The future is hybrid

Journal of Structural Biology 163 (2008) 186–195

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The future is hybrid Alasdair C. Steven a,*, Wolfgang Baumeister b a b

Editorial Office, Journal of Structural Biology, 525 B St., Ste. 1900, San Diego, CA 92101, USA Department of Structural Molecular Biology, Max Planck Institute for Biochemistry, 18a am Klopferspitz, 82152 Martinsried, Germany

a r t i c l e

i n f o

Article history: Received 2 June 2008 Accepted 2 June 2008 Available online 12 June 2008 Keywords: Hybrid methods Computational structural biology Structural systems biology Time-resolved imaging Variance analysis

a b s t r a c t On the occasion of the 50th anniversary of the Journal of Structural Biology, we review some of the major advances that have taken place in molecular and cellular structural biology over this timeframe and consider some current trends, as well as promising new directions. While the primary experimental techniques of X-ray diffraction, electron microscopy and NMR spectroscopy continue to improve and other powerful new techniques have come on-line, it appears that the most comprehensive analyses of large, dynamic, macromolecular machines will rely on integrated combinations of different methodologies, viz. ‘‘hybrid approaches”. The same prospect applies to the challenge of integrating observations of isolated macromolecules with data pertaining to their distributions and interaction networks in living cells. Looking ahead, computation in its diverse aspects may be expected to assume an increasingly important role in structural biology, as the prediction of molecular structures, the computation of dynamic properties, and quantitative time-resolved models of intracellular molecular populations (structural systems biology) move towards functional maturity. Ó 2008 Elsevier Inc. All rights reserved.

1. Roots and routes At this milestone, it is appropriate to review both the path followed by the Journal of Structural Biology through its first half-century as part of the scientific record and emerging directions in which the field—and the journal —are now heading. Its precursor, the Journal of Ultrastructure Research, was created largely at the initiative of Fritjöf Sjöstrand—in part, as Arvid Maunsbach’s memoir (this volume) relates—to provide a Eurocentric counterpoise to journals based in North America. Among other distinctions, JUR came to play an influential role during the formative years of cell biology. When Sjöstrand—and with him, the editorial office— moved from Stockholm to Los Angeles in 1959, the journal retained prominent representation from both sides of the Atlantic, on its editorial board as well as in its published papers. However, for JSB—as with JUR before it—such representation is by no means limited to the peri-Atlantic region; rather, we aim to provide an international scientific forum in the most inclusive sense of the term. It is noteworthy that the journal’s focus—as succinctly put in the Notice to Authors in the first issue of the JUR—has proved prescient and remains largely applicable today despite the transformations and diversifications—both instrumental and conceptual—that the field has undergone, viz. ‘‘. . . publishes papers dealing with the ultrastructural organization of biologic material as analyzed by means of electron microscopy, X-ray diffraction techniques, X-ray microscopy,

* Corresponding author. Fax: +1 619 699 6211. E-mail address: [email protected] (A.C. Steven). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.06.002

and polarization optical analysis. Papers dealing with techniques and instruments which are of importance for the development of the field will also be accepted. The field covered by the journal extends from the structure of molecules which are of biologic interest to the level of cell and tissue organization at the limit of the range of light microscopy.” While it may be argued that X-ray microscopy has yet to live up to this billing (but there is hope—Parkinson et al., 2008), the other techniques mentioned have developed remarkably over time and continue to play central roles in structural biology. They are now complemented by techniques that were not on the horizon in the 1950s, but have since become major players, including NMR spectroscopy, the probe microscopies (AFM/STM), ‘‘single molecule” methods, fluorescence techniques, and computational biology. The importance explicitly assigned to methods development in the 1957/8 manifesto is telling. While the primary goal of the journal is to determine structures and relate them to functions, we continue to recognize that paradigm-shifting observations tend to originate in methodological innovations. A second notable feature of JUR at its time of inception was its editorial board (Fig. 1). This group was small but distinguished. It included two eventual Nobel laureates (Claude, Ruska) and others of comparable stature. They were to make many seminal contributions, particularly to biological microscopy. 2. Relating structure to function Sjöstrand remained in charge of the journal’s affairs for two thirds of its first half-century until Ueli Aebi succeeded him as

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Fig. 1. The first editorial board of the Journal of Ultrastructure Research (1957/8).

