Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission electron microscopic techniques

CIS-01385; No of Pages 14 Advances in Colloid and Interface Science xxx (2014) xxx–xxx

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Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission electron microscopic techniques Hans v. Berlepsch a,b, Kai Ludwig a, Boris Schade a, Rainer Haag b,c, Christoph Böttcher a,⁎ a b c

Forschungszentrum für Elektronenmikroskopie, Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany Core Facility BioSupraMol an der Freien Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany Institut für Chemie und Biochemie — Organische Chemie, Freie Universität Berlin, Takustraße 3, 14195 Berlin, Germany

a r t i c l e

i n f o

Available online xxxx Keywords: Ribbon Tube Chirality Cryogenic transmission electron microscopy 3D reconstruction Cryo-electron tomography

a b s t r a c t The self-assembly of amphiphilic molecules into fibrous structures has been the subject of numerous studies over past decades due to various current and promising technical applications. Although very different in their head group chemistry many natural as well as synthetic amphiphilic compounds derived from carbohydrates, carbocyanine dyes, or amino acids tend to form fibrous structures by molecular self-assembly in water predominantly twisted ribbons or tubes. Often a transition between these assembly structures is observed, which is a phenomenon already theoretically approached by Wolfgang Helfrich and still focus point in current research. With the development of suitable sample preparation and electron optical imaging techniques, cryogenic transmission electron microscopy (cryo-TEM) in combination with three-dimensional (3D) reconstruction techniques has become a particular popular direct characterization technique for supramolecular assemblies in general. Here we review the recent progress in deriving precise structural information from cryo-TEM data of particularly fibrous structures preferably in three dimensions. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TEM imaging, image processing and 3D reconstruction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cryo-TEM — a direct characterization method for native supramolecular architectures . . . . . . . . . . . . . . . . . 2.2. Image processing methods and 3D reconstruction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Single particle analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Reconstruction of helical filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Cryo-electron tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Self-assembly of amphiphilic compounds towards fibrous aggregates and their characterization by electron microscopic methods 3.1. Morphological aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Amphiphilic glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Amphiphilic dye aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Amphiphilic amino acids, peptides and amyloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Amphiphilic amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Amphiphilic peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Amyloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at:. Forschungszentrum für Elektronenmikroskopie, Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 36a, 14195 Berlin, Germany. Tel.: + 49 30 83854934; fax: + 40 30 83856589. E-mail address: [email protected] (C. Böttcher). 0001-8686/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cis.2014.01.007

Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007

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1. Introduction The emergence of life on earth is to a great extent based on the ability of certain molecular building blocks to generate structurally precise and functional supramolecular architectures in a molecular selfassembly process [1]. The significance of a precise structure for a determined functionality therefore defines the superior scientific goal: the assignment of supramolecular structures at the highest possible spatial resolution with the objective of understanding the interdependency with their function. Nature has generated a number of different molecular players, which show self-assembling behavior, most importantly lipids, nucleic acids, proteins and carbohydrates. Depending on the individual molecular composition several driving forces determine the eventual threedimensional structural organization of supramolecular assemblies, which are solvophobic effects, inter- and intramolecular hydrogen bonding, π–π-stacking, multivalent ionic interactions (complexation), and chirality [2,3]. The multitude of potential molecular interactions is one of the reasons why often hierarchically organized complex structures form and it also clarifies the difficulties in understanding corresponding structure formation processes or in designing molecules for the construction of predetermined supramolecular architectures. A strategy to cope with this complexity is to create simpler molecular building blocks, which allow studying the impact of each of the above factors on structure formation either independently or at least in restricted combinations. In this context, a most suitable class of building blocks are amphiphilic molecules, which in their simplest form assemble by the interplay of hydrophilic and hydrophobic molecular domains due to the solvophobic or, in particular, the hydrophobic effect in an aqueous environment [4]. The interest in amphiphilic molecules and their ability to form supramolecular architectures by selfassembly dates back more than a hundred years and was initially focused on soaps, fats and oils [5] but continues up to date. Perhaps the most important naturally occurring amphiphiles are phospholipids. A balanced volume ratio of water-exposed hydrophilic head groups and hydrophobic tails, respectively, initiates the formation of molecular bilayers, which constitute the membranes of living cells. At first glance, one would expect that extended two-dimensional membranes or large spherical vesicles with minimum curvature would generally represent the lowest-energy state of amphiphilic bilayers. The variety of observed structures, however, is much more diverse and with the development of suitable preparation and imaging techniques structural investigations (which took its course in the 1980s) revealed various uncommon morphologies, including ring- and disk-like, as well as extended fibrous structures. By evaluating publications of recent decades which were dealing with self-assembly of amphiphilic compounds it was found that an overwhelming number of them reported on fibrous structures, i.e. threads, ribbons, tapes or hollow tubes [6–9]. This is noteworthy given that the studied materials significantly differed in their molecular building blocks and their dominating intermolecular forces. Particularly supramolecular fibrous assemblies exhibiting helical ultrastructural features predominantly formed by chiral molecules became of interest. Chirality is certainly one of nature's key strategies to trigger more complex ultrastructures [10] and, most importantly, enables stereospecific molecular recognition processes [11], but also plays a prominent role in pharmaceutical therapies [12]. A most-favored phenomenon with chiral amphiphiles is the interdependent formation of twisted ribbons, wound ribbons and tubules. These structures often coexist in preparations or their transition can be successively monitored by temperature or time-dependent preparation techniques. These observations have initiated a vivid interest to explain the relation between different types of anisotropic intermolecular interactions and the development of corresponding morphological features [13]. Theoretical descriptions of the forces driving the formation of helical ribbons and their maturation towards closed tubes were especially promoted by Helfrich [14,15]. Due to the importance of this

