complexes) that are difficult to prepare yet hold the key to central biomedical questions, the new technology is a major breakthrough. In the future, it may no longer be necessary to crystallize large, well-defined complexes such as ribosomes. Instead, their structures can be determined elegantly and quickly by cryo-EM.

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