Journal of Structural Biology 163 (2008) 186–195

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Journal of Structural Biology

journal homepage: www.elsevier .com/locate /y jsbi

The future is hybrid

Alasdair C. Steven a,*, Wolfgang Baumeister b

a Editorial Office, Journal of Structural Biology, 525 B St., Ste. 1900, San Diego, CA 92101, USAb 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 2008Accepted 2 June 2008Available online 12 June 2008

Keywords:Hybrid methodsComputational structural biologyStructural systems biologyTime-resolved imagingVariance analysis

1047-8477/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.jsb.2008.06.002

* Corresponding author. Fax: +1 619 699 6211.E-mail address: jsb@elsevier.com (A.C. Steven).

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 majoradvances that have taken place in molecular and cellular structural biology over this timeframe and con-sider some current trends, as well as promising new directions. While the primary experimental tech-niques of X-ray diffraction, electron microscopy and NMR spectroscopy continue to improve and otherpowerful 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 isolatedmacromolecules with data pertaining to their distributions and interaction networks in living cells. Look-ing ahead, computation in its diverse aspects may be expected to assume an increasingly important rolein structural biology, as the prediction of molecular structures, the computation of dynamic properties,and quantitative time-resolved models of intracellular molecular populations (structural systems biol-ogy) move towards functional maturity.

� 2008 Elsevier Inc. All rights reserved.

1. Roots and routes

At this milestone, it is appropriate to review both the path fol-lowed by the Journal of Structural Biology through its first half-cen-tury as part of the scientific record and emerging directions inwhich the field—and the journal —are now heading. Its precursor,the Journal of Ultrastructure Research, was created largely at the ini-tiative of Fritjöf Sjöstrand—in part, as Arvid Maunsbach’s memoir(this volume) relates—to provide a Eurocentric counterpoise tojournals based in North America. Among other distinctions, JURcame to play an influential role during the formative years of cellbiology. When Sjöstrand—and with him, the editorial office—moved from Stockholm to Los Angeles in 1959, the journal retainedprominent representation from both sides of the Atlantic, on itseditorial board as well as in its published papers. However, forJSB—as with JUR before it—such representation is by no means lim-ited to the peri-Atlantic region; rather, we aim to provide an inter-national scientific forum in the most inclusive sense of the term.

It is noteworthy that the journal’s focus—as succinctly put in theNotice to Authors in the first issue of the JUR—has proved prescientand remains largely applicable today despite the transformationsand diversifications—both instrumental and conceptual—that thefield has undergone, viz. ‘‘. . . publishes papers dealing with the ultra-structural organization of biologic material as analyzed by means ofelectron microscopy, X-ray diffraction techniques, X-ray microscopy,

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and polarization optical analysis. Papers dealing with techniques andinstruments which are of importance for the development of the fieldwill also be accepted. The field covered by the journal extends from thestructure of molecules which are of biologic interest to the level of celland 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 tothis billing (but there is hope—Parkinson et al., 2008), the othertechniques mentioned have developed remarkably over time andcontinue to play central roles in structural biology. They are nowcomplemented by techniques that were not on the horizon in the1950s, but have since become major players, including NMR spec-troscopy, the probe microscopies (AFM/STM), ‘‘single molecule”methods, fluorescence techniques, and computational biology.The importance explicitly assigned to methods development inthe 1957/8 manifesto is telling. While the primary goal of the jour-nal is to determine structures and relate them to functions, wecontinue to recognize that paradigm-shifting observations tendto originate in methodological innovations.

A second notable feature of JUR at its time of inception was itseditorial board (Fig. 1). This group was small but distinguished. Itincluded two eventual Nobel laureates (Claude, Ruska) and othersof comparable stature. They were to make many seminal contribu-tions, particularly to biological microscopy.

2. Relating structure to function

Sjöstrand remained in charge of the journal’s affairs for twothirds of its first half-century until Ueli Aebi succeeded him as

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

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Editor-in-chief in 1990. In his endeavors, Ueli was ably supportedby Robert (Bob) Glaeser as Associate Editor. Both of them have re-mained closely associated with and supportive of JSB since we tookover as Editors in 1996, and both have contributed articles to thisSpecial Issue. About the time of the editorial transition, the journalunderwent two name changes, reflecting the growing tendency tothink of biological processes—even at the cellular level—in molec-ular terms. In 1986, it became the Journal of Ultrastructural andMolecular Structure Research and, in 1990, assumed its presentname, the Journal of Structural Biology. In addition, an effort wasmade to place somewhat greater emphasis on quantitative as com-pared to descriptive approaches, to broaden the experimental ba-sis, and to further develop the functional implications of

structural observations (already a strength of JUR). We have con-sciously continued these trends.