Editor-in-chief in 1990. In his endeavors, Ueli was ably supported by Robert (Bob) Glaeser as Associate Editor. Both of them have remained closely associated with and supportive of JSB since we took over as Editors in 1996, and both have contributed articles to this Special Issue. About the time of the editorial transition, the journal underwent two name changes, reflecting the growing tendency to think of biological processes—even at the cellular level—in molecular terms. In 1986, it became the Journal of Ultrastructural and Molecular Structure Research and, in 1990, assumed its present name, the Journal of Structural Biology. In addition, an effort was made to place somewhat greater emphasis on quantitative as compared to descriptive approaches, to broaden the experimental basis, and to further develop the functional implications of

structural observations (already a strength of JUR). We have consciously continued these trends. In 1996, a high priority for JSB was to increase the frequency of issues from bimonthly to monthly, as expeditious publication— then as now—was a high priority in this competitive and fast-moving field and the now-ubiquitous practice of on-line pre-publication had yet to gell. One measure undertaken to facilitate this expansion was to launch a series of Special Issues on cutting-edge topics. These projects are entrusted to Guest Editors who conceive how their topic should be addressed, invite participants, handle manuscript review, and generally guide their projects to fruition. Overall, they have been outstandingly successful and Special Issues remain an important component of the JSB agenda today. A full list

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Table 1 Special Issues of the Journal of Structural Biology, 1998–2008 1996 (January) 1997 (March) 1997 (July) 1997 (December) 1998 (February) 1998 (May) 1998 (December) 1999 (April) 1999 (June) 1999 (September) 1999 (December) 2000(April) 2000(June) 2001 (February) 2001 (May) 2001 (August) 2002 (January) 2002 (April) 2002 (October) 2003 (April) 2003 (October) 2004 (January) 2004 (March) 2004 (July) 2004 (September) 2006 (July) 2006 (August) 2006 (October) 2006 (November) 2007 (January) 2007 (March) 2007 (May) 2007 (August) 2007 (December) 2008 (March) a

116 (1) 118 (2) 119 (2) 120 (3) 121 (2) 122 (1/2) 124 (2/3) 125 (2/3) 126 (3) 127 (2) 128 (1) 129 (2/3) 130 (2/3) 133 (2/3) 134 (2/3) 135 (2) 137(1/2) 138 (1/2) 140 (1–3) 142 (1) 144 (1/2) 145 (1/2) 146 (1/2) 147 (1) 147 (3) 155 (1)a 155 (2) 156 (1) 156 (2)a 157 (1) 157 (3) 158 (2) 159 (2) 160 (3) 161 (3)

Bridget Carragher and Ross Smith Wah Chiu Andreas Engel and Hermann Gaub Abraham Koster and David Agard Joerg Kistler and Andreas Engel David Parry and John Squire Martin Kessel and Alasdair Steven Ross Smith, Andreas Engel and Alasdair Steven Stephen Weiner and Stephen Mann James Hainfeld Eva Nogales and Wah Chiu Ivan Raska and David Spector Daniel Kirschner, David Teplow and Ana Damas Steven Ludtke and Wah Chiu Andrei Lupas, Robert Russell and Andreas Engel Jose Carrascosa and Jose Valpuesta David Parry and John Squire Bruce McEwen and Abraham Koster Ivan Raska and Ueli Aebi Alexander McPherson Bridget Carragher and Pawel Penczek Clinton Potter, Yuabxin Zhu and Bridget Carragher Michael Maurizi and Alasdair Steven Philippe Bastiaens and Stefan Hell Joachim Frank and Ilme Schlichting Gayle Woloschak David Parry and John Squire Andrei Lupas, Joerg Martin and Peter Zwickl Steven Zimmerman Bridget Carragher, Clinton Potter and Fred Sigworth Helmut Grübmuller and Klaus Schulten Dorit Hanein Andreas Engel and Fritz Winkler Ben Hankamer, Robert Glaeser and Henning Stahlberg Michael Marko, Mark Ellisman and Ohad Medalia

