Structure - Approaches

Introduction

The migration machinery is composed of several, large multi molecular complexes that are highly dynamic and highly regionalized. Examples include the lamellipodia and filopodia at the cell front and adhesions that form at the leading edge and disassembly behind the leading lamella and in retracting regions at the cell rear. All of these structures are composed of many different kinds of molecules and are part of dynamic processes.

How can one determine the structure of such complex assemblies? At one extreme, methods like X-ray and NMR provide high resolution structures of individual molecules. At the other extreme, light microscopy reveals molecular dynamics during migration but only at a low resolution. The Structure Initiative is integrating high and low resolution structural approaches to determine the high resolution structures of the large, multi-molecular assemblies that mediate migration. In order to provide a seamless structural net, we have integrated "bottom-up" approaches at the atomic and molecular level, including in vitro assembly and structural characterization of focal contacts and lamellipodia, with "top-down" approaches involving living cells and cryo-EM techniques of cells frozen in a particular migratory state (see figure below).

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Bottom Up Approach

X-Ray Crystallography

Recent advances in crystallographic techniques, computing power and synchrotron radiation, have meant that the rate-limiting step to achieving atomic resolution is typically the production of suitable crystals. While there are many unknown factors in the "crystallizability" of a protein, one key factor is its rigidity. Although the size of a protein or complex is not a limiting factor, per se, larger proteins do tend to have multiple domains, and if these are joined by flexible linkers, this will reduce crystallizability. Fortunately, an emerging theme in the regulation of migration proteins is head-tail intra molecular association that holds the protein in an inactive conformation that obscures binding sites for other migration proteins, either sterically or allosterically. This insight suggests that in these cases the inactive form is the more rigid and more likely to crystallize. By contrast the "active" conformation is often an extended flexible conformation (e.g. the "beads-on-a-string" seen in EM images of vinculin), which will in general be unsuited to high resolution studies, but should be accessible to EM techniques.

The key to successful crystallization is thus to capture the molecule in a rigid homogeneous form. The inactive conformation may be stabilized by phosphorylation (e.g. Src), or it may be disrupted by phosphorylation (e.g. moesin) or PIP₂ (e.g. vinculin). In the case of vinculin (MW 120 kDa), expression in E.coli led to the purification of a crystallizable full-length protein that diffracts to 3.5 Å resolution (see below), since here the inactive form is unmodified. By contrast, crystallization of a major fragment of Src required expression in insect cells in order that appropriate phosphorylation of the tail occurred; by careful purification it was possible to isolate stoichiometrically and uniquely phosphorylated protein in sufficient quantities for structural work (Xu et al., 1997).

In many cases, the full-length molecule needs to be trimmed or modified in some way, or must have a ligand or antibody bound, to enhance rigidity. Many proteins have unstructured termini, transmembrane segments, glycosylation, and other flexible regions, which can be removed biochemically or by mutagenesis. An outstanding example of such an approach is the structure determination of the HIV surface glycoprotein, gp120: here, surface loops and glycosylation sites were removed by mutagenesis, and both the ligand (CD4) and a monoclonal antibody were used for co-crystallization to enhance solubility and rigidity (Kwong et al., 1998). In other examples, a covalent linkage was used to "bypass" the transmembrane region of the glutamate receptor (Armstrong et al., 1998); whole domains were removed or ignored (Lee et al., 1998; Pearson et al., 2000); and surface cysteines, which created problems of aggregation at the high concentrations used for solution and crystallization studies, were mutated to alanine (Sun et al., 1999). The bottom line here is that biological insight, combined with much careful biochemistry and molecular biology, must often be performed in order to yield suitable samples for structural studies. Furthermore, these proteins are some of the least likely to yield to automated high-throughput approaches.

As an alternative to heterologous expression, some proteins are abundant in particular tissues and may be purified from source in sufficient quantities for structural studies. For example, the integrin alpha IIb beta3 from human platelets when subjected to limited proteolysis specifically cleaves the C-terminal tails and transmembrane regions, yielding a water soluble 200 kDa fragment suitable for crystallization trials. This offers the possibility of producing large quantities (readily 50-100 mg from platelets) of protein with authentic post-translational modifications.