phenomenon and the fact that morphological transitions from ribbons to tubes are frequently observed independently of the molecular structure, we will particularly highlight systems in this contribution, that favor such kind of assembly process. The allocation of precise experimental data is of fundamental importance to understand self-assembly processes. Moreover, accurate structural characterization is the precondition for a reproducible manufacture of molecular assemblies for technical applications. Due to the limitation that a vast majority of assemblies never crystallize (or adopt a different spatial or conformational organization in the crystalline state, see Section 3.2.), which, however, is a prerequisite for X-ray structure analysis, electron microscopy (TEM as well as SEM), especially in combination with cryo-fixating preparation techniques, has gained increased significance for the elucidation of supramolecular structures. Thanks to image processing and three-dimensional (3D) reconstruction techniques, including cryo-electron tomography, and in synergy with complementary analytical methods like NMR spectroscopy, scanning probe microscopy (AFM, STM), optical microscopy, X-ray fiber diffraction or various spectroscopic methods the characterization of supramolecular assemblies on the molecular and even the atomic level is in closer reach (see Section 2). From this point of view we review on the latest advances in the fabrication and structural characterization of defined fibrous supramolecular assemblies and provide an update on the technical progress which became available in recent years to elucidate 3D molecular architectures with high resolution. Appropriate examples of amphiphilic glycolipids, dyes, amino acids, and peptides are chosen. Studies on biological materials (in particular protein structures), however, would go far beyond the scope of this review and are therefore not included. 2. TEM imaging, image processing and 3D reconstruction techniques 2.1. Cryo-TEM — a direct characterization method for native supramolecular architectures The goal of this section is not to provide a general introduction to the field of electron microscopy, covering detailed aspects of electron optics, image formation, etc. For these aspects, the reader is referred to dedicated monographs or reviews cited below. In fact, the synergistic effects comprising novel approaches of sample preparation, instrumentation and image analysis techniques are the focus of this review, all of which have significantly improved our knowledge about structural aspects of supramolecular assembly processes in recent years. It should be noted that these strategies have long been successfully implemented in the methodical canon of structural biology. With the progress in synthetic supramolecular chemistry, which has produced a growing number of structurally precise supramolecular architectures, these established methods become applicable and promise to provide a much better understanding of the factors governing molecular selfassembly. Transmission electron microscopy has the huge advantage over all alternative direct and indirect structure characterization methods that it can provide structure information at very high spatial resolution in the native assembly state by employing suitable preparation and imaging methods. Moreover, due to the fact that electrons transmit the sample, projection images (comparable to the recording of a radiography where all densities of a 3D object are accumulated in a 2D image) are generated from which internal structural information can be elucidated by the application of image processing and 3D reconstruction methods. A more detailed description of corresponding strategies is given in Section 2.2.1. Low contrast and radiation sensitivity are the main obstacles to overcome in transmission electron microscopy in order to obtain high resolved structural information of biological or organic supramolecular architectures. The use of contrast agents, which aims to embed objects in an electron dense matrix of heavy metal salts, helps to increase

Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007

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contrast and is a simple and fast preparation method, but prone to the generation of artifacts [16]. This is due to structurally interfering chemical interactions between stain and sample as well as due to the air drying process preceding the transfer of the sample in the high vacuum of the microscope. Moreover, most carbonaceous samples in the mentioned context quite sensitively react against electron irradiation by a sequential loss of their structural details [17,18], which might prevent the recording of high resolution details of these materials. Cryo-fixation is the method of choice to overcome the limitations of contrast agents and/or sample drying and even increases the tolerance for electron irradiation [18,19]. The most elegant approach, which delivers direct structural information of the samples in almost native state of aggregation (most importantly in aqueous solution, but preparation in selected organic solvents such as toluene is also possible, see Section 3.4.1.) is the vitrification of ultrathin sample layers (200–300 nm) by a fast transfer into a cryogen such as liquid ethane or propane [20–22], a procedure widely known as the cryo-TEM method. The efficient heat transfer from the sample towards the cryogen allows for a high cooling rate and the generation of an amorphous glass-like state of the sample layer. Changes in the density of the water due to the formation of crystalline ice (which might damage the native structure) can thus be avoided.

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The benefit of thin vitrified samples is that they can be directly observed in the TEM (by employing special cryo-sample holders and transfer units) through making use of a phase contrast imaging method and exploiting density differences between objects and embedding solvent matrix. In short, small amplitude differences, which are per se too small to produce sufficient contrast for the visual discrimination of structural details (this is the case when a TEM is focused, where the contrast is the lowest) can be increased by defocusing of the microscopes' objective lens. This strategy, however, complicates the interpretation of the image data due to the consequences of a contrast modulation (i.e. the contrast transfer function (ctf) of the microscope, Fig. 1) imposed on the data [23]. Several dedicated monographs and reviews are available [24–27] for a detailed and fundamental introduction into the physics and optics of transmission electron microscopy. Despite the adumbrated electron optical complications the physical phenomena are well understood and by the employment of adequate correction strategies (e.g., automated ctf correction for single particle data) [28], structural information can in principle be extracted with very high resolution. It is worth to emphasize already at this point, that the quality of the sample preparation is often more critical than the optical performance of the microscope. On the one hand, there are manifold reports on structure determination of ordered and radiation resistant materials at atomic or even subatomic resolution, but on the

Fig. 1. (Above left) Test image (Siemens star) providing a graphical object with a continuous decrease of spacings from the periphery towards its center. The pixel resolution of the image corresponds to a TEM recorded micrograph at a primary magnification of 150,000. (Top right) Test image convolved with the contrast transfer function (ctf) of a transmission electron microscope (below) as generated at an accelerating voltage of UB = 160 kV (defocus of Δz = −300 nm; CS = 2.0 mm). It becomes evident that the test image information is significantly altered by the optical characteristics of the microscope, which leads to contrast reversals and multiple zeroes as a function of space frequencies. Figures on top are reproduced from Ref. [23] with permission. Copyright (2012) Wiley.

Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007

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other hand still much less examples on radiation sensitive materials in the resolution regime of less than 1 nm are reported. Through the allocation of direct and native structural data, cryo-TEM has developed, however, a unique status among the analytical methods involving radiation sensitive materials. No other method offers more direct information about the sample status by overcoming the phase problem [29] and providing real space images in contrast to other indirect diffraction based methods. By the complementary use of alternative, mostly indirect analytical methods, however, a very broad range of additional information can be accumulated. Moreover, the complementary analysis of X-ray crystallographic and cryo-TEM data proved a very satisfying structural agreement supporting the capacity of the image based method [30,31]. The recent advances in image analysis and 3D characterization techniques (single particle method, tomography) have boosted our knowledge of the structural diversity of biological supramolecular assemblies in general. These studies also give us valuable information about the potential as well as the limitation of the method. The following section will provide some insights about the status of the methods and the capacities to elucidate structural data in the field of supramolecular chemistry. 2.2. Image processing methods and 3D reconstruction techniques 2.2.1. Single particle analysis Raw cryo-TEM image data are noisy and of low contrast. Nevertheless, if the supramolecular structure is distributed in multiple copies over a vitrified matrix, the summing of extracted and aligned images of equal typical projection views of different individual objects can in principle result in a nearly noise-free class-sum image, as the structural features buried in the noise contribute density to the class sum image, whereas the statistically distributed noise is averaged out (e.g., Fig. 3 in Section 3.2.). This approach is known as the single particle analysis method. Several software packages are currently available which offer useful routines to extract, filter, analyze, align and classify corresponding data sets (e.g. IMAGIC [32], Spider [33], EMAN [34], RELION [35], among others). Moreover, they also comprise modules to calculate 3D structures from class-sum images by determining their spatial relationship (i.e. the determination of Euler angles) or by exploiting the known angular relation of tomographic series. A comprehensive and informative review covering the relevant aspects of the current state of instrumentation, imaging, image analysis and alignment, statistical analysis etc. is given by Orlova and Saibil [27]. Particularly in regard to aspects of 3D structure determination the book of Frank is a valuable contribution in the field [36]. Many biological structures have been solved in recent decades by the use of the single-particle method with unprecedented resolution. In many cases the resolution values were well beyond 1 nm for asymmetrical structures (e.g. ribosomal structures at ≈ 5 Å resolution [37,38]) or close to a few Ångström for symmetrical structures (e. g. a symmetric virus at 3.3 Å resolution [39]), which values has so far only been achievable by the use of X-ray crystallography. Moreover, the high reliability of the image based structure analysis increasingly allows synergetic hybrid approaches combining cryo-TEM data with X-ray crystallographic or NMR data [40]. The high degree of structural identity especially in proteins provides a fabulous play-ground for the 3D reconstruction technique as the random spatial distribution of the protein molecules in the vitrified sample provides different views of the structure, and although the structure of the particles itself is assumed to be identical, the projection images under different view angles are not. The crux of the matter is a statistical analysis (i.e. multivariate statistical analysis, MSA [41,42]) of data sets usually comprising several thousand individual objects which identifies the main structural differences and helps to separate projection images of particles (a procedure

