Archives of Biochemistry and Biophysics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Editorial
Electron microscopy: the coming of age of a structural biology technique Electron microscopy (EM) has been instrumental in the development of cellular and molecular biology over the last decades. A constant underlying condition in the use of EM in biology is its dependence on appropriate sample preparation methods. Indeed, the most important qualitative steps forward have relied on the incorporation of new techniques, with the cryomethods illustrating a clear example of this trend. After a lengthy period of steady changes, however, a revolution is taking place in the application of EM to structural biology. Based on instrument developments and improved computer implementations, it is leading microscopy to the forefront of structural analyses. The moment is now well suited for a comprehensive overview of how we reached this point, the facts, our expectations and the problems remaining to be addressed. With this purpose, we have organized an ABB Special Issue on the recent advances and trends in EM, which appears in 2015, the International Year of Light. The issue opens with a comprehensive overview of the history of EM development for the study of macromolecular complexes. Robin Harris reviews the field from its pioneers to the present day, with emphasis on the development of sample preparation methods [1]. He stresses the virtues of complementary approaches that have brought many successful applications, and shows how the incorporation of updated image analysis procedures helped lead us to the present bright situation. The special characteristics and relevance of current EM applications for structural biology are discussed by Richard Henderson, who offers a fundamental overview of the present and future status of single particle electron cryomicroscopy [2]. This is the area in which the most far-reaching changes have emerged, giving rise to the three-dimensional resolution of macromolecular complexes to atomic resolution. This quantum leap was due mainly to better microscopes, better detectors and better data processing methods, but to attain the theoretical upper limit for these applications we have still to deal with a number of problems. The most relevant developments in the present EM revolution are reviewed in three consecutive articles that deal with developments in instrumentation, the update of three-dimensional reconstruction methods, and the validation of results. Rasmus Schroeder discusses the importance of improved instrumentation for successful EM; the incorporation of energy filters, aberration correctors and phase plates, among other advances, has led to higher quality data [3]. Together with automation procedures and (most important) new recording devices, this awakens expectations for considerable advances in the near future. The importance of new methods for the quantitative reconstruction of macromolecular volumes from sets of improved two-dimensional EM images is discussed in depth by José M. Carazo et al. [4], while http://dx.doi.org/10.1016/j.abb.2015.06.018 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
Niels Volkmann and Xiao-Ping Xu present the methods required to exploit low resolution EM volumes by fitting atomic resolution components, and stress the need for standardized procedures in quality assessment as a critical aspect for correct validation of these fits [5]. Besides the advances in single particle reconstruction, other areas of three-dimensional EM reconstruction are also living a golden age. Classical applications for the study of helical particles, icosahedral virus reconstruction, and two-dimensional crystals are reviewed, respectively, by Edward Egelman, Carmen San Martín, Andreas Engel and Hennig Stahlberg [6,7,8]. These well-known experts in their respective areas present the evolution from the historical background of the pioneering publications to the incorporation of atomic resolution in recent papers, and highlight EM’s unique possibilities for the resolution of large, very complex macromolecular complexes. The study of variability in helical assemblies and of non-symmetric components in viruses, as well as the exploitation of crystallinity to obtain higher resolutions are immediate objectives in these areas. As a general method for three-dimensional reconstruction in EM, cryo-electron tomography has established itself as a most important approach, and is also combined with single particle methods for structural cellular biology. Ohad Medalia and coworkers discuss the advantages and limitations of tomography, which was recently boosted by the use of phase plates and direct detectors to reveal structures of macromolecular complexes in their physiological environment inside eukaryotic cells [9]. As an example of how EM-based contributions can complement one other to solve a complex problem, Juan Fontana and Alasdair Steven present the study of