Advances in computational image processing for microscopy Biophysics of microtubules Imaging and manipulating biological structures with scanning probe microscopies Electron tomography Membrane channels Fibrous proteins Macromolecular complexes of the bacterial cell Modern visualization software Biomineralization: structural questions at all length scales Heavy metal cluster labeling Electron crystallography of biological macromolecules Functional organization of the cell nucleus Twist and sheet: variations on the theme of amyloid Single-particle analysis Structural bioinformatics Chaperonins: folding in the hole Coiled-coils, collagen and co-proteins Electron tomography Functional organization of the cell nucleus Macromolecular crystallization in the structural genomics era Analytical methods and software tools for macromolecular microscopy Automated particle selection for cryo-electron microscopy AAA+ proteins Recent advances in light microscopy Time-resolved imaging of macromolecular processes and interactions X-ray fluorescence imaging Fibrous protein structure AAA+ proteins Bacterial nucleoid Software tools for macromolecular microscopy Advances in molecular dynamics simulations Structural analysis of supramolecular assemblies by hybrid methods Structure, dynamics and function of proteins in biological membranes Electron crystallography of membrane proteins Electron tomography

Focus issues.

is given in Table 1. Although many have been notable, we should not fail to credit the first Special Issue—vol. 116 (1), edited by Bridget Carragher and Ross Smith. Appearing at a critical juncture in the development of computer-enhanced electron microscopy, this collection of papers gave a major stimulus to an important emerging field, as reflected in their impressive citation records. The trilogy of Special Issues on coiled-coils and fibrous proteins, edited by David Parry and John Squire in 1998, 2002, 2006, and anchored on a series of Conferences that they organize at regular intervals—always at the Böglerhof hotel in Alpbach, Austria—have also had great impact. Articles stemming from both of these projects are part of this anniversary Issue. 3. The troika of molecular structural biology The first high resolution structures of proteins were solved by X-ray crystallography in the 1950s. They represented remarkable accomplishments, given the technology available at the time. Since then, crystallographic studies have gone from strength to strength, boosted by cloning and expression technology, progressively more powerful computational methods, novel phasing approaches, synchrotron-based beam-lines, and the Protein Data Bank. However—and fortunately—major challenges remain: they include membrane proteins and large macromolecular machines; and the ticklish question of relating crystal structures (solidstate) to physiological structures (solution-state)—of which, more anon. NMR spectroscopy of macromolecules—a relative newcomer— has become a major player in structural biology. Not only is NMR capable of determining folds but it affords unique insights into

the dynamic properties of macromolecules in solution. NMR also remains a field in flux where one limitation to be overcome is the rather restrictive size limit -40 kDa, or so—for molecules to be amenable to complete analysis. Promising emerging directions include application of NMR to local regions of larger complexes either directly (Szymczyna et al., 2007) or by selective deuteration (Sprangers and Kay, 2007), and solid-state NMR of otherwise refractory specimens such as membrane proteins (De Angelis et al., 2006; Schneider et al., 2008) and amyloids (Petkova et al., 2006; Wasmer et al., 2008). The electron microscope has always played a central role in the affairs of JUR/JSB and its role in biological imaging has evolved beyond recognition (so to speak) over the past 50 years. The transmission instrument was invented in the 1930s (for an account of the early history of EM, see Ruska (1979) or, even better, Sjostrand (1988)), but the first macromolecular applications appeared only in the late 1950s and early 1960s with the introduction of negative staining and metal shadowing. Solutions to the long-standing impediments of radiation damage and dehydration were achieved with the introduction of low-dose imaging in the 1970s and vitrification in the 1980s (see the articles by Glaeser and by Glaeser and Taylor, this volume). Vitrification was demonstrated and key contributions made to adapting electron microscopes and specimenhandling techniques to the imaging of those least ideal of specimens—ice-embedded proteins—by researchers at the EMBL in Heidelberg during the 1980s (Dubochet et al., 1988). It soon became apparent that cryo-EM data not only need image processing but also offer an opportunity to realize the full potential of threedimensional reconstruction techniques, such as those previously developed at the MRC Laboratory in Cambridge (Crowther et al.,