termed classification) representing different orientations of the same structure. For a reconstructed 3D structure the angular relation of many of such views has to be determined, which is in principle possible due to the inherent structural relation of projection images of a 3D object that can be obtained from Fourier analysis (i.e. angular reconstitution techniques [43]) of the images. We should recapitulate at this point that X-ray crystallography requires the growth of crystals in order to store multiple copies of a structure within a spatially restricted volume. Crystal growth is not necessarily successful and even can be counterproductive if crystal and supramolecular structures differ [23]. Coherent image averaging requires structural identity. This is a rare issue in the field of synthetic supramolecular chemistry. Nature turns out to be the much better chemist by far, indicated by the mere number of publications which report on biological ultrastructures in three dimensions (not to mention their functional significance) and which outnumber the reports on organic supramolecular structures which were suitable to be treated by the single particle method. Even though several theoretical approaches are available, which aim to predict the approximate geometry of supramolecular architectures based on molecular specifications (Section 3.2.), we basically still lack the knowledge to predict a molecular structure, which responds the dedicated supramolecular assembly process. Though still rare, there are, however, reports on structurally defined ultrastructures, which can be treated in much the same way as reported for biological materials. In some of these cases even 3D structures were determined [44,45], in particular of helical structures, which usually show a high degree of structural precision and can in principle be reconstructed in three dimensions from single projection images. Details of the different approaches for the determination of helix structures are given in the following section. More examples of successfully reconstructed supramolecular helical structures are given in Section 3. 2.2.2. Reconstruction of helical filaments The reconstruction of helical 3D structures from individual 2D projection images can in principle be performed either in real space or in Fourier space. For the latter a modified version of the inverse Fourier approach (Fourier Bessel reconstruction) is widely used [46,47]. The high symmetry of a helical object is very advantageous in the sense that all symmetry related projections are combined in one projection image of the helix. Therefore, in the ideal case one single helix segment is sufficient to describe the complete 3D structure. Using polar coordinates in Fourier space (Bessel functions) a set of Z-planes is obtained in which the amplitudes are represented as concentric rings with phases alternating between 0° and 180° describing the helical pitch. Inversion of the 3D Fourier transform eventually gives the 3D structure in real space [48]. The situation is complicated if the helical segments are distorted or bent due to structural flexibility or disorder. Here to some extent a computational straightening can help to overcome limitations [49], but problems might occur due to deviations from the helical symmetry or ambiguous indexing due to an insufficient resolution in the image data. Real space approaches on the other hand have the advantage to circumvent the problems of flexibility and disorder as undisturbed helix segments can be chosen from micrographs under optical control. The 3D reconstruction strategy is based on the finding that one obtains the geometrical equivalent if one either follows the helix' long-axis over a complete pitch or if one turns the helix through 360°. Thus, once having determined the pitch of a helix the 3D volume can be recalculated from one individual projection image. The first example for a 3D reconstruction of a helical structure based on single particle real space image processing was performed with stacked bilayered filaments of a synthetic aldonamide amphiphile [50]. More details are given in Section 3.2. and additional examples for helical dye structures are described in Section 3.3. The approach has been later extended and refined by Egelman [51–53], in particular for

Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007

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the reconstruction of helically organized proteins or amyloids (see Section 3.4.3.). 2.2.3. Cryo-electron tomography In those cases, where 3D information of vitrified specimen cannot be obtained by the single particle approach, which is certainly valid for most synthetic macromolecular structures due to inhomogeneity in their spatial organization, the recording of image series at systematically varied tilt-angles (tomographic series) is an elegant methodical alternative, provided that the accumulation of electron dose does not cause severe damage on the specimen structure. With the availability of highly electron sensitive CCD (charged coupled device) cameras, which allow to record image series of individual biological or organic supramolecular assemblies without seriously damaging of the structure, electron tomography and particularly cryogenic electron tomography (cryo-ET) [54] has experienced an enormous scientific stimulus. Most of the modern TEM's can be equipped with additional tomography software for the controlled recording of tomographic series. Moreover, all major image processing software packages possess evaluation modules for the reconstruction of 3D volumes from tomographic image series, although special tomography packages are also available [55]. The main task of the tomographic reconstruction step is to align the images of a tilt-series with respect to a common tilt axis and to correct for distortions which might have occurred during data acquisition by mechanical instabilities of the tomographic specimen holder. The limitation of the electron dose allowed to be accumulated per individual image might, however, lead to a poor signal-to-noise ratio, which renders the exact alignment of the image series difficult. Here, the addition of fiducial markers (e.g. colloidal gold particles), which provide a high contrast due to strong electron scattering even at low electron dose, can help to obtain a reliable alignment of the image series. The benefit of a reconstruction from a tomographic series therefore is (i) that the angular relation between images is known and (ii) that, similarly to the creation of class sum images the signal-to-noise ratio which is poor in individual images can be improved by the combination of multiple images in the reconstruction. The calculation of the corresponding 3D volume follows the same strategy as applied in the context of the single particle method, where the common algorithm of weighted backprojections is used [56–58]. There is certainly an urgent need for the characterization of samples in three dimensions as the evaluation of single untilted images often opens space for ambiguous interpretations due to the misleading overlap of 3D object features. A vivid example is given in Section 3.4.2., where tomography reveals a surprising simple solution for a complex problem raised by individual untilted projection images. A general overview of the benefits but also limitations by the use of cryo-ET with radiation sensitive soft matter systems is given by Nudelman et al. [59]. A more general review [60] considers relevant aspects for material science and also outlines the potential of X-ray tomography.