In 1996, a high priority for JSB was to increase the frequency ofissues from bimonthly to monthly, as expeditious publication—then as now—was a high priority in this competitive and fast-mov-ing field and the now-ubiquitous practice of on-line pre-publica-tion had yet to gell. One measure undertaken to facilitate thisexpansion was to launch a series of Special Issues on cutting-edgetopics. These projects are entrusted to Guest Editors who conceivehow their topic should be addressed, invite participants, handlemanuscript review, and generally guide their projects to fruition.Overall, they have been outstandingly successful and Special Issuesremain an important component of the JSB agenda today. A full list

Table 1Special Issues of the Journal of Structural Biology, 1998–2008

1996 (January) 116 (1) Bridget Carragher and Ross Smith Advances in computational image processing for microscopy1997 (March) 118 (2) Wah Chiu Biophysics of microtubules1997 (July) 119 (2) Andreas Engel and Hermann Gaub Imaging and manipulating biological structures with scanning probe microscopies1997 (December) 120 (3) Abraham Koster and David Agard Electron tomography1998 (February) 121 (2) Joerg Kistler and Andreas Engel Membrane channels1998 (May) 122 (1/2) David Parry and John Squire Fibrous proteins1998 (December) 124 (2/3) Martin Kessel and Alasdair Steven Macromolecular complexes of the bacterial cell1999 (April) 125 (2/3) Ross Smith, Andreas Engel and Alasdair Steven Modern visualization software1999 (June) 126 (3) Stephen Weiner and Stephen Mann Biomineralization: structural questions at all length scales1999 (September) 127 (2) James Hainfeld Heavy metal cluster labeling1999 (December) 128 (1) Eva Nogales and Wah Chiu Electron crystallography of biological macromolecules2000(April) 129 (2/3) Ivan Raska and David Spector Functional organization of the cell nucleus2000(June) 130 (2/3) Daniel Kirschner, David Teplow and Ana Damas Twist and sheet: variations on the theme of amyloid2001 (February) 133 (2/3) Steven Ludtke and Wah Chiu Single-particle analysis2001 (May) 134 (2/3) Andrei Lupas, Robert Russell and Andreas Engel Structural bioinformatics2001 (August) 135 (2) Jose Carrascosa and Jose Valpuesta Chaperonins: folding in the hole2002 (January) 137(1/2) David Parry and John Squire Coiled-coils, collagen and co-proteins2002 (April) 138 (1/2) Bruce McEwen and Abraham Koster Electron tomography2002 (October) 140 (1–3) Ivan Raska and Ueli Aebi Functional organization of the cell nucleus2003 (April) 142 (1) Alexander McPherson Macromolecular crystallization in the structural genomics era2003 (October) 144 (1/2) Bridget Carragher and Pawel Penczek Analytical methods and software tools for macromolecular microscopy2004 (January) 145 (1/2) Clinton Potter, Yuabxin Zhu and Bridget Carragher Automated particle selection for cryo-electron microscopy2004 (March) 146 (1/2) Michael Maurizi and Alasdair Steven AAA+ proteins2004 (July) 147 (1) Philippe Bastiaens and Stefan Hell Recent advances in light microscopy2004 (September) 147 (3) Joachim Frank and Ilme Schlichting Time-resolved imaging of macromolecular processes and interactions2006 (July) 155 (1)a Gayle Woloschak X-ray fluorescence imaging2006 (August) 155 (2) David Parry and John Squire Fibrous protein structure2006 (October) 156 (1) Andrei Lupas, Joerg Martin and Peter Zwickl AAA+ proteins2006 (November) 156 (2)a Steven Zimmerman Bacterial nucleoid2007 (January) 157 (1) Bridget Carragher, Clinton Potter and Fred Sigworth Software tools for macromolecular microscopy2007 (March) 157 (3) Helmut Grübmuller and Klaus Schulten Advances in molecular dynamics simulations2007 (May) 158 (2) Dorit Hanein Structural analysis of supramolecular assemblies by hybrid methods2007 (August) 159 (2) Andreas Engel and Fritz Winkler Structure, dynamics and function of proteins in biological membranes2007 (December) 160 (3) Ben Hankamer, Robert Glaeser and Henning Stahlberg Electron crystallography of membrane proteins2008 (March) 161 (3) Michael Marko, Mark Ellisman and Ohad Medalia Electron tomography

a Focus issues.