influenza virus-mediated membrane fusion, combining a variety of methods [10]. Cryo-tomography, conventional EM of thin sections, single particle cryo-EM, subtomogram averaging and X-ray crystallography are used to solve different aspects of the problem, and are merged to offer insight into native biological complexes in physiological environments. Recent advances in EM have generated new interfaces with other techniques such as light and X-ray microscopies, allowing us to extend structural analysis beyond the inherent sample size and thickness limits imposed by the use of electrons. Correlative methods are developing rapidly to offer an extended resolution approach, ranging from EM to light/fluorescence domains. Céline L. Fonta and Bruno Humbel present the advantages of this combination to study biological events at the submicrometer level by reviewing different protocols, instruments and software for improving correlative microscopy [11]. Also in this resolution domain, soft X-ray microscopy has recently been incorporated as a major new method. Carolyn Larabell et al. describe how cryo
2
Editorial / Archives of Biochemistry and Biophysics xxx (2015) xxx–xxx
X-ray tomography is able to offer three-dimensional visualization of whole cells in close-to-native state, permitting quantitative analysis by structural microscopy [12]. The combination of this technique with light fluorescence to locate specific molecules in the three-dimensional reconstructed cells is a major goal in the development of this approach. In this rapidly progressing resolution domain, advances in focused ion beam instruments are a potent alternative and a complement to other tomographic microscopies. Alexander Rigort and Jurgen Plitzko offer a comprehensive discussion of how cryo-preparation methods combined with ion milling and scanning electron microscopy give an alternative with which to address large and thick samples, such as tissues or cellular aggregates, by sequential imaging of milled block faces [13]. This milling approach can also be used to provide thin vitrified specimens with thickness appropriate for electron tomography studies. The combination and correlation of these new instrument and processing capabilities, together with the robotization and automation of sample preparation, data acquisition and processing steps, clearly offers a real possibility of extending the structural analysis of biomolecules from the atomic all the way to the cellular scale, which has long been an aspiration of biologists.
[4] J.M. Carazo, C.O. Sorzano, J. Oton, R. Marabini, J. Vargas, Three-dimensional reconstruction methods in single particle analysis from transmission electron microscopy data, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/ j.abb.2015.05.003. this issue. [5] X.P. Xu, N. Volkmann, Validation methods for low-resolution fitting of atomic structures to electron microscopy data, Arch. Biochem. Biophys. (2015), http:// dx.doi.org/10.1016/j.abb.2015.06.017. this issue. [6] E.H. Egelman, Three-dimensional reconstruction of helical polymers, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.04.004. this issue. [7] C. San Martı´n, Transmission electron microscopy and the molecular structure of icosahedral virases, Arch. Biochem. Biophys. (2015), http://dx.doi.org/ 10.1016/j.abb.2015.06.001. this issue. [8] H. Stahlberg, N. Biyani, A. Engel, 3D reconstruction of two-dimensional crystals, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb. 2015.06.006. this issue. [9] A. Dubrovsky, S. Sorrentino, J. Harapin, T. Sapra, O. Medalia, Developments in cryo-electrontomography for in situ structural análisis, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.04.006. this issue. [10] J. Fontana, A.C. Steven, Influenza virus-mediated membrane fusion: structural insights from electron microscopy (2015), http://dx.doi.org/10.1016/ j.abb.2015.04.011. this issue. [11] C.L. Fonta, B.M. Humbel, Correlative microscopy, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.05.017. this issue. [12] M. Do, S.A. Isaacson, G. McDermott, M.A. Le Gros, C.A. Larabell, Imaging and characterizing cells using tomography, Arch. Biochem. Biophys. (2015), http:// dx.doi.org/10.1016/j.abb.2015.01.011. this issue. [13] A. Rigort, J.M. Plitzko, Cryo-focused-ion-beam applications in structural biology, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb. 2015.02.009. this issue.
References [1] R.J. Harris, Transmission electron microscopy in molecular structural biology: a historical survey, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/ j.abb.2014.11.011. this issue. [2] R. Henderson, Overview and future of single particle electron cryomicroscopy, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.02.036. thisissue. [3] R.R. Schroeder, Advances in electron microscopy: a qualitative view of instrumentation development for macro molecuar imaging and tomography, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.05.010. this issue.