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Nuclear Magnetic Resonance (NMR)

NMR methods can currently generate co-ordinates ab initio for proteins up to around 50 kDa in molecular weight. For proteins larger than around 15 kDa, however, the method is relatively slow compared to crystallography, and isotope labeling is required. NMR has a number of important uses that are complementary to crystallography. It is particularly good at studying relatively small proteins or domains, which are often difficult to crystallize. It can also be used to help with the "phase" problem, by providing structural information about domains within a larger protein. It can also identify flexible regions in a protein that may be causing problems with crystallization or difficulties with chain tracing. NMR has a unique ability to study unfolded proteins at the atomic level and it can play a significant role as an analytical tool - defining the purity and stability of protein preparations. It can also be used to identify the relative flexibility of domains in a multi-domain protein (Ulmer et al., 2002).

NMR technology is under intense development, and continues to make significant advances. Better resolved spectra can be obtained with very high fields (900 MHz) and techniques like TROSY (Riek et al., 2000); cold probes can give better sensitivity, and improved structural restraints can be obtained using residual dipolar coupling and relaxation anisotropy (Schwarz-Linek et al, 2003).

Sample preparation is also a key component for NMR studies. Isotope labeling with ¹⁵N, ¹³C and ²H is often required. Methods for doing this with E.coli are well established. Pichia pastoris has also been successfully used for the production of labeled protein (Bright et al., 2000). A promising method currently being explored is cell-free synthesis, using new isotope labeling strategies; for example, chemical ligation technology is being investigated, where domains within a larger protein can be differentially labeled (Staunton et al., 2003). Such tools will be extremely powerful for exploring the biological role of modular proteins in various contexts.

The Module Approach

In parallel with studies of the intact proteins, often the classic reductionist approach is taken to define biochemically the minimal domains or modules within a pair of proteins that are involved in specific complex formation, since this is most likely to produce protein that is highly soluble, mono-disperse and stable, and thus suitable for crystallographic or NMR studies. By combining information on whole proteins crystallized in their "inactive" form, EM images of full-length active forms, and the structures of isolated domain:domain complexes, at the very least, strong clues to the nature of binding and regulation can be obtained (Pickford & Campbell, 2004).

The complex web of interactions amongst migration proteins means that it may be impractical to determine 3 dimensional structures for every domain pair. Instead, examples are selected from each class. This process may be simplified because many interactions can be reduced to a domain:peptide pair, for which we already have many examples at atomic resolution for signal transduction proteins. This class of interaction may turn out to be the dominant one amongst migration proteins, and it is likely that the nature of many of these interactions will be understand at the atomic level obviating the need for determining further structures. Determining the thermodynamic and kinetic properties of each example will be ultimately required however, if cell migration is to be modeled from first principles.

The first step in the module approach is the determination of domain boundaries. The best case is where a homologous domain structure is already known, so that the boundaries can be accurately estimated by sequence alignment. This is an area where recent Structural Genomics initiatives, whose explicit goals are to determine one example of each domain type in the human genome will have a significant and favorable impact. In cases where no homolog exists, there are some established general procedures, including trial-and-error with a range of overlapping constructs, limited proteolysis of "loose ends" of a preformed complex, and in silico approaches (e.g. secondary structure prediction).

The next step is to define interaction pairs. Many biochemical and genetic data on domain pairs already exist in the literature and many more are expected to be generated from the proteomics investigations. In most cases, the information falls short of defining the precise lengths of the interaction pairs. All of the approaches use well established technologies, but are time-consuming, and require biochemical and biological insight.

There are five potential classes of interactions that are relevant to cell migration:
Peptide:peptide interactions. There appear to be many cases where intracellular proteins are essentially in random coil formation until they form a complex. In most cases the complex will be formed between a folded protein and an unfolded peptide. In some cases, however, there may be interactions between two peptides that are initially unfolded. Although integrins are large, mainly extracellular, proteins their short cytoplasmic tails play an important role. A large research effort has been expended to determine whether direct interactions between these tails contribute significantly to the different affinity states of integrins. Some groups have observed in vitro interactions (Vinogradova et al., 2002) while other groups have not (Ulmer et al., 2001). This exact role of direct integrin tail interactions remains an area of considerable interest.