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is given in Table 1. Although many have been notable, we shouldnot fail to credit the first Special Issue—vol. 116 (1), edited byBridget Carragher and Ross Smith. Appearing at a critical juncturein the development of computer-enhanced electron microscopy,this collection of papers gave a major stimulus to an importantemerging 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 an-chored on a series of Conferences that they organize at regularintervals—always at the Böglerhof hotel in Alpbach, Austria—havealso had great impact. Articles stemming from both of these pro-jects are part of this anniversary Issue.

3. The troika of molecular structural biology

The first high resolution structures of proteins were solved byX-ray crystallography in the 1950s. They represented remarkableaccomplishments, given the technology available at the time.Since then, crystallographic studies have gone from strength tostrength, boosted by cloning and expression technology, progres-sively more powerful computational methods, novel phasing ap-proaches, synchrotron-based beam-lines, and the Protein DataBank. However—and fortunately—major challenges remain: theyinclude membrane proteins and large macromolecular machines;and the ticklish question of relating crystal structures (solid-state) to physiological structures (solution-state)—of which, moreanon.

NMR spectroscopy of macromolecules—a relative newcomer—has become a major player in structural biology. Not only is NMRcapable of determining folds but it affords unique insights into

the dynamic properties of macromolecules in solution. NMR alsoremains a field in flux where one limitation to be overcome isthe rather restrictive size limit -�40 kDa, or so—for molecules tobe amenable to complete analysis. Promising emerging directionsinclude application of NMR to local regions of larger complexeseither directly (Szymczyna et al., 2007) or by selective deuteration(Sprangers and Kay, 2007), and solid-state NMR of otherwiserefractory specimens such as membrane proteins (De Angeliset 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 theaffairs of JUR/JSB and its role in biological imaging has evolved be-yond recognition (so to speak) over the past 50 years. The trans-mission instrument was invented in the 1930s (for an account ofthe early history of EM, see Ruska (1979) or, even better, Sjostrand(1988)), but the first macromolecular applications appeared only inthe late 1950s and early 1960s with the introduction of negativestaining and metal shadowing. Solutions to the long-standingimpediments of radiation damage and dehydration were achievedwith the introduction of low-dose imaging in the 1970s and vitri-fication in the 1980s (see the articles by Glaeser and by Glaeser andTaylor, this volume). Vitrification was demonstrated and key con-tributions made to adapting electron microscopes and specimen-handling techniques to the imaging of those least ideal of speci-mens—ice-embedded proteins—by researchers at the EMBL in Hei-delberg during the 1980s (Dubochet et al., 1988). It soon becameapparent that cryo-EM data not only need image processing butalso offer an opportunity to realize the full potential of three-dimensional reconstruction techniques, such as those previouslydeveloped at the MRC Laboratory in Cambridge (Crowther et al.,

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1970). Stephen Fuller, then at EMBL, and Tim Baker, then at PurdueUniversity, 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 nowreached the point where we see three-dimensional reconstructionsof free-standing (‘‘single”) particles to ever-improving resolutions,already at the 4–5 Å level (e.g., Ludtke et al., 2008) and beginningto allow chain tracing, some high resolution structures from elec-tron crystallography of two-dimensional crystals (e.g., Nogaleset al., 1998; Gonen et al., 2004), and applications of cryo-electrontomography (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 andprocessing of the large data sets that high resolution analyses re-quire (Steven and Belnap, 2008).

4. Pros and cons of image variability: molecular mobility andmovements

Another area of current interest involves systematic analysisof variability in image sets. At first sight, such variability appearsto be an exasperating resolution-limiting phenomenon but, on asecond look, provides a potential source of insight into multipleconformations and, through variance analysis (Fig. 2), to localiz-ing and quantifying mobile elements (Ishikawa et al., 2004;Penczek et al., 2006). Discriminating between several discreteconformations simultaneously present in a molecular population(multiple particle analysis or multi-reference analysis) poses astiff challenge to image classification techniques in which vari-ability from this source must be distinguished from variabilityarising from viewing geometry, ‘shot’ noise, and imaging condi-tions (e.g., defocus, magnification). In favorable circumstances,when the conformational distinctions are large and the confor-mations resolved may be assigned to a time-ordered sequence,the resulting density maps may be used to compile time-resolvedmovies of dynamic processes, such as virus maturation (Hey-mann et al., 2004; Fig. 3). In this context, a basic and still openquestion concerns how conformations are captured in the freez-ing process: as the vitrification front sweeps through a specimen,are macromolecules instantaneously immobilized in one of theconformational states sampled in solution, or does the freezingprocess—while overall benign—result in a coarser sampling ofthese options?