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In many cases the bilayer profile in the supramolecular assemblies of amphiphiles can be resolved by cryo-TEM. Even more, if the assembly behavior of more complex molecules such as dendritic amphiphilic calixarene or fullerene amphiphiles is studied, where space demanding head groups and hydrophobic alkyl chains are attached to a rigid platform, structurally persistent micellar architectures with a well resolved core-shell structure can be observed [44]. This implies that density differences between molecular components e. g. the hydrophilic head groups and the hydrophobic core exist so that a structural discrimination can be achieved. The differences in the assembly behavior can be often explained by the packing concept of Israellachvilli et al. [61], who have theoretically rationalized for the first time the interrelation of the amphiphiles' molecular structure and the supramolecular packing geometry. These authors introduced a geometric packing parameter, P (critical packing parameter), relating hydrocarbon core volume v per amphiphile, surface area ao per amphiphile at the hydrocarbon-water interface, and a critical length, lc, roughly equal to but less than the fully extended length (all-trans conformation) of the hydrocarbon chain by the formula P = v / aolc. Depending on the value of the critical packing parameter different supramolecular packing geometries are expected to be stable. Amphiphiles with a packing parameter P ≤ 1/3 should form spherical micelles. For 1/3 b P ≤ 1/2 cylindrical micelles are expected to appear, whereas P N 1/2 promotes the formation of bilayers. The latter condition is usually met for amphiphiles bearing two alkyl chains. Bilayered aggregates can exist in the aqueous solvent as 2D extended sheets with halfcylindrical rims avoiding the contact of hydrocarbon chains to the surrounding water, or they can form closed vesicles. The introduction of chirality in a molecular structure, however, has a considerable and visible impact on the morphology of the bilayered structures [65–68]. The most frequent morphologies of assembled chiral amphiphilic molecules are twisted or helically wound ribbons and cylindrical tubes [69,70], whereby the chirality on the molecular level is expressed by a correspondingly pronounced handedness on the supramolecular level. Usually the directionality of the ribbons twist is defined by the stereochemistry of the compound and assemblies of enantiomers often behave like image and mirror image. Bilayered ribbons or stacks of bilayered ribbons but without regular or preferential twist (with uniform or varying width) have also been found for achiral amphiphiles [65,71], but are less common. Fig. 2 presents sketches of the three principal morphologies preferentially observed with chiral amphiphiles: (i) twisted ribbons (left), characterized by a negative Gaussian curvature, (ii) helically wound (spiral) ribbons (center), characterized by both zero Gaussian curvature and significant mean curvature, and (iii) closed tubular assemblies either with smooth unstructured surfaces (right) or with helical markings that wind around the tube cylinder. The curvature of the tubes can be

3. Self-assembly of amphiphilic compounds towards fibrous aggregates and their characterization by electron microscopic methods 3.1. Morphological aspects Conventional achiral amphiphilic compounds such as single-chained detergents preferentially tend to form simply organized aggregates comprising spherical micelles, cylindrical micelles, or extended 2D bilayered sheets [61]. Although micelles are usually highly dynamic entities with monomer exchange rates to the order of milliseconds, they can, however, be cryo-fixated and visualized by cryo-TEM [62]. Under certain conditions even more complex structures such as ring-like or disk-like micellar assemblies can be found [63,64]. Amphiphiles bearing two alkyl chains usually form vesicles or extended bilayers.

Fig. 2. Principal morphologies of bilayered assemblies formed by chiral amphiphiles and the suggested pathway of their morphological transformation.

Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007

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very high, often exceeding that of vesicles. The geometric parameters of all the structures depend, in general, on the material and the particular experimental conditions of preparation. The extensive study on these highly curved structures started in the 1980s with chiral lipids [8,72]. Since that time different theoretical approaches on the basis of elastic theories were proposed by de Gennes [73], Helfrich [14], Helfrich and Prost [15], Selinger et al. [74], Oda et al. [65], Nyrkova and Semenov [75] and others to explain the mechanisms forcing a membrane composed of chiral molecules to develop a curved superstructure and to tune their properties. These models also tried to predict the supramolecular morphology, the geometric parameters of the structures, such as ribbon width, tilt angle or tube radius, and the conversion mechanism between twisted and helical ribbons, tubes, or vesicles. However, despite the extensive work and the progress in the experimental methods, many details about the structure and the mechanism of formation remained unanswered. Numerous experiments have indicated that the structural motifs represent different states of a time-dependant growth process. In some cases by employing coincident preparation steps these consecutive states of aggregation can be monitored. In other cases different assemblies in different states of aggregation coexist. Also temperature dependent growth processes were observed and appropriate preparation techniques employing controlled ambient conditions allowed to cryo-fixate intermediate morphologies (Section 3.2.). With respect to potential applications in different areas of nanoscience and nanotechnology [76,77], it is highly important to know the molecular factors playing a role in the tuning of shape and dimensions of structures. Therefore the explanation of the mentioned morphological transformations [78,79] is still of great current experimental and theoretical interest [80–86]. Moreover, it is an important prerequisite for applications (in template-directed synthesis, encapsulation, nanofluidic devices, or mesoscopic springs) to obtain assemblies with sufficient morphological stability upon exertion of force [87]. In particular peptide based amphiphiles have been recently shown to produce promising candidates in terms of structural persistence and stability (Section 3.4.). 3.2. Amphiphilic glycolipids Amphiphilic glycoconjugates such as carbohydrate-based surfactants or glycolipids are of particular interest due to the intriguing interaction patterns of their sugar head groups, which add a new quality of complexity to the process of self-aggregation [88–90]. The general molecular structures comprise a combination of saccharidic (e.g. mono-, dior oligo-saccharides with an open chain or cyclic form) and lipidic (single-chained fatty acids or double-chained glycerol glycolipids or sphingo glycolipids) components, that are naturally biosynthesized within living cells, e.g. sophorolipids [91,92] or are synthetically prepared in multiple combinations [88–90]. Sugar head groups with prominent or multiple polar groups undergo complex hydrogen bonding patterns and allow for a similar variety of assembly structures to be formed ranging from micelles, via ribbons and tubes towards vesicles. Moreover, the aspects of an easy accessibility, the biomimetic properties, the applicability for drug delivery or the non-viral gene vectors, as well as ecological compatibility and, last but not least the role in molecular recognition processes have intensely motivated the current research activities. Many of the glycolipids, in particular those with dialkyl chains tend to form liquid crystalline phases in the melt or in solution (nematic, lamellar, cubic, hexagonal phases) [88–90]. Liquid crystal formation, however, is a quite extensive field for glycolipids [90] and beyond the scope of this review. For this review we have singled out examples where individual structurally defined supramolecular assemblies with high structural precision were detected by electron microscopic techniques. In the past decades, the peculiar self-assembly of synthetic glycolipids in