Domain: peptide interactions. In many cases, the situation is simplified because one partner in the complex is a folded domain - such as an SH2, SH3, PTB or LIM domain - while the other partner is a short stretch of a larger protein that adopts no definite structure by itself but does so when bound to its partner. Domains can be expressed in E. coli, peptides can be synthesized chemically, and binding constants determined by surface plasmon resonance and/or isothermal titration calorimetry, demonstrating that a specific interaction exists in vitro, prior to structure determination. NMR is very powerful for this class of interaction, particularly when used to identify relatively weak ligand interaction sites (Schwarz-Linek et al., 2003) For example, ¹⁵N-labeled peptide may be titrated with unlabeled domain. Binding should lead to the adoption of a definite structure for the peptide, which should be readily detectable by the change in the HQSC spectrum. This information could then be used to synthesize a minimal length binding peptide for enhanced solubility, monodisperseness and crystallization. In some cases, it may be appropriate to engineer a covalent linkage between the interaction pairs (e.g. alpha- and beta-catenin (Pokutta and Weis, 2000)) in order to enhance the local concentration. In a recent example from this consortium talin/integrin tail interactions were studied both by crystallography and NMR (García-Alvarez et al., 2003).

Domain:domain interactions: Interactions between integrins and ECM proteins form a major family within this class. The complex between the integrin I domain and a collagen-like triple helical peptide provides a paradigm for this class of interaction, certainly for those integrins that contain an alpha-subunit I domain. For those that do not, and particularly for those that require binding surfaces on both the alpha- and beta-subunits (e.g. fibrinogen-alpha IIb beta3), a complex structure is a priority (see below). Intracellular examples are the WASP-Cdc42 interaction and PAK-Cdc42 interaction (Kim et al., 2000; Lei et al., 2000). Head-tail intramolecular interactions often involve domain:domain interactions. Examples include the moesin head-tail interaction and Liddington's work on vinculin (see below). In the case of moesin, the 100 residue tail is somewhere between a peptide and a domain, since it has secondary structure but no hydrophobic core.

Domain: membrane interactions: Several different types of membrane association need to be distinguished. One example is the radixin FERM domain (Hamada et al., 2000) - here the PIP₂ head group, IP₃, was co-crystallized with the FERM domain, and shown to bind to a specific site, leading to a model of an electrostatic interaction with PIP₂-containing membrane. A distinct example is vinculin - here several lines of evidence point to an insertion of the domain into the PIP₂-enriched membrane accompanied by a major conformational change on binding (Bakolitsa et al., 1999); this type of interaction is best studied by NMR.

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Cryo-EM

Cryo-EM is the principal method for solving the structures of large complexes that remain beyond the reach of NMR or x-ray crystallography. The field is in a state of rapid development on several fronts, and further methodological improvements are likely to come from automation of both data collection and image processing. Cryo-EM can image 2-D protein arrays, helical filaments, individual molecules, large assemblies and subcellular structures in situ (for recent reviews see (Baumeister et al., 1999; Chiu et al., 1999; Kuhlbrandt and Williams, 1999; Saibil, 2000). The technique utilizes a variety of image analysis approaches for producing 3-D reconstructions; these include single particle approaches, helical reconstruction, electron crystallography, and electron tomography. The specimen is typically analyzed in its fully hydrated state (Dubochet et al., 1988). Although mainly aiming at molecular resolution (10-30 Å), electron microscopy can provide near-atomic resolution e.g., tubulin (Nogales et al., 1998), aquaporin (Murata et al., 2000), light-harvesting complex (Kuhlbrandt et al., 1994), and bacteriorhodopsin (Henderson et al., 1990). Time-resolved techniques offer the possibility of studying conformations that are inaccessible to X-ray crystallography. For example, the activated state of the nicotinic acetylcholine receptor, which has a lifetime of 10 ms, was studied at 4.6-9 Å resolution, using three-dimensional helical reconstruction of tubular membrane crystals (Miyazawa et al., 1999; Unwin, 1995). Conformational changes in motor proteins (myosin, kinesin, NCD) are also currently under study (for reviews see Vale and Milligan, 2000; Volkmann and Hanein, 2000), by using helical reconstruction techniques of decorated actin or microtubules in different nucleotide states that are not attainable by crystallography.