Other approaches to time-resolved imaging of the growth ormovements of macromolecular complexes have also been success-ful and offer considerable potential for further development (seethe 2004 Special Issue, edited by Joachim Frank and Ilme Schlich-ting). They include AFM (Fig. 4) and LM of polymers differentiallylabeled with fluorescent tags (DePace and Weissman, 2002).

Fig. 2. Variance analysis of a preparation of 70S E. coli ribosomes (reproduced from (Penoverlaid with a variance map calculated by a ‘‘bootstrap” method. The region of highest vapartial occupancy (�36%) of this site by the elongation factor, EF-G.

5. Hybrid approaches

Powerful as single methodologies may be, they are enhancedwhen applied in judiciously integrated combinations. The proto-typic ‘‘hybrid” approach unites X-ray crystallography and cryo-EM (Rayment et al., 1993; Stewart et al., 1993; Wang et al.,1992). An account of developments in applying hybrid approachesto problems in structural virology is given in the article by JackJohnson (this volume).

When the object of interest is a complex for which a cryo-EMdensity map has been obtained at moderate resolution (say, 10–25 Å) and high resolution structures for individual subunits areavailable, the resolution of the complex may be leveraged by fitting(‘‘docking”) the components into the density map. Under favorablecircumstances, the residual uncertainty may be no more than 1–2 Å RMSD—an order of magnitude more precise than the resolutionof the density map (Baker and Johnson, 1996; Conway et al., 2003).While this may sound too good to be true, we may recall that thesame principle—of using data that are of relatively low resolutionbut have a high signal-to-noise ratio to localize components in adefined frame of reference with surprisingly high precision—hasappeared before in other contexts. For instance, the centers of massof features resolved in negatively stained projections of two-dimensional protein arrays could pinpointed to within 2–3 Å de-spite the image resolution being at the 25–30 Å level, and usedto demonstrate global conformational changes (Steven et al.,1976). A second example is given by the recently introduced tech-nique of PALM (photoactivated localization microscopy; Betziget al., 2006), whereby the center-of-mass coordinates of featuresin fluorescence images are determined with an estimated precisionof 25–250 Å—one to two orders of magnitude finer than the reso-lution of the primary images. In all these cases, there is no essentialcontradiction between the precision of the coordinates extractedand resolution of the images because one is not resolving per se:rather, one is localizing in light of a priori information or assump-tions about the object.

Returning to the hybridization of cryo-EM and X-ray crystallog-raphy, most studies to date have treated the fitted components asrigid bodies; however, as the resolution of the cryo-EM maps im-proves, discrepancies are beginning to be encountered that requireadjustments in the components (‘‘flexible fitting”—Fig. 5); that is,structures captured in crystals, may not always represent confor-mations assumed in solution or in physiological complexes.

More generally, numerous hybrid approaches involving a widerset of base techniques have been pioneered. They represent an areaof research that JSB is strongly committed to fostering and havealready been the subject of one Special Issue, in 2007 by DoritHanein, and a major part of another, in 1999 by Eva Nogales andWah Chiu (Table 1).

czek et al., 2006), with permission). A cryo-EM density map is shown in A. In B, it isriance is shown in red. The major source of this variability was inferred to arise from

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 proteolyticdisruption 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 ofthe 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 inprocapsid assembly, then switch to a reinforcing clamp-like role in the mature capsid. Freshly isolated procapsids mature spontaneously in vitro over several days. Such apreparation 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 time-ordered sequence (bottom panel). The outer surfaces of four species are compared in the top panel. Note the progressive regularization of the hexamer protrusions (yellowarrows). 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.