water and organic solvents has been extensively studied. Hierarchical architectures including low molecular-weight gels (LMWG's) and liquid crystals, twisted and helical nanofibers and nanotubes were reported already 25 years ago. Pioneering work in their characterization by the use of electron microscopic methods has been conducted by Pfannemüller and Welte [93], Nakazawa et al. [94], and, in particular, by the Fuhrhop group [95–99]. Recently, the fabrication of artificial nanohelices via supramolecular self-assembly of a synthetic sugar-lipid was reported [100], that could be used as soft template to create silica nanohelices [101]. A comprehensive book dealing with self-assembled fibrillar networks was edited by Weiss and Terech [7]. Of particular interest in this focus area are N-alkyl-aldonamides, a family of open chain glycolipids introduced by Pfannemüller and Welte [93], which were also theoretically examined by Helfrich [14,15]. These compounds combine important molecular features in a very simple and elegant way namely amphiphilicity, chirality and amide (peptide) bonding, all of which play an eminent role in nature for the generation of structurally precise supramolecular assemblies. All three features alone can trigger strong intermolecular interactions leading to self-assembly either through the hydrophobic effect and or by directed hydrogen bonds. The specific benefit of this family of compounds is that the impact of very small changes in the head group stereochemistry on the assembly behavior can be systematically studied. Moreover, in particular the compound n-octyl-D or L-gluconamide provides a very unique and rare example of forming structurally very precise supramolecular architectures. Here for the first time the use of an image based helical reconstruction method allowed for the determination of the 3D supramolecular organization of a helical assembly structure with unprecedented precision (Fig. 3) [50]. Because of this unique status a few more details of the complex assembly behavior of this intriguing compound will be summarized here. The enantiomeric n-octyl-D or L-gluconamides are only soluble at elevated temperature (N 68 °C) in water and form solid gels upon cooling to ambient temperature, respectively. Cryo-TEM images revealed the gels to consist of fibrous assemblies with a structurally complex but very precise periodic structure element. The corresponding image average, calculated by summing roughly 100 aligned individual images, is shown in Fig. 3 [50]. In combination with an independent determination of the fibers' helicity (left-handedness for the D- and right-handedness for the L-enantiomer were found by AFM or by platinum shadowing of the fiber surface) and the length of the helical pitch, a 3D structure was calculated following the image based reconstruction of helices described in Section 2.2.2. The 3D electron density map revealed that the periodic projection motif is generated by a uniformly twisted bundle of four stacked bilayer ribbons. An alternative approach to determine surface morphologies, which are difficult to determine solely from TEM projection images is the use of scanning electron microscopic techniques, in particular in combination with cryo-fixation preparation. Moreover, this technique is most useful for the characterisation of supramolecular assemblies in organic solvents [67], which are not accessible by cryo-TEM. The compound's particular aggregation behavior facilitated kinetic studies [23]. A time-dependent preparation on the minutes scale allowed for the successive visualization of the structural conversion process from spherical micelles over stacked ribbons, helically structured tubes, towards crystals. The studies showed that the stacked helices represent an intermediate state in a multi-step aggregation process, which comprises a slow transition within minutes starting from ordered lamellar structures at elevated temperatures via spherical micelles and stacked ribbons towards helical tubules, before eventually crystallization of the material occurred within a timescale of a few hours. It is worth mentioning that the crystal structure, which had been determined by X-ray crystallography, revealed a completely different packing (head to tail orientation) and even different molecular conformation (all-trans) [102,103] compared to the structural organization in supramolecular assemblies (tail-to-tail orientation and 2G sickle

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Fig. 3. (Left) Formula of n-octyl-D-gluconamide and its prominent molecular features. Right panel: (Top left) Image average of 100 aligned repetitive fiber motifs obtained from cryo-TEM data of n-octyl-D-gluconamide gel (formula left). (Right) Stereoimage of the three-dimensionally reconstructed helical fiber based on the image average shown top left (for more technical details of an image based helix reconstruction see Section 2.2.2.). The left-handedness and the pitch were independently determined by AFM or by TEM imaging of platinum shadowed fiber surfaces, respectively. (Bottom, left) The calculated back projection image of the reconstructed helix bundle demonstrates the conformity with the experimental data top left. Scale is 12 nm. Panel reproduced from Ref. [50] with permission. Copyright (1996) Elsevier Science B. V.

conformation), which was proven by 15N and 13C REDOR spectroscopy studies [104]. In contrast, the stereochemically only slightly different n-octyl-Dgulonamide diastereomer (the main difference is that the 1,3 synhydroxyl groups at C2,C4 position which were responsible for the extraordinary behavior of the glucose isomer is located more to the outer edge of the gulon moiety towards C3,C5 position) which is also completely soluble in water at elevated temperature, does not show an evident tendency to undergo a complex aggregation process, e.g. gel formation. Instead, spontaneous precipitation towards crystals is observed [97]. These clear differences in aggregation behavior can only be explained by stereochemical effects since all other molecular features are identical. It turned out that an intramolecular hydrogen bond between neighboring 1,3-hydroxyl groups, that was only present close to the amide bond in the glucose head group, caused a gauge kink at the C2–C3 bond. The kink enables a spatial displacement between molecules in the assembly, which probably introduces the helical pitch in the assembly structure. The analogous interaction pattern between 1,3-syn-hydroxyl groups at the more outer C3 and C5 position in the case of the gulon derivative can obviously not trigger an corresponding assembly behavior and prevents cooperative interactions towards linear fibrous aggregates. 3.3. Amphiphilic dye aggregates Dyes are conjugated unsaturated organic compounds comprising chromophoric and auxochromic groups. The chromophore is the part of the dye molecule that is responsible for its color, whereas auxochromic groups modify the ability of the chromophore to absorb light. In aqueous solution enthalpically driven attractive interactions between the π-systems of neighbored dye molecules give rise to the formation of stacks of molecules with often dramatic consequences for the optical properties. For 1,1′-diethyl-2,2′-cyanine chloride (pseudoisocyanine chloride, PIC), the prototypical example of the class of cyanine dyes, Jelly [105] and Scheibe [106] were the first to report a new and sharp optical absorption band upon self-assembly in aqueous solution, which is shifted to longer wavelengths with respect to that of monomers. From the appearance of viscoelasticity and the ability to orient the assemblies by flow Scheibe and Kandler [107] concluded a threadlike morphology. These dye assemblies were named J-(Jelly) or Scheibe aggregates in honor of their discoverers. The molecular exciton theory later supplied the explanation for the observed optical properties. Today many synthetic [108,109] molecular dye aggregates are known. However, similar aggregates also appear as functional units in nature, such as in the absorption and energy

transferring parts of light-harvesting complexes in plants or certain types of bacteria (chlorosomes) [110]. In spite of the consensus that the dye aggregates formed in solution are composed of well-ordered stacks of monomers the exact packing on molecular scale remained the subject of much speculation and controversy. Only one early study exists, where TEM (using the negative staining sample preparation technique) was employed to visualize the mesoscopic aggregates of a thiacarbocyanine dye grown from aqueous solution [111]. In the 1990's the group of Tiddy [112,113] described the formation of lamellar and columnar liquid-crystalline phases (chromonic phases) by dilute aqueous cyanine and azo dyes. Here, the equilibrium superstructures of self-assembled solutions of Jaggregates were determined by X-ray diffraction, polarized-light optical microscopy, and NMR, and their architecture was compared with postulated aggregate models. In contrast, Barbara et al. [114] used near-field imaging experiments of polyelectrolyte-bound PIC J-aggregates and detected bundles of fibers with lengths in the micrometer range and ≈160 nm widths composed of smaller fibers of ≈30 nm width. Motivated by the success in the structural characterization of selfassembled amphiphilic aggregates von Berlepsch and Böttcher started to investigate the microstructure of cyanine dye aggregates by cryoTEM [115,116]. Meanwhile also aggregates of porphyrins [117,118] and semi-synthetic water-soluble bacteriochlorophyll (BChl) c [119] could be characterized in this way. Although cryo-TEM cannot generally resolve the aggregates internal molecular organization, it often gives valuable quantitative information on the nanoscale that can be used for the theoretical structure modeling. The achievable resolution is generally better than that of AFM but due to the restriction of cryo-TEM sample preparations to aqueous solutions (with some exceptions [64,120]) dye aggregates formed in organic solution can often only be characterized by AFM on surfaces after removal of the solvent [121–123]. Using cryo-TEM the rod-like morphology of single PIC J-aggregates and for the first time their cross-sectional diameter of ≈2.3 nm could be determined [116]. Moreover, systematic cryo-TEM studies of the morphology of aggregates of several differently substituted derivatives of the 5,5′,6,6′-tetrachlorobenzimidacarbocyanine (TBC) chromophore were performed with the dyes in pure form, after the addition of alcohols, surfactants or other TBC derivatives, and in dependence of preparation conditions, respectively [124]. The studies revealed a rich diversity of aggregate morphologies, ranging from soft 2D molecular layers, quasi-one-dimensional threadlike assemblies up to tubular aggregates. The structures proved often to be hierarchically organized to form more complex superstructures. One of the most fascinating aspects among these dye systems was the appearance of optically active

