The Resolution Revolution Werner Kühlbrandt Science 343, 1443 (2014); DOI: 10.1126/science.1251652
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Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6178/1443.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6178/1443.full.html#related This article cites 10 articles, 4 of which can be accessed free: http://www.sciencemag.org/content/343/6178/1443.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on April 2, 2014
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PERSPECTIVES BIOCHEMISTRY
Advances in detector technology and image processing are yielding high-resolution electron cryo-microscopy structures of biomolecules.
The Resolution Revolution Werner Kühlbrandt
CREDIT: PANEL B ADAPTED WITH PERMISSION FROM (2); PANEL C ADAPTED FROM (3)
P
recise 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 is what Amunts et al. have done in their research article on page 1485 of this issue (1), reporting 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 (2–4) (see the figure), 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 proteinRNA complexes that translate the linear genetic code into three-dimensional proteins. Mitochondria—semi-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 (5, 6). In determining the highresolution structure of the mitochondrial ribosome by cryo-EM, Amunts et al. achieve something that, less than a year ago, few would have thought possible. 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. Department of Structural Biology, Max Planck Institute of Biophysics, 60538 Frankfurt, Germany. E-mail: werner. [email protected]
A
B
C
Near-atomic resolution with cryo-EM. (A) The large subunit of the yeast mitochondrial ribosome at 3.2 Å reported by Amunts et al. In the detailed view below, the base pairs of an RNA double helix and a magnesium ion (blue) are clearly resolved. (B) TRPV1 ion channel at 3.4 Å (2), with a detailed view of residues lining the ion pore on the four-fold axis of the tetrameric channel. (C) F420-reducing [NiFe] hydrogenase at 3.36 Å (3). The detail shows an α helix in the FrhA subunit with resolved side chains. The maps are not drawn to scale.
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. Some 10 years ago, Henderson and Faruqi realized that it should be possible to design a sensor that detects electrons directly and that combines the advantages of CCD cameras and film (7). They and two competing teams (8) have since 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 radiationhard. 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 struc-
tures. 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 similarly dramatic as that of the Hubble telescope in astronomy, although the blurring is caused by different effects in the two cases. 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 (9). Averaging of recognizable molecular features in tomographic volumes is already revealing subnanometer detail even with standard CCD cameras (10). The new detectors are bound to make an enormous difference in this area.
www.sciencemag.org SCIENCE VOL 343 28 MARCH 2014 Published by AAAS
1443
PERSPECTIVES Concurrently with the new cameras, powerful maximum likelihood image processing routines became available. These routines define reliable and objective criteria for averaging tens or hundreds of thousands of single-particle images, as is necessary to achieve high resolution (11). 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 cryoEM images. Does the resolution revolution in cryo-EM mean that the era of x-ray protein crystallography (12) 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. These are exciting times. References and Notes 1. A. Amunts et al., Science 343, 1485 (2014). 2. M. Liao, E. Cao, D. Julius, Y. Cheng, Nature 504, 107 (2013). 3. M. Allegretti et al., eLife 3, e01963 (2014). 4. X. Li et al., Nat. Methods 10, 584 (2013). 5. N. Ban et al., Science 289, 905 (2000).
6. B. T. Wimberly et al., Nature 407, 327 (2000). 7. A. R. Faruqi, R. Henderson, Curr. Opin. Struct. Biol. 17, 549 (2007). 8. Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) formed a consortium with engineers at the Rutherford Appleton Laboratory and scientists at the Max Planck Society to fund and develop a first prototype. The consortium then joined forces with the electron microscope manufacturer FEI to roll out and market the new design. At about the same time, Gatan Inc. of Pleasanton, California came out with a similar detector designed by Peter Denes (Lawrence Berkeley National Laboratory) and David Agard (University of California, San Francisco). A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company (San Diego, California). 9. B. Daum, A. Walter, A. Horst, H. D. Osiewacz, W. Kühlbrandt, Proc. Natl. Acad. Sci. U.S.A. 110, 15301 (2013). 10. F. K. Schur et al., J. Struct. Biol. 184, 394 (2013). 11. S. H. Scheres, J. Struct. Biol. 180, 519 (2012). 12. Special section on Crystallography at 100, Science (7 March 2014). 10.1126/science.1251652
APPLIED PHYSICS
Molecular Tuning of Quantum Plasmon Resonances
The tuning of nanostructure plasmon resonances with bridging molecules offers opportunities for both plasmonics and molecular electronics.