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1970). Stephen Fuller, then at EMBL, and Tim Baker, then at Purdue University, were to the fore in this enterprise (Baker et al., 1988; Fuller, 1987). Building on this basis over the past 20 years, cryo-EM has now reached the point where we see three-dimensional reconstructions of free-standing (‘‘single”) particles to ever-improving resolutions, already at the 4–5 Å level (e.g., Ludtke et al., 2008) and beginning to allow chain tracing, some high resolution structures from electron crystallography of two-dimensional crystals (e.g., Nogales et al., 1998; Gonen et al., 2004), and applications of cryo-electron tomography (see Section 7 below) to macromolecular complexes, (e.g., Walz et al., 1997; Nitsch et al., 1998). Growth areas include ‘‘higher throughput” methods to streamline the acquisition and processing of the large data sets that high resolution analyses require (Steven and Belnap, 2008). 4. Pros and cons of image variability: molecular mobility and movements Another area of current interest involves systematic analysis of variability in image sets. At first sight, such variability appears to be an exasperating resolution-limiting phenomenon but, on a second look, provides a potential source of insight into multiple conformations and, through variance analysis (Fig. 2), to localizing and quantifying mobile elements (Ishikawa et al., 2004; Penczek et al., 2006). Discriminating between several discrete conformations simultaneously present in a molecular population (multiple particle analysis or multi-reference analysis) poses a stiff challenge to image classification techniques in which variability from this source must be distinguished from variability arising from viewing geometry, ‘shot’ noise, and imaging conditions (e.g., defocus, magnification). In favorable circumstances, when the conformational distinctions are large and the conformations resolved may be assigned to a time-ordered sequence, the resulting density maps may be used to compile time-resolved movies of dynamic processes, such as virus maturation (Heymann et al., 2004; Fig. 3). In this context, a basic and still open question concerns how conformations are captured in the freezing process: as the vitrification front sweeps through a specimen, are macromolecules instantaneously immobilized in one of the conformational states sampled in solution, or does the freezing process—while overall benign—result in a coarser sampling of these options? Other approaches to time-resolved imaging of the growth or movements of macromolecular complexes have also been successful and offer considerable potential for further development (see the 2004 Special Issue, edited by Joachim Frank and Ilme Schlichting). They include AFM (Fig. 4) and LM of polymers differentially labeled with fluorescent tags (DePace and Weissman, 2002).

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5. Hybrid approaches Powerful as single methodologies may be, they are enhanced when applied in judiciously integrated combinations. The prototypic ‘‘hybrid” approach unites X-ray crystallography and cryoEM (Rayment et al., 1993; Stewart et al., 1993; Wang et al., 1992). An account of developments in applying hybrid approaches to problems in structural virology is given in the article by Jack Johnson (this volume). When the object of interest is a complex for which a cryo-EM density map has been obtained at moderate resolution (say, 10– 25 Å) and high resolution structures for individual subunits are available, the resolution of the complex may be leveraged by fitting (‘‘docking”) the components into the density map. Under favorable circumstances, the residual uncertainty may be no more than 1– 2 Å RMSD—an order of magnitude more precise than the resolution of the density map (Baker and Johnson, 1996; Conway et al., 2003). While this may sound too good to be true, we may recall that the same principle—of using data that are of relatively low resolution but have a high signal-to-noise ratio to localize components in a defined frame of reference with surprisingly high precision—has appeared before in other contexts. For instance, the centers of mass of features resolved in negatively stained projections of twodimensional protein arrays could pinpointed to within 2–3 Å despite the image resolution being at the 25–30 Å level, and used to demonstrate global conformational changes (Steven et al., 1976). A second example is given by the recently introduced technique of PALM (photoactivated localization microscopy; Betzig et al., 2006), whereby the center-of-mass coordinates of features in fluorescence images are determined with an estimated precision of 25–250 Å—one to two orders of magnitude finer than the resolution of the primary images. In all these cases, there is no essential contradiction between the precision of the coordinates extracted and resolution of the images because one is not resolving per se: rather, one is localizing in light of a priori information or assumptions about the object. Returning to the hybridization of cryo-EM and X-ray crystallography, most studies to date have treated the fitted components as rigid bodies; however, as the resolution of the cryo-EM maps improves, discrepancies are beginning to be encountered that require adjustments in the components (‘‘flexible fitting”—Fig. 5); that is, structures captured in crystals, may not always represent conformations assumed in solution or in physiological complexes. More generally, numerous hybrid approaches involving a wider set of base techniques have been pioneered. They represent an area of research that JSB is strongly committed to fostering and have already been the subject of one Special Issue, in 2007 by Dorit Hanein, and a major part of another, in 1999 by Eva Nogales and Wah Chiu (Table 1).