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6. The stuff of cells

Compared to the macromolecules of which they are composed,cells represent a higher order of complexity, mandating differentvisualization methods. The common (but by no means universal)misconception of the cell as ‘‘just a bag of enzymes” has been re-placed by recognition that cells contain specific albeit stochasti-cally varying populations of molecules whose distributions arefundamental to defining cellular properties (Robinson et al.,2007). (Fundamental and still largely open questions are how thesedistributions are regulated and what bounds of spatial and demo-graphic variability a given cell type may tolerate). Cellular struc-tural biology has seen (sic) revolutionary advances in lightmicroscopy whose highlights include confocal instruments andlive cell imaging with green fluorescent protein and other tags.Other fluorescence-based methods such as FRET have growing im-pact. Although recent advances, discussed in a 2004 JSB Special Is-sue edited by Philippe Bastiaens and Stefan Hell, are capable ofretrieving some information at resolutions beyond the diffractionlimit of visible photons, it is still to electron microscopy that onemust resort for visualizations that are close to the molecular level.

With cells as with isolated molecules, preservation of nativestructure when specimens are prepared for electron microscopyhas long been recognized as a major stumbling-block, with dehy-dration identified as the most destructive step when plastic blockembeddings are prepared prior to sectioning. Many key contribu-tions to the development of this methodology have appeared inJUR or JSB: they include the embedding protocol of Ryter and

Kellenberger (Ryter and Kellenberger, 1958) and the developmentof water-tolerant Lowicryl resins by Kellenberger and co-workers(Carlemalm et al., 1985). The Ryter–Kellenberger paper appearedin one of the first issues of JUR (in French, as was still a commonpractice) and has become a citation classic. A micrograph from thispaper showing a thin section of an Escherichia coli cell infected withbacteriophage T2 is reproduced in Fig. 6. The DNA-filled heads ofthe progeny virions are recognizable as 1000 Å-long translucentovals occupying much of the cytoplasm. For comparison, we alsoreproduce in Fig. 7 a recent cryo-EM reconstruction (Fokine et al.,2006) that depicts the intricate molecular anatomy of the closelyrelated 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-substitutionhas been demonstrated to provide substantially improved preser-vation over traditional liquid-phase dehydration and is in wide-spread use. Recently, the final step—to total avoidance ofdehydration, by cryo-sectioning vitrified samples—has been madeand, although still technically demanding, is beginning to be ap-plied to cellular and tissue samples (Al-Amoudi et al., 2004). A re-cent cryo-section of E. coli (Fig. 8) may be compared with theoriginal plastic section of 1958 (Fig. 6).

7. Electron tomography—a generally applicable three-dimensional 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

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 fromValle et al., 2003, with permission). Cryo-EM density is represented by the pinktranslucent surface and molecular models as ribbon diagrams. When the crystalstructure of EF-G was fitted as a rigid body into the cryo-EM density (left panel), theleft-most domains remained outside. When these domains were allowed to moveflexibly 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).

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their thickness. Thus, co-projection of multiple molecules compli-cates the already thorny issue of interpreting electron micrographsof 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 auto-mated microscope control and data collection (Koster et al.,1997) have made ET into a practicable technique, particularly forradiation-sensitive vitrified specimens. Thus, although furthertechnical progress is anticipated, ET is already having a major im-pact as a three-dimensional imaging tool. In addition to serving asan adjunct to cryo-EM that is uniquely well suited for analyzinginhomogenous populations of isolated particles (e.g., Grünewaldet al., 2003), we may distinguish three areas of application in a cel-lular context: (i) tomography of plastic sections of freeze-substi-tuted material. These specimens are relatively radiation-resistantand their contrast is enhanced by staining, although lingeringdoubts about the (non)preservation of native structure persist;(ii) cryo-tomography of whole cells, which have near-native pres-ervation of structure but is currently limited to the smallest andthinnest of cell types (Medalia et al., 2002); and (iii) tomographyof vitrified sections (Al-Amoudi et al., 2007; Gruska et al., 2008;Hsieh et al., 2002) which has an essentially unlimited range ofapplicability but remains, technically, very difficult.

Electron tomography represents another emerging area that JSBis committed to fostering and has already been the subject of threeSpecial Issues: in 1997, by Abraham Koster and David Agard; 2002,by Bruce McEwen and Abraham Koster; and 2008, by Michael Mar-ko, Mark Ellisman, and Ohad Medalia—see Table 1.