Docking: One of the most exciting aspects of electron cryomicroscopy is the possibility of fitting atomic models derived from crystallography, NMR or theoretical modeling into EM reconstructions. Several methodologies are being pursued in a number of laboratories. Many researchers still rely on interactive, manual manipulation in front of the computer screen to obtain a fit between the atomic model and the EM density (see for example Beroukhim and Unwin, 1995; Gilbert et al., 1999; Hewat et al., 1998; Hoenger et al., 1998; Voges et al., 1994; Yu et al., 2000). Objective scoring functions have also been used to assess the quality and to refine the initial manual fit (see for example Che et al., 1998; Chen et al., 2001). A particularly well suited refinement procedure is based on the use of real-space methods (Chapman, 1995; Chen et al., 2001). Recently, a number of objective, global search based docking algorithms were introduced that do not require the manual fitting step (Roseman, 2000; Volkmann and Hanein, 1999; Wriggers et al., 1999). One of these algorithms (Volkmann and Hanein, 1999) recently revealed actin-bound myosin conformations that are distinct from any of the crystal structures of unbound myosin (Volkmann et al., 2000), enabling a structural mechanism for the release of myosin from actin to be proposed. This methodology has also led to an atomic model of actin-fimbrin complex and the structural basis of its regulation by calcium (Hanein et al., 1998).

F-actin and associated complexes: There are a large number of interactions between migration proteins and the actin cytoskeleton. Actin filaments are challenging to decipher at atomic resolution, since they are not amenable to crystallization. However, they provide promising specimens for electron cryomicroscopy and image analysis. The major image analysis tool for actin filaments is helical (otherwise known as Fourier-Bessel) 3-D reconstruction. In principle, taking advantage of the helical symmetry of actin filaments, one can use a single projection to generate a 3-D reconstruction (DeRosier and Klug, 1968). Actin filaments (Belmont et al., 1999; De La Cruz et al., 2000; Egelman and Orlova, 1995; Orlova and Egelman, 2000; Steinmetz et al., 2000); actin filaments bound to the capping proteins gelsolin (McGough and Way, 1995) and cofilin (McGough et al., 1997); actin complexed to domains of cytoskeletal proteins such as alpha-actinin (McGough et al., 1994), fimbrin (Hanein et al., 1997; Hanein et al., 1998), utrophin (Moores et al., 2000), and calponin (Hodgkinson et al., 1997) have all been studied through EM and helical reconstructions techniques.

2-D crystallization on lipid monolayers:

For proteins or conformations that do not form three-dimensional crystals, an alternative approach is to make two-dimensional arrays on a suitable substrate. Kornberg and colleagues (Kornberg and Ribi, 1987; Ribi et al., 1988; Schultz et al., 1990) developed the method of forming 2-D arrays of proteins bound to lipid layers, which is based on the interaction of a water soluble protein with a specific lipid bound ligand that is inserted into a fluid lipid monolayer. Bound macromolecules can then spontaneously arrange into a regular lattice with identical orientations. The method has wide applicability. For example, electrostatic interactions instead of ligand binding can be used to partition charged proteins to the monolayer (Darst et al., 1988). This is illustrated by the crystallization of annexin V (Brisson et al., 1991), alpha-actinin (Taylor and Taylor, 1993) and the heavy meromyosin fragment of smooth muscle myosin II (Wendt et al., 1999). Monolayer crystallization has provided 3-D reconstructions at 15 -20 Å resolution of alpha-actinin (Tang et al., 2001) and HMM (Wendt et al., 2001), both using ice-embedded specimens. These specimens have so far defied numerous attempts to crystallize in a form suitable for X-ray crystallography.

The essential ingredient for lipid monolayer crystallization is the binding of the soluble protein to the lipid. For crystallization to occur, the protein must bind to the monolayer. Binding can be achieved by using a cationic surfactant if the protein is acidic, or an anionic surfactant if the protein is basic. A general technique for crystallization of expressed proteins purified via a histidine tag has been developed using Ni-chelating lipids (Kubalek et al., 1994). Most recently, ceramide lipids, which form lipid tubes, have been introduced as a vehicle for helical crystallization (Ringler et al., 1997; Wilson-Kubalek et al., 1998), which depending on the helical indexing can provide 3-D images from a single micrograph. In this case it is possible to introduce positively or negatively charged lipids as well as lipid ligands into the ceramide "carrier" lipid.