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helical J-aggregates although the constituting dye monomers were achiral [125]. Besides that, the predominating helix handedness of the pure aggregates could be tuned by the addition of chiral alcohols, as circular dichroism measurements suggested and electron micrographs (after heavy metal shadowing) directly proved [126]. Octyl side chains attached to TBC chromophores introduce an amphiphilic character to the dye molecule. In this way double-layered molecular stacks are generated upon self-assembly in aqueous environment, whereas the octyl side chains form the hydrophobic inner core domain and the charged substituents point towards the solvent. Two main morphologies were observed, namely narrow twisted bilayer stacks and double-layered nanotubes. The absorption spectrum of the latter aggregates is highly complex and up to now is not fully understood. Here, better structural data are desirable and could improve the theoretical description. For the tubular C8S3 (3,3′-bis(2-sulfopropyl)5,5′,6,6′-tetrachloro-1,1′-dioctylbenzimidacarbocyanine) aggregate prominent helical ultrastructural details were detectable in the cryoTEM raw data, which allowed for a more detailed structural differentiation using image averaging and 3D reconstruction techniques [124] as described in Section 2.2.2. Fig. 4 shows a representative cryo-TEM image of the tubular C8S3 J-aggregates (panel a) and a corresponding sum image (panel b) calculated from 180 extracted individual motifs. Following the long axis of the 13 nm wide tubule centro-symmetric cross-like striations at a regular distance of 2 nm are most notable. The tubule wall is characterized by a typical bilayer density profile of 3.6 nm width with an undulating repeat of 2 nm. These geometrical relations suggest that the aggregate is composed of 2 nm wide bilayered ribbons helically enwrapping a central water filled channel. In panels (c) and (d) the reconstructed volume of the helical tubule is shown. For a non-obscured view of the inner layer one quarter of the outer layer was removed. The molecular dimensions remain, however, still well below the resolution achieved, but the detected helical

Fig. 4. 3D reconstruction of the tubular C3S3 J-aggregate based on the real image approach described in Section 2.2.2. (a) Original cryo-TEM raw image data of tubular assemblies. (b) Class sum image based on alignment and averaging of several tens of structurally identical segments chosen from different tubular assemblies shown in (a). (c,d) Surface rendered oblique (c) and side (d) view (outer layer removed for a detailed view) of the reconstructed 3D volume. Reproduced from Ref. [124] with permission. Copyright (2012) World Scientific, Singapore.

arrangement of the dye molecules represents a sound basis for the theoretical modeling of the optical properties [127,128]. Chlorosomes are natural light-harvesting systems found in green sulfur bacteria. They contain heterogeneous molecular mixtures of bacteriochlorophyll (BChl) c, d, or e with different side chain modifications and different stereochemistry. Because a heterogeneous structure cannot be determined by crystallographic methods, NMR and TEM techniques were used for a structure analysis. In particular cryo-TEM studies provided a wealth of new knowledge about the chlorosomes' structure in recent years. The first highly resolved cryo-TEM images pointed to a lamellar organization of BChls [129]. Later studies revealed that the BChl layers are organized as coaxial cylinders or rolled-up sheets [130–132]. These conflicting models emphasize the need for further structural data to obtain a consistent model that can explain the molecular ordering and resulting spectroscopic signals. 3.4. Amphiphilic amino acids, peptides and amyloids 3.4.1. Amphiphilic amino acids Amphiphilic amino acids represent an ionic complement to the above reviewed non-ionic aldonamides (Section 3.2.) due to a pH sensitive carboxylate functionality. A prominent example is N-dodecanoylserine, which has been extensively examined by cryo-TEM. Serine provides an additional hydroxyl group in C2 position close to the stereo center capable of forming additional intermolecular hydrogen bonds. The terminal carboxylate group ensures sufficient water solubility and is adjustable by the pH. Interestingly, N-dodecanoyl-serine not only self-assembles in aqueous media but also indicates self-assembly by forming gels in toluene [133,134]. Toluene is one of the few organic solvents which can be vitrified by cooling in liquid nitrogen forming a glass-like solid matrix very much like vitrified water and thus allowing direct investigations by cryo-TEM [120]. In this way the assembly process in solvents with reversed polarity can be compared (Fig. 5). Even though multilayered tubes are eventually generated in both solvents, where inverted packing geometries (tail-to-tail orientation in aqueous and head-to-head orientation in apolar solvent) can be assumed, the initially formed assembly structures are different. Whereas spherical multi-layered vesicles are formed in toluene, twisted multi-layered ribbons, however, are represented as precursor structure observed in water. It can be surmised that directional growth by formation of water moderated intermolecular hydrogen-bonds is crucial in the polar environment but not in apolar toluene, where vesicle formation due to a phase separation is dominant. Currently, small amphiphilic molecules gain vast importance as lowmolecular-weight organogelators (LMOG's) [7]. LMOG's are potential candidates for applications in pharmaceutical, food and cosmetic formulations, catalysis or the controlled synthesis of nanostructured materials [135,136]. However, as Barclay et al. [137] put it “yet the predictability of structure function relationship is still at the rudimentary level for the complex nature of the subject.” Therefore, today's researchers are still trying to work out the general principles of supramolecular aggregation phenomena, sometimes falling back on compounds being as “simple” as N-dodecanoyl-serine [135–140]. 3.4.2. Amphiphilic peptides Amphiphilic peptides attracted immense interest in recent years due to their outstanding potential for biomedical and bio-nanotechnological applications. Two main classes of peptides can be thereby considered: (i) pure peptidic systems with amphiphilic properties arising from specific sequences of hydrophobic and hydrophilic amino acids and (ii) peptides modified by the synthetical introduction of hydrophobic chains, termed peptide amphiphiles (PAs). Several tens of amphiphilic peptide systems have been synthesized until now and their selfassembly behavior has been reviewed several times [141,142]. Fibrils are the most commonly observed self-assembled structures

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Fig. 5. Cryo-TEM micrographs of dodecanoyl-L-serine assemblies in water (a–c) and in toluene (d). The aqueous solution shows different states of aggregation starting from multi-layered ribbons (a), which subsequently grow in width (b) towards closed multi-layered tubes (c). In toluene multi-layered ribbons appear next to smooth multi-layered tubes. The repetitive layer thickness agrees in all cases with the dimension of a molecular bilayer. Adapted with permission from Ref. [133]. Copyright (2001) American Chemical Society.