8. Mapping molecular populations in cells

Few macromolecules are individually large enough and distinc-tive enough in appearance to be recognizable in electron micro-graphs of sectioned cells. However, the important developmentof immuno-gold labeling techniques has enabled the detailed map-

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 majorcapsid protein, gene product 23 (gp23�); in mauve, the pentameric minor capsidprotein, gp24�; in yellow, monomers of gp.hoc (hoc, highly antigenic outer capsidprotein); 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 vertexwhere the tail attaches, the capsid architecture conforms to icosahedral symmetrybut elongated along a 5-fold axis, with a triangulation number T of 13 laevo and a Q-number of 20. In the T4 capsid, the mechanism of quasi-equivalence is based ongene duplication, with different but related proteins occupying the vertices (gp24�)and the remainder of the surface lattice (gp23�). Gp23� and gp24� are derived fromtheir precursors by post-assembly proteolysis, which facilitates a major conforma-tional change that allows gp.hoc and gp.soc to bind. Recently, a crystal structure hasbeen determined for gp24 (Fokine et al., 2005): it shows that the externalprotrusions represent an insertion domain included in gp24� and gp23� relative tothe 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 sequenceidentity (�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 togp23� and not to gp24� (Iwasaki et al., 2000). Gp.soc overlies the 3-fold sitesbetween capsomers and serves as a molecular clamp that enables the capsid toresist 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 ispreserved without buckling and the outer membrane (OM), peptidoglycan sacculus(PG) and inner membrane (IM) are resolved. The envelope varies in apparentthickness around the cell as a consequence of cutting-induced compression in thedirection bottom left to top right. Bar = 0.2 lm.

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ping of populations of antigenically defined macromoleculesin situ. Here, a key consideration has been to preserve the specific-ity of the antigen-antibody reaction despite the rigors of tissueembedding, and the cryogenic techniques pioneered by Tokoyasuare now widely used (Lucic et al. in press).

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The overall distributions of molecules in living cells may bemapped by light microscopy of sections treated with fluorescentlylabeled antibodies or in live cells by expressing fusions of the mole-cule of interest with GFP. In the latter case, it is assumed that the chi-meric proteins distribute in the same way as the native molecules.

Following the success of GFP-related applications in LM, it isclearly an attractive proposition to develop ‘‘clonable labels” foruse in EM. Although appended domains and even peptide motifsmay be detected in ‘‘difference” SPA imaging of isolated complexes,in a cellular context, they must be rendered visible by treating withhigh-affinity colloidal metal particles (usually gold) or acting asnucleation sites for metal salts (Mercogliano and DeRosier, 2006).In general, it will be difficult to employ such treatments forin vivo labeling without poisoning or otherwise perturbing the cellsduring uptake. However, they are more readily applicable to sec-tioned material, which provides an additional incentive for perse-vering with cryo-sections.

9. Correlative microscopy: another hybrid approach

Although the mapping techniques outlined above are in wide-spread and productive use, they are subject to the limitation thatonly one or a few different proteins may be simultaneously

Fig. 9. Correlative microscopy. Illustrative example of an LM/EM correlation where lighelectron tomographic visualization. In each case, the white arrow points to the area blowneurons (neurons were labeled with FM1-43). The arrow points to the spot with a high Fwhere the tomogram was taken (arrow). (D) Tomographic slice showing two neuronal profrom the other one. An endocytotic invagination on the protrusion is visible. (E) Surfprocesses (gray), connections to them (yellow and orange) and some vesicle-bound moleside open in order to expose the complexes in its interior. (Figure courtesy of J. Plitzko,

mapped, as distinguished in LM by fluorescence at different wave-lengths 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 thoseobserved, remain undetected and unidentified. Some alternativeapproaches to identifying the molecular inhabitants of particularintracellular neighborhoods are discussed in Section 10 below.

A second limitation is that the LM and EM observations tend tobe 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 EMso as to achieve imaging of the self-same features with both tech-niques (e.g., Fig. 9). Although it is, in general, difficult to home in onspecific areas of interest (designated as such by LM surveillance)after the specimen has been subjected to conventional preparationfor EM, this has been accomplished in a number of studies (e.g.,Nakata et al., 1998). More recent approaches have correlated fluo-rescent labeling LM with immuno-gold EM (Mironov et al., 2001)or introduced genetically engineered proteins that fluoresce forLM visualization and are subsequently treated to nucleate elec-tron-dense deposits for EM detection (Gaietta et al., 2002). Afurther line of development is based on observing vitrified, specif-ically fluorescent, specimens on a cryogenic light microscope and

t/fluorescence microscopy at progressively higher magnifications feed into a cryo-n up in the following panel. (A) Phase-contrast image and (B) fluorescence image ofM1-43 fluorescence. (C) Cryo-EM image of neuronal processes surrounding the areacesses, an extracellular vesicle connected to one of the processes and to a protrusion

ace rendering showing the extracellular vesicle (blue), two neighboring neuronalcular complexes (external—green, and internal—red). The vesicle is shown with onebased on data published by Lucic et al., 2007).