Fig. 2. Variance analysis of a preparation of 70S E. coli ribosomes (reproduced from (Penczek et al., 2006), with permission). A cryo-EM density map is shown in A. In B, it is overlaid with a variance map calculated by a ‘‘bootstrap” method. The region of highest variance is shown in red. The major source of this variability was inferred to arise from partial occupancy (36%) of this site by the elongation factor, EF-G.

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Fig. 3. Time-resolved cryo-EM visualization of herpes simplex virus capsid maturation (adapted from Heymann et al., 2003). Like tailed bacteriophages and, presumably, other herpesviruses, the HSV capsid first assembles as procapsid that undergoes major conformational changes as it matures. Maturation is promoted by proteolytic disruption of the interaction between the internal scaffolding shell (not shown) and the capsid shell. The HSV capsid is a T=16 icosahedron with hexamers and pentamers of the major capsid protein, VP5 (blue), with ‘‘triplexes”—heterotrimers of VP19c and VP23 (green)—at the 3-fold sites. The triplexes play a morphogenic scaffolding role in procapsid assembly, then switch to a reinforcing clamp-like role in the mature capsid. Freshly isolated procapsids mature spontaneously in vitro over several days. Such a preparation was sampled for cryo-EM at four time-points, and the data analyzed to discriminate 17 morphologically distinct capsid species, which are arranged in a timeordered sequence (bottom panel). The outer surfaces of four species are compared in the top panel. Note the progressive regularization of the hexamer protrusions (yellow arrows). Capsid diameter = 1250 Å. Movies conveying various aspects of the maturation dynamics are archived at http://www.nature.com/nsmb/journal/v10/n5/suppinfo/ nsb922_S1.html.

6. The stuff of cells Compared to the macromolecules of which they are composed, cells represent a higher order of complexity, mandating different visualization methods. The common (but by no means universal) misconception of the cell as ‘‘just a bag of enzymes” has been replaced by recognition that cells contain specific albeit stochastically varying populations of molecules whose distributions are fundamental to defining cellular properties (Robinson et al., 2007). (Fundamental and still largely open questions are how these distributions are regulated and what bounds of spatial and demographic variability a given cell type may tolerate). Cellular structural biology has seen (sic) revolutionary advances in light microscopy whose highlights include confocal instruments and live cell imaging with green fluorescent protein and other tags. Other fluorescence-based methods such as FRET have growing impact. Although recent advances, discussed in a 2004 JSB Special Issue edited by Philippe Bastiaens and Stefan Hell, are capable of retrieving some information at resolutions beyond the diffraction limit of visible photons, it is still to electron microscopy that one must resort for visualizations that are close to the molecular level. With cells as with isolated molecules, preservation of native structure when specimens are prepared for electron microscopy has long been recognized as a major stumbling-block, with dehydration identified as the most destructive step when plastic block embeddings are prepared prior to sectioning. Many key contributions to the development of this methodology have appeared in JUR or JSB: they include the embedding protocol of Ryter and