Once the arrays are formed, they can be recovered from the monolayer surface using another hydrophobic surface. The most efficient method of monolayer recovery utilizes reticulated carbon films mounted on a standard EM grid. These films can be purchased or made in the lab. Kits are available for producing reticulated films with uniform hole sizes. The disadvantage of using reticulated films is their poor flatness, which can limit the resolution of the final 3-D reconstruction.

Arrays can be examined in the EM preserved in negative stain or in their native state by rapidly freezing the specimens in a slush of liquid ethane. The 3-D image is obtained by tilting the arrays in the microscope and collecting images. These different "projections", since they are of the same molecule in the same crystal lattice, can be combined into a single 3-D image. Generally, when analyzing images of 2-D arrays, the data from different images is combined in "Fourier" space and an inverse Fourier transform is applied to obtain the 3-D image.

In vitro assembly of adhesion complexes

In vitro assembly lies at the interface between "bottom-up" and "top-down" approaches because it will produce 3-D images that can be used to identify similar structures in focal contacts that are imaged in situ. A lipid monolayer system is being developed to use as a vehicle for in vitro assembly of focal adhesion complexes. Most transmembrane receptors involved in cell adhesion, i.e. integrins and cadherins, are characterized by a large extracellular domain, a single transmembrane helix and a small cytoplasmic domain. The bottleneck for structural studies is the transmembrane helix. This helix can be cut out of the system using lipid monolayers composed of a nickel chelating lipid, which can be purchased from Avanti Polar Lipids, with a phosphatidyl choline carrier lipid. The extracellular and cytoplasmic domains are then obtained with a his6 tag at their N- or C-terminus (C-terminus for extracellular, N-terminus for cytoplasmic domains). The extracellular assemblies and the cytoplasmic assemblies can then be studied independently using a specimen that is highly favorable for electron microscopy. As these specimens are likely to be disordered or paracrystalline at best, the structure analysis will utilize electron tomography for producing the 3-D image.

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Top Down Approaches

This section will deal with "top-down" technologies. The techniques described here analyze the dynamic and structural organization of lamellipodia and filopodia; and explore the feasibility of studying at high resolution, by cryo-EM, isolated and reconstituted adhesive sites. Ultimately, the aim is to combine the information provided from the high-resolution structural information of the domains (NMR, X-ray, 2-D arrays) together with the in-situ characterization, to formulate atomic models for the assembly and dynamics of the cell contacts.

Correlative Microscopy

Correlative microscopy involves high temporal resolution analysis of fluorescent features in a living cell followed by high resolution spatial analysis of the same features in the same cell as captured in the electron microscope. The technique has demonstrated that myosin II in fibroblast cells exists as clusters of bipolar filaments which form initially behind the leading edge and then become part of a contracting network involved in cell body translocation (Verkhovsky et al., 1995). It has also been used to show the branched nature of the actin filament network at the leading edge and that the Arp2/3 complex was positioned at the branch points (Svitkina and Borisy, 1999; Svitkina et al., 1997) - key evidence supporting the dendritic nucleation/array tread milling model of cellular protrusion (Maly & Borisy , 2002; Mullins et al., 1998).

The success of correlative microscopy imposes demands at both the light and electron microscopic level. For fluorescence light microscopy, a suitable fluorescent probe must be prepared either by chemical derivatization, or by molecular biological conjugation to green fluorescent protein or one of its cognates. The activity of the derivatized protein needs to be established either by biochemical assays in vitro or functional rescue in vivo. The derivatized protein is introduced into the cell either by micro injection or by expression after transfection. Issues of photobleaching, photodamage and phototoxicity need to be attended to, generally through low light level imaging and the use of sensitive cameras. At present, a variety of chemical and molecular biological procedures are available for fluorescently labeling specific components of the cytoskeleton, and other members of the Consortium will prepare further novel fluorescent derivatives.