194 A.C. Steven, W. Baumeister / Journal of Structural Biology 163 (2008) 186–195

then transferring them into an electron microscope for cryo-tomo-graphic analysis (Sartori et al., 2007; Schwartz et al., 2007).Software-based methods can assist with the still incompletelysolved problem of locating specific areas of interest for EM or ETanalysis (Lucic et al., 2007).

10. Marrying the power of visualization with the power ofidentification

A cryo-electron tomogram of a cell affords a three-dimensionalimage of its entire proteome; if interpretable as such, it provides asnapshot of the network of interactions underlying the gamut ofcellular functions. However, the adverse signal-to-noise ratio ofthe tomograms and the crowded nature of the cytoplasm renderssuch interpretation a challenging task. The most direct approachto interpreting cellular cryo-tomograms in molecular termsemploys pattern recognition methods that rely on two sources ofa priori information: a library of three-dimensional templates forall of its particulate components; and knowledge of the cell’smolecular inventory (Baumeister, 2005). The library may be usedto sift through the tomographic volume to map the population ofeach recognizable component by cross-correlation techniques, inan approach akin to the fitting of subunit crystal structural incryo-EM density maps (Bohm et al., 2000; see Section 5). Whilethe level of success in such an experiment will, in general, dependon both the completeness of the reference library and the resolu-tion and signal-to-noise ratio of the tomogram, promising resultshave already been obtained with in situ mappings of bacterial ribo-somes (Ortiz et al., 2006; Seybert et al., 2006).

However, interpretation is a more feasible proposition if inven-tory data are available, and such data may be acquired in a compre-hensive manner by mass spectrometry. In the traditional ‘‘slice anddice” approach, cellular cultures and tissue samples are homoge-nized prior to mass spectrometry, thus destroying valuable infor-mation about the compositions of individual cells. This loss ofinformation may be avoided if it is possible to develop effectiveprocedures for operating at the level of individual cells, or evensubcellular volumes. In this context, Laser Capture Microdissection(LCM) has considerable potential for isolating distinct cell typesfrom heterogeneous populations, as have dual beam SEM/FIB sys-tems, to excise defined subvolumes under visual control (Burge-meister, 2005). Ideally, pipelines will be assembled in whichcomplex samples are ‘navigated’ by (cryo)-fluorescence micros-copy, targets identified, and then isolated via micromanipulationand micromachining methods for analysis by both mass spectrom-etry and electron microscopy/tomography. Needless to say, thisenvisages a highly hybrid approach.

11. All roads lead to Rome

A priori, it is clear that a complete molecular atlas of a singlecell—which, of itself, would represent a prodigious technicalaccomplishment—would yield only limited functional insight. Forthis information to be fully exploited, such an atlas would haveto be compiled for a great many (how many?) similar cells andthe trends of co-association and compartmentation deduced onstatistical grounds. This proposition represents just one of manyareas where computational analysis moves into center-stage notonly for data acquisition and processing but also in integrationand analysis. Indeed, the computer is also the only medium inwhich direct structural data may be cohesively integrated withother sources of information from diverse biochemical and bio-physical approaches to formulate three- or four-dimensional mod-els of a system of interest. A good example of integrative analysis ofthis kind is given in recent work on the nuclear pore complex

(Alber et al., 2007a,b). Structurally oriented bioinformatics is animportant part of this general movement and Andrei Lupasappraises its trends in his contribution to this Special Issue. If, look-ing out over the next decade or two, we may anticipate at least apartial solution to the protein folding problem, a comprehensivegrasp of intermolecular interactions and conformational changes,and a functionally validated basis for molecular dynamics, itappears likely that the destiny of structural biology is to morphinto a branch of computer science—arguably, the most interestingbranch and the one most relevant to the human condition.

Acknowledgments

We thank Drs. D. Belnap, B. Heymann, V. Lucic and J. Plitzko fortheir contributions to figures for this paper, and Drs. J. Frank, C.Goldsbury, V. Matias, and M. Rossmann for permission to repro-duce their work.

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