Kellenberger (Ryter and Kellenberger, 1958) and the development of water-tolerant Lowicryl resins by Kellenberger and co-workers (Carlemalm et al., 1985). The Ryter–Kellenberger paper appeared in one of the first issues of JUR (in French, as was still a common practice) and has become a citation classic. A micrograph from this paper showing a thin section of an Escherichia coli cell infected with bacteriophage T2 is reproduced in Fig. 6. The DNA-filled heads of the progeny virions are recognizable as 1000 Å-long translucent ovals occupying much of the cytoplasm. For comparison, we also reproduce in Fig. 7 a recent cryo-EM reconstruction (Fokine et al., 2006) that depicts the intricate molecular anatomy of the closely related T4 capsid. One may conclude that, after an excellent start, there has been some progress in the past 50 years. Returning to cellular electron microscopy, freeze-substitution has been demonstrated to provide substantially improved preservation over traditional liquid-phase dehydration and is in widespread use. Recently, the final step—to total avoidance of dehydration, by cryo-sectioning vitrified samples—has been made and, although still technically demanding, is beginning to be applied to cellular and tissue samples (Al-Amoudi et al., 2004). A recent cryo-section of E. coli (Fig. 8) may be compared with the original plastic section of 1958 (Fig. 6). 7. Electron tomography—a generally applicable threedimensional imaging modality Thin sections are really not so thin—usually 80 nm or more— and their resolution in the third dimension can be no better than

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Fig. 4. Visualization by time-lapse AFM of growing amyloid filaments (reproduced from (Stolz et al., 2000), with permission). Single fibrils (protofibrils) of human amylin— two examples are marked by the white and black arrowheads in (a)—attached to a mica surface elongated during this portion of an assembly experiment. The two larger (white) structures are aggregates. Panels (d) and (e), respectively, quantitate the number of fibrils and the cumulative fibril length in 4 lm by 4 lm area as a function of time.

Fig. 5. Flexible fitting of elongation factor EF-G into cryo-EM density (adapted from Valle et al., 2003, with permission). Cryo-EM density is represented by the pink translucent surface and molecular models as ribbon diagrams. When the crystal structure of EF-G was fitted as a rigid body into the cryo-EM density (left panel), the left-most domains remained outside. When these domains were allowed to move flexibly with respect to the rest of the model, a much better fit was obtained (bottom). Figure courtesy of D. Belnap, using Chimera (Pettersen et al., 2004).

their thickness. Thus, co-projection of multiple molecules complicates the already thorny issue of interpreting electron micrographs of sections. Here, then, is a major opportunity for electron tomog-

raphy (ET). Although the concept dates back to the 1960s (Hart, 1968), it is only quite recently that technical advances in automated microscope control and data collection (Koster et al., 1997) have made ET into a practicable technique, particularly for radiation-sensitive vitrified specimens. Thus, although further technical progress is anticipated, ET is already having a major impact as a three-dimensional imaging tool. In addition to serving as an adjunct to cryo-EM that is uniquely well suited for analyzing inhomogenous populations of isolated particles (e.g., Grünewald et al., 2003), we may distinguish three areas of application in a cellular context: (i) tomography of plastic sections of freeze-substituted material. These specimens are relatively radiation-resistant and their contrast is enhanced by staining, although lingering doubts about the (non)preservation of native structure persist; (ii) cryo-tomography of whole cells, which have near-native preservation of structure but is currently limited to the smallest and thinnest of cell types (Medalia et al., 2002); and (iii) tomography of vitrified sections (Al-Amoudi et al., 2007; Gruska et al., 2008; Hsieh et al., 2002) which has an essentially unlimited range of applicability but remains, technically, very difficult. Electron tomography represents another emerging area that JSB is committed to fostering and has already been the subject of three Special Issues: in 1997, by Abraham Koster and David Agard; 2002, by Bruce McEwen and Abraham Koster; and 2008, by Michael Marko, Mark Ellisman, and Ohad Medalia—see Table 1. 8. Mapping molecular populations in cells Few macromolecules are individually large enough and distinctive enough in appearance to be recognizable in electron micrographs of sectioned cells. However, the important development of immuno-gold labeling techniques has enabled the detailed map-

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Fig. 6. Thin section electron micrograph of E. coli cells infected with bacteriophage T2 embedded in the polyester Vestopal. Reproduced from (Ryter and Kellenberger, 1958).