for amphiphilic peptides, but tape-like, tubular and vesicular structures have also been reported. For the class of peptide amphiphiles self-assembly towards β-sheet containing nanofibrils is particularly common. The folding (or refolding) of peptide chains into highly specific and functional three-dimensional protein structures by cooperative electrostatic and hydrophobic interactions is the most versatile example of biological self-assembly [143]. Many functional proteins can, however, spontaneously misfold e.g. into β-sheet tapes, referred to as amyloids that are associated with a range of increasingly prevalent clinical disorders, including Alzheimer's disease, type II Diabetes, transmissible spongiform encephalopathy or Parkinson's diseases. This is an eminently important area of current scientific activity in life and medical sciences [144]. With respect to their aggregation behavior and typical structural motifs synthetic amphiphilic peptides and natural amyloids show many similarities. Though the formation process of amyloids is still not fully clear, the de novo design of peptides forming amyloidlike structures [145] has proved to be an elegant strategy for studying the principles of amyloidosis. The characterization of amyloids is the subject of a separate section (Section 3.4.3.). The potential of the new peptide-based materials for applications in bio-nanotechnology and the importance of amyloids in medical science have considerably stimulated the structural research. Hereby, cryo-TEM has emerged as the premier technique for real-space investigations. Some questions, such as the morphological transition from ribbons to tubes have recently been resumed and will therefore be reviewed in the following paragraphs. Hamley and co-workers [146] investigated small amphiphilic peptides containing the fragment KLVFF (for convenience, the peptide sequence is denoted with the established single letter abbreviation for the amino acids) of the amyloid β-peptide that has been identified as a key sequence involved in β-sheet formation. They synthesized an end-capped heptapeptide (CH3CONH–βAβAKLVFF–CONH2) which shows six different intermediate states over a period of four weeks from the early aggregation into isotropic micelles, the growth towards flat ribbon-like structures, twisting and subsequent coiling into helical topologies and the eventual closure into nanotubes. These transient states could be resolved as a function of incubation time using cryoTEM, AFM and SAXS. The morphological investigations revealed that the end-capping of the KLVFF fragment had a strong influence on the self-assembly of this peptide. AAKLVFF, the KLVFF fragment extended

by two alanine residues, self-assembled into twisted fibers in aqueous solution [147]. The addition of NaCl produced large crystalline nanotapes [148]. In contrast to AAKLVFF, the addition of large amounts of NaCl to βAβAKLVFF enhanced the fibril's twist and induced the formation of nanotubes [148]. A further capped version of βAβAKLVFF showed the formation of flat ribbons at intermediate salt concentrations due to the screening of electrostatic interactions [149]. The Stupp group synthesized hexadecanyl peptide amphiphiles, C16PAs, which form nanofibers upon self-aggregation [150]. For such a PA, containing three phenylalanine residues, a morphological transition was detected by cryo-TEM from short twisted ribbon segments into long twisted ribbons and later into helical ribbons [151]. An alternating sequence of a hydrophobic (V) and a charged amino acid (E) in the PA (C16-(VE)2) produced non-twisted ribbons, however, with dimensions of over 100 nm in width, 5 nm in thickness, and several micrometers in length [152]. Elongating the amino acid sequence by two further units ((VE)4) gave ribbon-like structures [153] that were weakly twisted and showed only ≈40 nm in width. The respective PA with six units ((VE)6) forms a mixture of cylinders and narrow ribbons. Here the transition point between flat and curved supramolecular structures was obviously crossed. All these studies revealed a definite relationship between pitch and width of ribbons. Quite recently a C16-KKFFVLK peptide amphiphile was designed [154] that self-assembles into nanotubes and helical ribbons at room temperature, but remarkably, showed an unwinding transition upon heating that led to the formation of twisted tapes. Upon cooling, the nanotubes and ribbons reformed. Ziserman et al. [155,156] introduced a new library of so called pseudopeptides consisting of alternating acyl chains and the cationic lysine termed oligo-acyl-lysines (OAKs) [157] and performed time-lapse cryo-TEM studies on the nanotube formation. The OAKs comprise all prime motifs required for nanotubes formation, i.e. amphiphilicity, chirality, capability of forming hydrogen bonds, as well as hydrophobic chains that, depending on the solution conditions, may behave as hydrophobic spacers. The cryo-TEM investigations revealed unequivocal evidence for the link between ribbon width and subsequently evolving structure. In agreement with expectations from theory [81,158] narrow chiral ribbons favor Gaussian saddle-like twisted curvature, while the cylindrical curvature becomes favorable for wider ribbons. The twisted ribbons proved to be the precursors for coiled ribbons that subsequently develop into nanotubes as sketched in Fig. 1.

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As frequently mentioned, precise supramolecular structures are rare, so that we usually have to stick to image interpretation of singular events. In particular the latter aspect is prone to misinterpretation if the structure is complex and additional spatial information is not obtainable. In this case, tomographic information can be helpful. An instructive example is given in the context of studies on the biosufactant surfactin [159,160], a highly surface active natural PA with a cyclic hepta peptide head group linked to a β-hydroxy fatty acid, which strongly interacts with membranes. Biosurfactants in general are a subject of intense research, because of their potential for unique biotechnological and medical applications [161–165]. Surfactin forms on its own spherical micelles in aqueous solution [166–169]. The concentration dependent activity of surfactin on the DMPC vesicle membrane structure was recently studied by cryo-TEM [170,171]. In the lower concentration range, the phospholipid vesicle membrane is first degraded towards membrane fragments [170], but at higher concentrations a multitude of different structures (Fig. 6), such as rings, entangled networks and rod-like assemblies appeared [171]. Cryo-electron tomography of the latter, however, revealed that all these putative different structures are different spatial views of one supramolecular motif, namely flexible linear fibrous assemblies with a constant diameter of 6.5 nm, probably consisting of a surfactin/DMPC mixture which tends to form loop-like assemblies of different size and shape. Fig. 6 provides a series of three selected images taken from a reconstructed volume at different tilt-angles based on a corresponding tomographic series. Cryo-ET reveals that the variations in the loop diameter and the loop flexibility both obscure the common motif present in all assemblies. 3.4.3. Amyloids Although their soluble precursor proteins widely differ in the primary structure, insoluble amyloid fibrils share some common features [172]. They all appear to be straight and unbranched with diameters of several nanometers and lengths in the range of micrometers. Early morphological studies using TEM [173] or AFM [174] showed that amyloid fibrils commonly consist of a small number of protofilaments with typical diameters ranging from 20 Å up to 50 Å. Circular dichroism spectroscopy reveals a high β-sheet content of the fibrils. X-ray fiber diffraction shows a characteristic anisotropic pattern, the so-called cross-β diffraction pattern, consisting of a meridional reflection at 4.7 Å, corresponding to the hydrogen-bonding distance between the β-strands in a β-sheet, and an equatorial reflection around 8–12 Å, attributed to the spacing between the β-sheets stacked within the fibril. Recent X-ray crystallographic studies on a crystallizing seven-residue fibril-forming peptide have revealed so-called steric zippers [175,176]. Steric zippers consist of a pair of two cross-β sheets with interdigitated side chains and constitute the structural spine of the amyloid fibrils. A thorough understanding of the hierarchical assembly of peptides and proteins