Peter Nordlander A C etallic nanoparticles D exhibit plasmon resoD nances, which are colEgap = Eext dg lective, coherent oscillations of their conduction electrons that can dg couple very efficiently to light. Originally a subfield of condensedB matter physics, the past decade has ε FL (t) ε RF(t’) e– e– ε RF (t) ε FL(t’) seen tremendous growth of plasmonics as an interdisciplinary field spanning chemistry, materials science, and biology. On page 1496 of this issue, Tan et al. (1) discuss an experiment that will almost certainly further fuel this Plasmon-induced electron tunneling. (A) The electric potential associated with the incident radiation field (black dashed growth—the coupling of plasmon line) is screened by the nanoparticles and creates an enhanced local field in the gap (solid red line) determined by the strucexcitations to molecular conduc- tural parameters (blue) D and dg. (B) The field in the junction shifts the relative positions of the Fermi levels εF of the left and tion. The merging of plasmonics right particles as a function of time t and enables electrons to transfer between nanoparticles in each cycle of the incident with molecular electronics prom- radiation. (C) A schematic of the molecular junction used in the present experiment shows the planar junction geometry and ises both novel fundamental dis- interrogation by a light beam (green) that allows a large number of molecules to serve as conduction channels for electrons. coveries and new applications. The energy of the plasmon resonances ties of plasmonic nanoparticles, perhaps the Nanostructures with sharp protrusions depends strongly on nanostructure shape and most mature is the use of the large plasmon- naturally induce large local field enhancecomposition. Given the emergence of highly induced enhancements in local electromag- ments, but the optimal structures for local precise nanofabrication methods, it is possi- netic fields near the surfaces of the nanoparti- field enhancements consist of a pair of metalble to design nanostructures that have plas- cles for surface-enhanced spectroscopies (2). lic nanoparticles separated by a nanometermon resonances ranging from the ultraviolet Amplified local fields can enhance the prob- scale gap (a “plasmonic dimer”). Several into the infrared. Among the many important abilities for molecular transitions by many studies have demonstrated that the local field applications of the light-harvesting proper- orders of magnitude. In nonlinear spectros- enhancements in the “hot spots” in the gaps copies such as Raman scattering, the signal of dimers can be sufficiently strong that the scales as the fourth power of the local electric Raman scattering from an individual molDepartment of Physics and Astronomy, Rice University, field across the molecule. ecule can be detected (3). Classical electroHouston, TX 77005, USA. E-mail: [email protected]
M
1444
28 MARCH 2014 VOL 343
SCIENCE www.sciencemag.org
Published by AAAS
This copy is for your personal, non-commercial use only.
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here.
Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6178/1443.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/343/6178/1443.full.html#related This article cites 10 articles, 4 of which can be accessed free: http://www.sciencemag.org/content/343/6178/1443.full.html#ref-list-1 This article appears in the following subject collections: Biochemistry http://www.sciencemag.org/cgi/collection/biochem
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on April 2, 2014
The following resources related to this article are available online at www.sciencemag.org (this information is current as of April 2, 2014 ):
PERSPECTIVES BIOCHEMISTRY
Advances in detector technology and image processing are yielding high-resolution electron cryo-microscopy structures of biomolecules.
The Resolution Revolution Werner Kühlbrandt
CREDIT: PANEL B ADAPTED WITH PERMISSION FROM (2); PANEL C ADAPTED FROM (3)
P
recise 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 is what Amunts et al. have done in their research article on page 1485 of this issue (1), reporting 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 (2–4) (see the figure), 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 proteinRNA complexes that translate the linear genetic code into three-dimensional proteins. Mitochondria—semi-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 (5, 6). In determining the highresolution structure of the mitochondrial ribosome by cryo-EM, Amunts et al. achieve something that, less than a year ago, few would have thought possible. 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. Department of Structural Biology, Max Planck Institute of Biophysics, 60538 Frankfurt, Germany. E-mail: werner. [email protected]
A
B
C
Near-atomic resolution with cryo-EM. (A) The large subunit of the yeast mitochondrial ribosome at 3.2 Å reported by Amunts et al. In the detailed view below, the base pairs of an RNA double helix and a magnesium ion (blue) are clearly resolved. (B) TRPV1 ion channel at 3.4 Å (2), with a detailed view of residues lining the ion pore on the four-fold axis of the tetrameric channel. (C) F420-reducing [NiFe] hydrogenase at 3.36 Å (3). The detail shows an α helix in the FrhA subunit with resolved side chains. The maps are not drawn to scale.