Fig. 7. Cryo-EM reconstruction of the outer surface of the prolate T4 capsid at 26– 28 Å resolution (from Fokine et al., 2006). Shown in blue is the hexameric major capsid protein, gene product 23 (gp23); in mauve, the pentameric minor capsid protein, gp24; in yellow, monomers of gp.hoc (hoc, highly antigenic outer capsid protein); and in white, trimers of gp.soc (soc, small outer capsid protein); in green, the azimuthally averaged upper portion of the tail. Apart from the portal vertex where the tail attaches, the capsid architecture conforms to icosahedral symmetry but elongated along a 5-fold axis, with a triangulation number T of 13 laevo and a Qnumber of 20. In the T4 capsid, the mechanism of quasi-equivalence is based on gene duplication, with different but related proteins occupying the vertices (gp24) and the remainder of the surface lattice (gp23). Gp23 and gp24 are derived from their precursors by post-assembly proteolysis, which facilitates a major conformational change that allows gp.hoc and gp.soc to bind. Recently, a crystal structure has been determined for gp24 (Fokine et al., 2005): it shows that the external protrusions represent an insertion domain included in gp24 and gp23 relative to the prototypic ‘‘tailed phage” capsid protein—that of HK97 (Wikoff et al., 2000). Gp23 is likely to have a very similar fold to gp24, despite modest sequence identity (21%), because a single site point mutation in gene 23 produces a protein (byp24) that can form the pentamers as well as the hexamers (Fokine et al., 2006). Nevertheless, they are sufficiently different that gp.hoc and gp.soc, bind only to gp23 and not to gp24 (Iwasaki et al., 2000). Gp.soc overlies the 3-fold sites between capsomers and serves as a molecular clamp that enables the capsid to resist extremes of alkaline pH or temperature. Gp.hoc has no known function. Bar = 100 Å.

Fig. 8. Cryo-electron micrograph of a vitrified section of E. coli strain K12 (reproduced from (Matias et al., 2003), with permission). The cell envelope is preserved without buckling and the outer membrane (OM), peptidoglycan sacculus (PG) and inner membrane (IM) are resolved. The envelope varies in apparent thickness around the cell as a consequence of cutting-induced compression in the direction bottom left to top right. Bar = 0.2 lm.

ping of populations of antigenically defined macromolecules in situ. Here, a key consideration has been to preserve the specificity of the antigen-antibody reaction despite the rigors of tissue embedding, and the cryogenic techniques pioneered by Tokoyasu are now widely used (Lucic et al. in press).

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The overall distributions of molecules in living cells may be mapped by light microscopy of sections treated with fluorescently labeled antibodies or in live cells by expressing fusions of the molecule of interest with GFP. In the latter case, it is assumed that the chimeric proteins distribute in the same way as the native molecules. Following the success of GFP-related applications in LM, it is clearly an attractive proposition to develop ‘‘clonable labels” for use in EM. Although appended domains and even peptide motifs may be detected in ‘‘difference” SPA imaging of isolated complexes, in a cellular context, they must be rendered visible by treating with high-affinity colloidal metal particles (usually gold) or acting as nucleation sites for metal salts (Mercogliano and DeRosier, 2006). In general, it will be difficult to employ such treatments for in vivo labeling without poisoning or otherwise perturbing the cells during uptake. However, they are more readily applicable to sectioned material, which provides an additional incentive for persevering with cryo-sections. 9. Correlative microscopy: another hybrid approach Although the mapping techniques outlined above are in widespread and productive use, they are subject to the limitation that only one or a few different proteins may be simultaneously

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mapped, as distinguished in LM by fluorescence at different wavelengths and in EM experiments by gold labels of different sizes. Thus the majority of other macromolecules in the same vicinity, whose presence may affect the distribution and activity of those observed, remain undetected and unidentified. Some alternative approaches to identifying the molecular inhabitants of particular intracellular neighborhoods are discussed in Section 10 below. A second limitation is that the LM and EM observations tend to be correlated only indirectly on the basis of statistical trends, e.g., association with or proximity to a particular cellular compartment. In correlative microscopy, LM is systematically combined with EM so as to achieve imaging of the self-same features with both techniques (e.g., Fig. 9). Although it is, in general, difficult to home in on specific areas of interest (designated as such by LM surveillance) after the specimen has been subjected to conventional preparation for EM, this has been accomplished in a number of studies (e.g., Nakata et al., 1998). More recent approaches have correlated fluorescent labeling LM with immuno-gold EM (Mironov et al., 2001) or introduced genetically engineered proteins that fluoresce for LM visualization and are subsequently treated to nucleate electron-dense deposits for EM detection (Gaietta et al., 2002). A further line of development is based on observing vitrified, specifically fluorescent, specimens on a cryogenic light microscope and