into fully-formed fibrils has been limited so far by the absence of a complete atomic-resolution structural analysis. Structural analysis is difficult because crystals are usually not obtained and the amyloid fibrils display structural polymorphism even on the structural level of steric zippers [175,176]. During the last decade cryo-TEM and solid-state NMR spectroscopy (ssNMR) have become standard methods for the structural characterization of amyloids with the potential to determine the structure at atomic or near-atomic resolution. ssNMR yields atomic constraints that can be used for the construction of models with atomic precision. This technique, however, averages over different coexisting polymorphic structures. Cryo-TEM combined with 3D reconstruction techniques, on the other hand, enables a quantitative characterization of specific fibril morphologies even for a few individual fibrils and thus avoids problems of intra-sample polymorphism. Recent studies comprise the amyloid fibrils formed by the SH3 domain of phosphatidylinositol-3′-kinase [177], insulin [178], β-PrP′ [179], HET-s (218–289) [180,181], α-synuclein [182], β2-microglobulin [183], and the two β-amyloid alloforms Aβ(1–40) [184–186] and Aβ(1–42) [187,188]. While the resolution of the electron density map is normally lower than the distance information provided by ssNMR [189], nearatomic resolution of 0.5 Å has been reached for the first time in a study combining magic angle spinning NMR, X-ray fiber diffraction, cryoTEM, SEM, and AFM for amyloid polymorphs of an 11-residue fragment of the protein transthyretin TTR (105–115) [190]. The Fändrich group intensively studied amyloid fibrils of the β-amyloid (Aβ) peptide (known as a component of amyloid plaques which are associated with Alzheimer's disease) under physiological pH conditions. Based on the best data sets a resolution of 0.8–3 nm was reached, which provides information about the global fibril architecture, protofilament substructure, location of the cross-β structure within the fibril helix and allows construction of structure models (cf. reviews [191,192]). According to their investigations, mature Aβ fibrils often appear to be twisted, generally left-handed and with a two-fold axial symmetry. Although sample solution preparation conditions are identical, the structural polymorphism is immense. More than 10 differently structured fibril reconstructions were received just for Aβ(1–40). Moreover, there are differences between the protofilaments of its two alloforms Aβ(1–40) and Aβ(1–42). In general, three distinct types of polymorphism were observed: The fibrils differ in (i) the number of protofilaments, (ii) their relative orientation, and (iii) in the internal protofilament substructure. Fig. 7 shows two structure models of Aβ amyloid fibrils derived from cryo-TEM data. In contrast to the ribbon-like fibrils described by Fändrich and coworkers another cryo-TEM study [188] revealed fibrils with tubular shape and hollow core for Aβ(1–42) at low pH. By simulating tubular models and fitting NMR data [189] to the electron density maps atomic

Fig. 6. Mixture of surfactin (30 mol%) and DMPC. Three images at different tilt angles (−18°,0°, +18°) selected from a 3D volume reconstruction based on a tomographic tilt-series. The images were slightly band-pass filtered for a better visibility. The individual images show structures of different size and complexity, such as rings (straight arrow), networks (dashed arrow) or rods (dotted arrow). Following the image series it becomes apparent that despite the structural differences all assemblies consist of micellar threads with a diameter of 6.5 nm which form loops of different size and flexibility. A more accurate resolution can be observed in an appropriate presentation of the complete reconstructed volume. Scale bar is 100 nm. Reproduced from Ref. [171] with permission. Copyright (2010) Elsevier Science B. V.

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Fig. 7. Structural models of Aβ amyloid fibrils derived from cryo-TEM images. Surface representation of two reconstructed electron density maps (gray) and structural models of the cross-β sheets (color). Left: views of cross-sectional slices of the electron density; right: side views. Reproduced from Ref. [192] with permission. Copyright (2011) Elsevier Science B. V.

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ultrastructure with high spatial resolution and, by the use of the cryoTEM method, even in the native state of aggregation. With the recent developments in TEM instrumentation and methodology as well as advances in generating structurally more precise supramolecular assemblies the possibility arose for a more detailed characterization of their 3D structure down to the nanometer scale by analyzing data sets consisting of a few thousand copies of an assembly in different spatial orientations. The approach is easiest for fibrous structures possessing a helical ultrastructure, because only one noise-free projection image is required due to the axial symmetry. For the 3D structure determination of inhomogeneous assembly populations, however, tomographic approaches have experienced an enormous propagation in recent time. Based on selected examples, where structural information has been retrieved with unprecedented precision, this review illustrates how TEM has revolutionized our knowledge about the formation and mesoscopic structure of diverse fibrous supramolecular systems. The selection ranges from gel-forming helical lipid aggregates over tubular dye architectures up to molecular structures of cross-β amyloid fibrils. Acknowledgment

models of the tubular filament could be obtained [193]. Only models with C termini facing the external surface of the fibril retained the hollow core with similar dimensions to those observed in the experiment. The simulations further revealed that although the tubular structure was also stable at physiological pH, it was less populated than other polymorphic states. A quite recent negative stain TEM study revealed a tubular helical structure in Aβ protofibrils [194]. Three-dimensional analysis of single particle and tomography data generated a triple helix of density wound around a hollow core. The reconstruction differs from that of matured tubular Aβ amyloid fibrils [188]. Both have a similar width, but differ in the helical pitch. This newly discovered tubular order in Aβ protofibrils could have relevance for Aβ toxicity in Alzheimer's disease. Besides for native and synthetic amyloid fibrils cryo-TEM has also been used to reveal the nanostructure of different non-amyloid fiber forming peptide systems. Thus, micrometer long and only few nanometer wide α-helical coiled coil nanofibers have been detected that are formed by self-assembly of short de novo designed model peptides [195,196]. The peptides can find applications as bio-inspired materials in different fields of bio- and nanotechnology and some proved to be useful model systems to study the inhibition of amyloid aggregation [197]. Among them is a so-called self-assembling fiber system (SAF), which was designed to form thick fiber bundles upon aggregation [198]. The quasi-crystalline packing of constituent α-helical fibers allowed a 3D reconstruction based on cryo-TEM data and the determination of the packing structure at an approximate resolution of 0.8 nm. Valuable quantitative measurements from amyloid assemblies can be retrieved by the use of dedicated scanning transmission electron microscopes (STEM). The linear relationship between the number of elastically scattered electrons and the mass of an irradiated area for example allows for visualization as well as measurement of the mass-per-length (MPL) of individual filaments. This approach sheds light on the polymorphism of fibrils and yields valuable quantitative packing constraints [199,200].

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Please cite this article as: Berlepsch H, et al, Progress in the direct structural characterization of fibrous amphiphilic supramolecular assemblies in solution by transmission el..., Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.01.007