José M. Valpuesta José L. Carrascosa Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain E-mail address: [email protected] (J.L. Carrascosa) Available online xxxx
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Editorial
Electron microscopy: the coming of age of a structural biology technique Electron microscopy (EM) has been instrumental in the development of cellular and molecular biology over the last decades. A constant underlying condition in the use of EM in biology is its dependence on appropriate sample preparation methods. Indeed, the most important qualitative steps forward have relied on the incorporation of new techniques, with the cryomethods illustrating a clear example of this trend. After a lengthy period of steady changes, however, a revolution is taking place in the application of EM to structural biology. Based on instrument developments and improved computer implementations, it is leading microscopy to the forefront of structural analyses. The moment is now well suited for a comprehensive overview of how we reached this point, the facts, our expectations and the problems remaining to be addressed. With this purpose, we have organized an ABB Special Issue on the recent advances and trends in EM, which appears in 2015, the International Year of Light. The issue opens with a comprehensive overview of the history of EM development for the study of macromolecular complexes. Robin Harris reviews the field from its pioneers to the present day, with emphasis on the development of sample preparation methods [1]. He stresses the virtues of complementary approaches that have brought many successful applications, and shows how the incorporation of updated image analysis procedures helped lead us to the present bright situation. The special characteristics and relevance of current EM applications for structural biology are discussed by Richard Henderson, who offers a fundamental overview of the present and future status of single particle electron cryomicroscopy [2]. This is the area in which the most far-reaching changes have emerged, giving rise to the three-dimensional resolution of macromolecular complexes to atomic resolution. This quantum leap was due mainly to better microscopes, better detectors and better data processing methods, but to attain the theoretical upper limit for these applications we have still to deal with a number of problems. The most relevant developments in the present EM revolution are reviewed in three consecutive articles that deal with developments in instrumentation, the update of three-dimensional reconstruction methods, and the validation of results. Rasmus Schroeder discusses the importance of improved instrumentation for successful EM; the incorporation of energy filters, aberration correctors and phase plates, among other advances, has led to higher quality data [3]. Together with automation procedures and (most important) new recording devices, this awakens expectations for considerable advances in the near future. The importance of new methods for the quantitative reconstruction of macromolecular volumes from sets of improved two-dimensional EM images is discussed in depth by José M. Carazo et al. [4], while http://dx.doi.org/10.1016/j.abb.2015.06.018 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
Niels Volkmann and Xiao-Ping Xu present the methods required to exploit low resolution EM volumes by fitting atomic resolution components, and stress the need for standardized procedures in quality assessment as a critical aspect for correct validation of these fits [5]. Besides the advances in single particle reconstruction, other areas of three-dimensional EM reconstruction are also living a golden age. Classical applications for the study of helical particles, icosahedral virus reconstruction, and two-dimensional crystals are reviewed, respectively, by Edward Egelman, Carmen San Martín, Andreas Engel and Hennig Stahlberg [6,7,8]. These well-known experts in their respective areas present the evolution from the historical background of the pioneering publications to the incorporation of atomic resolution in recent papers, and highlight EM’s unique possibilities for the resolution of large, very complex macromolecular complexes. The study of variability in helical assemblies and of non-symmetric components in viruses, as well as the exploitation of crystallinity to obtain higher resolutions are immediate objectives in these areas. As a general method for three-dimensional reconstruction in EM, cryo-electron tomography has established itself as a most important approach, and is also combined with single particle methods for structural cellular biology. Ohad Medalia and coworkers discuss the advantages and limitations of tomography, which was recently boosted by the use of phase plates and direct detectors to reveal structures of macromolecular complexes in their physiological environment inside eukaryotic cells [9]. As an example of how EM-based contributions can complement one other to solve a complex problem, Juan Fontana and Alasdair Steven present the study of influenza virus-mediated membrane