Fig. 9. Correlative microscopy. Illustrative example of an LM/EM correlation where light/fluorescence microscopy at progressively higher magnifications feed into a cryoelectron tomographic visualization. In each case, the white arrow points to the area blown up in the following panel. (A) Phase-contrast image and (B) fluorescence image of neurons (neurons were labeled with FM1-43). The arrow points to the spot with a high FM1-43 fluorescence. (C) Cryo-EM image of neuronal processes surrounding the area where the tomogram was taken (arrow). (D) Tomographic slice showing two neuronal processes, an extracellular vesicle connected to one of the processes and to a protrusion from the other one. An endocytotic invagination on the protrusion is visible. (E) Surface rendering showing the extracellular vesicle (blue), two neighboring neuronal processes (gray), connections to them (yellow and orange) and some vesicle-bound molecular complexes (external—green, and internal—red). The vesicle is shown with one side open in order to expose the complexes in its interior. (Figure courtesy of J. Plitzko, based on data published by Lucic et al., 2007).

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then transferring them into an electron microscope for cryo-tomographic analysis (Sartori et al., 2007; Schwartz et al., 2007). Software-based methods can assist with the still incompletely solved problem of locating specific areas of interest for EM or ET analysis (Lucic et al., 2007). 10. Marrying the power of visualization with the power of identification A cryo-electron tomogram of a cell affords a three-dimensional image of its entire proteome; if interpretable as such, it provides a snapshot of the network of interactions underlying the gamut of cellular functions. However, the adverse signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm renders such interpretation a challenging task. The most direct approach to interpreting cellular cryo-tomograms in molecular terms employs pattern recognition methods that rely on two sources of a priori information: a library of three-dimensional templates for all of its particulate components; and knowledge of the cell’s molecular inventory (Baumeister, 2005). The library may be used to sift through the tomographic volume to map the population of each recognizable component by cross-correlation techniques, in an approach akin to the fitting of subunit crystal structural in cryo-EM density maps (Bohm et al., 2000; see Section 5). While the level of success in such an experiment will, in general, depend on both the completeness of the reference library and the resolution and signal-to-noise ratio of the tomogram, promising results have already been obtained with in situ mappings of bacterial ribosomes (Ortiz et al., 2006; Seybert et al., 2006). However, interpretation is a more feasible proposition if inventory data are available, and such data may be acquired in a comprehensive manner by mass spectrometry. In the traditional ‘‘slice and dice” approach, cellular cultures and tissue samples are homogenized prior to mass spectrometry, thus destroying valuable information about the compositions of individual cells. This loss of information may be avoided if it is possible to develop effective procedures for operating at the level of individual cells, or even subcellular volumes. In this context, Laser Capture Microdissection (LCM) has considerable potential for isolating distinct cell types from heterogeneous populations, as have dual beam SEM/FIB systems, to excise defined subvolumes under visual control (Burgemeister, 2005). Ideally, pipelines will be assembled in which complex samples are ‘navigated’ by (cryo)-fluorescence microscopy, targets identified, and then isolated via micromanipulation and micromachining methods for analysis by both mass spectrometry and electron microscopy/tomography. Needless to say, this envisages a highly hybrid approach. 11. All roads lead to Rome A priori, it is clear that a complete molecular atlas of a single cell—which, of itself, would represent a prodigious technical accomplishment—would yield only limited functional insight. For this information to be fully exploited, such an atlas would have to be compiled for a great many (how many?) similar cells and the trends of co-association and compartmentation deduced on statistical grounds. This proposition represents just one of many areas where computational analysis moves into center-stage not only for data acquisition and processing but also in integration and analysis. Indeed, the computer is also the only medium in which direct structural data may be cohesively integrated with other sources of information from diverse biochemical and biophysical approaches to formulate three- or four-dimensional models of a system of interest. A good example of integrative analysis of this kind is given in recent work on the nuclear pore complex

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