A suitable electron microscopic procedure should satisfy the following requirements: (a) reproducibility; (b) high resolution, contrast and clarity of images; (c) compatibility with high-quality light microscopy (i.e., cells should be grown on coverslips in suitable environmental chambers); (d) compatibility with immunogold labeling procedures; (e) compatibility with specific EM protein labeling procedures such as myosin S1 decoration of actin filaments to determine their identity and polarity; and, finally (f) high yield. Yield is essential because detailed observation of individual living cells places a high investment of investigator time and effort in a single cell.

Specimen preparation procedures have been developed specifically for correlative analysis. The procedure involves detergent extraction, chemical fixation, critical point drying, and transmission EM of platinum replicas (Svitkina and Borisy, 1998; Svitkina et al., 1995). Although no single step is unique to this procedure, and each of them is not without pitfalls, the combination of steps employed together with specific modifications have resulted in an improved procedure for visualization of the supramolecular organization of the cytoskeleton appropriate for correlative microscopy. The quality of the images approaches that of the Heuser procedure and thus is sufficient for addressing numerous questions at the supramolecular level.

Two additional problems should be noted. One is the potential for artifact. To make the cytoskeleton available to both immuno EM reagents and to metal coating, the cell membrane must be removed. However, immediately upon removal of the cell membrane, the potential for extraction and artifact arises. Consequently, the composition of the extraction solution is extremely important and extraction procedures need to be evaluated for each component to be analyzed. A key advantage of correlative microscopy is the possibility of such direct evaluation. Fluorescent features seen in the living cell can be examined at each step in a preparative procedure and finally compared with the structures observed at the EM level in the same cell. Close similarity of spatial organization may then be taken as a measure of the fidelity of the procedure. In this way, specific non-perturbing extraction conditions have been developed for myosin II and microtubules (Svitkina and Borisy, 1998; Svitkina et al., 1997; Verkhovsky et al., 1995).

The second major problem for correlative investigations of the migratory machinery arises from the abundance of actin filaments interfering with the visualization of minor cytoskeletal components. As a solution to this problem, an optional procedure has been developed in which actin is depleted from detergent-extracted cells with the actin-severing protein, gelsolin, under conditions that do not perturb other cytoskeletal components. Removal of the actin filaments in this way renders the minor components visible. This approach was successfully used for the visualization in cultured cells of bipolar filaments of non muscle myosin II, which had previously not been seen in situ by any other technique. It has also been used to discover that the molecule, plectin, cross-links intermediate filaments with microtubules (Svitkina et al., 1996). For analysis of actin filament organization or direct actin binding proteins, the depletion procedure is omitted.

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3-D Organization

In order to complete our understanding of the migratory machinery at the cellular level, correlative microscopy of whole cell preparations needs to be supplemented with high-resolution three-dimensional data. One way of doing this would be the application of high resolution 3-D imaging to preparations of large macromolecular migratory assemblies isolated from whole cells. Application of electron tomography in conjunction with labeling, image analysis and pattern recognition techniques will allow interpretation of these 3-D complex structures in the context of cell migration.

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Electron Tomography

Electron tomography is a powerful tool for elucidating the 3-D architecture of large biological complexes at medium resolution and is the only molecular resolution imaging method available for imaging unique structures (Baumeister et al., 1999; Kornberg & Ribi, 1987; Penczek et al., 1995; Ribi et al., 1988; Schultz et al., 1990). It cannot, however, take advantage of image-averaging techniques for noise reduction unless the specimen itself consists of identical structures, that may be irregularly positioned in the field. Today, with the use of computer-controlled microscopes and the availability of large-area charge-coupled device (CCD) cameras, it has become possible to image large-scale structures at a resolution of 50 Å, with data sets comprising up to 150 projections with a cumulative dose as low as 5000 e- nm^-2 (Grimm et al., 1998). Tomography is undergoing considerable growth at the present time due to the realization that molecular information can be obtained from unstained, frozen hydrated whole cells and isolated organelles (Medalia et al., 2002; Nicastro et al., 2000).

Segmentation: Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratio. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. A segmentation approach that consists of a novel three-dimensional variant of the immersion-based watershed algorithm was recently introduced (Volkmann, 2002). The algorithm is applicable to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures.

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Additional Reading

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. 2000. The Protein Data Bank. Nucleic Acid Res. 28,235-242. PubMed

Weissig H, Bourne PE. 2002. Protein structure resources. Acta Crystallographia D58, 908-915. PubMed.

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