fusion, combining a variety of methods [10]. Cryo-tomography, conventional EM of thin sections, single particle cryo-EM, subtomogram averaging and X-ray crystallography are used to solve different aspects of the problem, and are merged to offer insight into native biological complexes in physiological environments. Recent advances in EM have generated new interfaces with other techniques such as light and X-ray microscopies, allowing us to extend structural analysis beyond the inherent sample size and thickness limits imposed by the use of electrons. Correlative methods are developing rapidly to offer an extended resolution approach, ranging from EM to light/fluorescence domains. Céline L. Fonta and Bruno Humbel present the advantages of this combination to study biological events at the submicrometer level by reviewing different protocols, instruments and software for improving correlative microscopy [11]. Also in this resolution domain, soft X-ray microscopy has recently been incorporated as a major new method. Carolyn Larabell et al. describe how cryo
2
Editorial / Archives of Biochemistry and Biophysics xxx (2015) xxx–xxx
X-ray tomography is able to offer three-dimensional visualization of whole cells in close-to-native state, permitting quantitative analysis by structural microscopy [12]. The combination of this technique with light fluorescence to locate specific molecules in the three-dimensional reconstructed cells is a major goal in the development of this approach. In this rapidly progressing resolution domain, advances in focused ion beam instruments are a potent alternative and a complement to other tomographic microscopies. Alexander Rigort and Jurgen Plitzko offer a comprehensive discussion of how cryo-preparation methods combined with ion milling and scanning electron microscopy give an alternative with which to address large and thick samples, such as tissues or cellular aggregates, by sequential imaging of milled block faces [13]. This milling approach can also be used to provide thin vitrified specimens with thickness appropriate for electron tomography studies. The combination and correlation of these new instrument and processing capabilities, together with the robotization and automation of sample preparation, data acquisition and processing steps, clearly offers a real possibility of extending the structural analysis of biomolecules from the atomic all the way to the cellular scale, which has long been an aspiration of biologists.
[4] J.M. Carazo, C.O. Sorzano, J. Oton, R. Marabini, J. Vargas, Three-dimensional reconstruction methods in single particle analysis from transmission electron microscopy data, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/ j.abb.2015.05.003. this issue. [5] X.P. Xu, N. Volkmann, Validation methods for low-resolution fitting of atomic structures to electron microscopy data, Arch. Biochem. Biophys. (2015), http:// dx.doi.org/10.1016/j.abb.2015.06.017. this issue. [6] E.H. Egelman, Three-dimensional reconstruction of helical polymers, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.04.004. this issue. [7] C. San Martı´n, Transmission electron microscopy and the molecular structure of icosahedral virases, Arch. Biochem. Biophys. (2015), http://dx.doi.org/ 10.1016/j.abb.2015.06.001. this issue. [8] H. Stahlberg, N. Biyani, A. Engel, 3D reconstruction of two-dimensional crystals, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb. 2015.06.006. this issue. [9] A. Dubrovsky, S. Sorrentino, J. Harapin, T. Sapra, O. Medalia, Developments in cryo-electrontomography for in situ structural análisis, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.04.006. this issue. [10] J. Fontana, A.C. Steven, Influenza virus-mediated membrane fusion: structural insights from electron microscopy (2015), http://dx.doi.org/10.1016/ j.abb.2015.04.011. this issue. [11] C.L. Fonta, B.M. Humbel, Correlative microscopy, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.05.017. this issue. [12] M. Do, S.A. Isaacson, G. McDermott, M.A. Le Gros, C.A. Larabell, Imaging and characterizing cells using tomography, Arch. Biochem. Biophys. (2015), http:// dx.doi.org/10.1016/j.abb.2015.01.011. this issue. [13] A. Rigort, J.M. Plitzko, Cryo-focused-ion-beam applications in structural biology, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb. 2015.02.009. this issue.
References [1] R.J. Harris, Transmission electron microscopy in molecular structural biology: a historical survey, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/ j.abb.2014.11.011. this issue. [2] R. Henderson, Overview and future of single particle electron cryomicroscopy, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.02.036. thisissue. [3] R.R. Schroeder, Advances in electron microscopy: a qualitative view of instrumentation development for macro molecuar imaging and tomography, Arch. Biochem. Biophys. (2015), http://dx.doi.org/10.1016/j.abb.2015.05.010. this issue.
José M. Valpuesta José L. Carrascosa Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049 Madrid, Spain E-mail address: [email protected] (J.L. Carrascosa) Available online xxxx