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. Some 10 years ago, Henderson and Faruqi realized that it should be possible to design a sensor that detects electrons directly and that combines the advantages of CCD cameras and film (7). They and two competing teams (8) have since 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 radiationhard. 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 struc-
tures. 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 similarly dramatic as that of the Hubble telescope in astronomy, although the blurring is caused by different effects in the two cases. 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 (9). Averaging of recognizable molecular features in tomographic volumes is already revealing subnanometer detail even with standard CCD cameras (10). The new detectors are bound to make an enormous difference in this area.
www.sciencemag.org SCIENCE VOL 343 28 MARCH 2014 Published by AAAS
1443
PERSPECTIVES Concurrently with the new cameras, powerful maximum likelihood image processing routines became available. These routines define reliable and objective criteria for averaging tens or hundreds of thousands of single-particle images, as is necessary to achieve high resolution (11). 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 cryoEM images. Does the resolution revolution in cryo-EM mean that the era of x-ray protein crystallography (12) 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. These are exciting times. References and Notes 1. A. Amunts et al., Science 343, 1485 (2014). 2. M. Liao, E. Cao, D. Julius, Y. Cheng, Nature 504, 107 (2013). 3. M. Allegretti et al., eLife 3, e01963 (2014). 4. X. Li et al., Nat. Methods 10, 584 (2013). 5. N. Ban et al., Science 289, 905 (2000).
6. B. T. Wimberly et al., Nature 407, 327 (2000). 7. A. R. Faruqi, R. Henderson, Curr. Opin. Struct. Biol. 17, 549 (2007). 8. Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) formed a consortium with engineers at the Rutherford Appleton Laboratory and scientists at the Max Planck Society to fund and develop a first prototype. The consortium then joined forces with the electron microscope manufacturer FEI to roll out and market the new design. At about the same time, Gatan Inc. of Pleasanton, California came out with a similar detector designed by Peter Denes (Lawrence Berkeley National Laboratory) and David Agard (University of California, San Francisco). A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company (San Diego, California). 9. B. Daum, A. Walter, A. Horst, H. D. Osiewacz, W. Kühlbrandt, Proc. Natl. Acad. Sci. U.S.A. 110, 15301 (2013). 10. F. K. Schur et al., J. Struct. Biol. 184, 394 (2013). 11. S. H. Scheres, J. Struct. Biol. 180, 519 (2012). 12. Special section on Crystallography at 100, Science (7 March 2014). 10.1126/science.1251652
APPLIED PHYSICS
Molecular Tuning of Quantum Plasmon Resonances
The tuning of nanostructure plasmon resonances with bridging molecules offers opportunities for both plasmonics and molecular electronics.
Peter Nordlander A C etallic nanoparticles D exhibit plasmon resoD nances, which are colEgap = Eext dg lective, coherent oscillations of their conduction electrons that can dg couple very efficiently to light. Originally a subfield of condensedB matter physics, the past decade has ε FL (t) ε RF(t’) e– e– ε RF (t) ε FL(t’) seen tremendous growth of plasmonics as an interdisciplinary field spanning chemistry, materials science, and biology. On page 1496 of this issue, Tan et al. (1) discuss an experiment that will almost certainly further fuel this Plasmon-induced electron tunneling. (A) The electric potential associated with the incident radiation field (black dashed growth—the coupling of plasmon line) is screened by the nanoparticles and creates an enhanced local field in the gap (solid red line) determined by the strucexcitations to molecular conduc- tural parameters (blue) D and dg. (B) The field in the junction shifts the relative positions of the Fermi levels εF of the left and tion. The merging of plasmonics right particles as a function of time t and enables electrons to transfer between nanoparticles in each cycle of the incident with molecular electronics prom- radiation. (C) A schematic of the molecular junction used in the present experiment shows the planar junction geometry and ises both novel fundamental dis- interrogation by a light beam (green) that allows a large number of molecules to serve as conduction channels for electrons. coveries and new applications. The energy of the plasmon resonances ties of plasmonic nanoparticles, perhaps the Nanostructures with sharp protrusions depends strongly on nanostructure shape and most mature is the use of the large plasmon- naturally induce large local field enhancecomposition. Given the emergence of highly induced enhancements in local electromag- ments, but the optimal structures for local precise nanofabrication methods, it is possi- netic fields near the surfaces of the nanoparti- field enhancements consist of a pair of metalble to design nanostructures that have plas- cles for surface-enhanced spectroscopies (2). lic nanoparticles separated by a nanometermon resonances ranging from the ultraviolet Amplified local fields can enhance the prob- scale gap (a “plasmonic dimer”). Several into the infrared. Among the many important abilities for molecular transitions by many studies have demonstrated that the local field applications of the light-harvesting proper- orders of magnitude. In nonlinear spectros- enhancements in the “hot spots” in the gaps copies such as Raman scattering, the signal of dimers can be sufficiently strong that the scales as the fourth power of the local electric Raman scattering from an individual molDepartment of Physics and Astronomy, Rice University, field across the molecule. ecule can be detected (3). Classical electroHouston, TX 77005, USA. E-mail: [email protected]
M
1444
28 MARCH 2014 VOL 343
SCIENCE www.sciencemag.org
Published by AAAS
