Modern X-ray scattering studies of complex biological systems

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Modern X-ray scattering studies of complex biological systems M Kornreich, R Avinery and R Beck X-ray scattering is one of the most prominent structural characterization techniques in biology. The key advantage of Xray scattering is its ability to penetrate and weakly interact with the bare studied materials. In addition, X-ray scattering does not require any tags, markers or modification to the sample under examination, and is not limited by the nature of the surrounding environment. The main handicapping limitation of X-ray scattering is the subject of particles polydispersity. However, the monodispersity in biological complexes and supra-molecular interactions makes them ideal for structural and interaction studies in particular when combined with higher (e.g. NMR) and/or lower resolution (e.g. optical microscopy) techniques. This review seeks to highlight some of the major recent achievements in the field of X-ray scattering as being implemented for complex biological systems. Address The Raymond and Beverly Sackler School of Physics and Astronomy, Tel-Aviv University, 69978 Tel Aviv, Israel Corresponding author: Beck, R ([email protected], [email protected]) Current Opinion in Biotechnology 2013, 24:716–723 This review comes from a themed issue on Nanobiotechnology Edited by Michael C Jewett and Fernando Patolsky For a complete overview see the Issue and the Editorial Available online 19th Junuary 2013 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.01.005

transform of the electron density, r (r), with the scattered transfer vector q [2]: Z Escattered ¼ A rðrÞeiqr d r Here, q is related to the scattered angle, A is a known coefficient which depends on polarization, particle concentration and experimental setup. X-ray detectors can only measure the intensity of the electric-field; therefore, the measured intensity is: Z 2 2 IðqÞ ¼ jEscattered j ¼ jAj j rðrÞeiqr d rj2 This brings an inherent problem to scattering experiments, as the phase information is lost in the diffracted image, and direct reconstruction of the electron density is not unique. However, as will be discussed in the last section, phase information can be retrieved through iterative algorithms and the use of a coherent X-ray source. Assuming mono-disperse particles, the average scattering amplitude can be simplified to: Iðq; cÞ ¼ N p V 2p Dr2 jFFðqÞj2 SFðq; cÞ Here, c is the particle concentration, Dr is the excess electron density in a particle and Np and Vp are the number of particles and single particle volume respectively. The FF (q) function is the form factor which represents the electron density distribution within the particles, and SF (q, c), known as the structure factor, represents the inter-particle correlation. For nonuniformly oriented particles the measured scattering intensity is an average over all orientations.

Overview Over a century after the discovery of Wilhelm Roentgen, and over half a century since its use by Watson and Crick, modern X-ray techniques are once again revolutionizing materials and biological sciences. Below, we will begin with a brief theoretical introduction to scattering theorem and contemporary data analysis. Then, we will continue by reviewing how scattering measurements are currently used to study bio-molecular interactions. Modern technological advances will follow. We will finish with a short introduction to the upmost promising techniques using coherent scattering, which opened a novel and exciting era to X-ray scattering techniques.

Scattering theorem At X-ray energies below 10–20 keV the dominant scattering mechanism is due to elastic scattering from the electrons in the sample. The scattered electric field is composed of both amplitude and phase through Fourier Current Opinion in Biotechnology 2013, 24:716–723

Data analysis Today, a plethora of analysis tools for processing scattering measurements exists. In most synchrotron beamlines, a general analysis scheme using these tools is automatically processed for the benefit of the non-expert user [3,4]. Common tool categories include datareduction [5], data-manipulation [6], data-analysis [7,8,9], and ab initio modeling [10,11] to name only a few. These tools and their use in analysis and modeling are elaborated in recent reviews [12–15]. Continuing growth of computing power gives rise to more computationally intensive analysis methods, this being a clear trend in modern analysis of X-ray scattering. For example, small angle X-ray scattering (SAXS) of dilute protein solutions contains almost featureless scattering intensity curves. Up until few years ago, only global average parameters, such as the radius of gyration and www.sciencedirect.com

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Figure 1

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Increasing magnesium concentration Current Opinion in Biotechnology

An illustration of scattering curves of charged particles with increasing amounts of screening magnesium. (a) Schematic particle picture. Left: some orientational order due to an overall repulsive interaction. (Center) Non-interacting particles are randomly distributed. (Right) Particles aggregate. (b) Corresponding scattering curves I (q) on the various particle distributions. The blue curve is the form factor scattering intensity, when normalized for concentration. Figure adapted from [23].

the protein MW, could be extrapolated from the scattering curves at zero angle. Recently developed methods implement an iterative optimization approach, which resolves scattering measurements of flexible proteins in terms of an ensemble of conformations traversed by the measured nano-scale complexes (e.g. proteins) [16–18]. These methods, which are of particular interest to the study of intrinsically disordered proteins, are thoroughly reviewed and discussed [19]. Similar ensemble description is also gained by coarse-grained molecular dynamics (MD) simulations, with reasonable computation time [20]. Extending this approach, X-ray scattering can be used to verify all-atom MD simulations [21]. Another novel experimental scheme implemented 3D computer tomography along with X-ray scattering to measure nm-scale repeating structures within a rat’s brain, thus generating a 3D map of known macromolecular structures [22]. The common denominator to all of the methods is the combination of some a priori structural information, or prediction, to directly fit the measured scattering curves to computed conformations. Using these methods, we gain additional statistical and structural information on the measured biocomplexes. We anticipate that the increasing computing power will provide the possibility to incorporate more accurate physical and structural constraints, thus promising new insights into www.sciencedirect.com

the structure of biological systems measured in solution in the near future.

Bio-molecular interactions Careful SAXS analysis can resolve both attractive and repulsive interactions, the structure factor and the form factor ([24–27] and Figure 1). For example, in dilute conditions the dominant scattering is that of the form factor. At higher concentrations, and for stable particles, the structure factor can be extracted as well [27]. Analysis of the structure factor at low angles reveals the overall intra-particle interaction through the 2nd virial coefficient, A2 [27]. This is used in studying aggregation processes [28], and in predicting crystallization [26]. SAXS study of IgG2 antibodies has lately shown to reveal conditions for attractive and repulsive interactions, both at short range and long range [28]. These findings were compared with several techniques, such as circular dichroism, fluorescence, size exclusion chromatography and dynamic light scattering. Alternatively, the structure factor can be calculated by the Ornstein–Zernike equation, coupled with a closure equation and chosen pair potentials [26,29,30]. The parameterized potentials are fitted to the experimental structure factor curves. Common potentials used include Current Opinion in Biotechnology 2013, 24:716–723

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combinations [30] of one or two Yukawa potentials [28,31]; Van der Waals attraction, repulsive screened coulombic interaction and hard spheres [30,32,33]. Among the parameters to be optimized in the fitting procedures are volume fraction, hard-sphere radius and surface charge. Most applications of the parameterized potentials are still limited to model systems, but are currently used to investigate the effects of many variables: pH [34], macromolecules concentration [31,32,35], pressure [36], ionic strength and specific salts [32,33,35]. By contrast, some novel methods do not pose the requirement of a priori interaction potential models [37]. Such methods produce the pair correlation function, g(r), using indirect Fourier transformation of the structure factor. This approach may be beneficial when the inter-particle potentials are more complex, as in the case of biological complexes.

An alternative approach is to examine samples under varying conditions, while deducing the forces and interactions directly from the resultant structural changes measured [1,38]. Variations include environmental modifications (such as pH, salts, temperature, solvent polarity) or sample composition (concentration of the different components, e.g. protein, lipids). Measured electron densities, phase diagrams and structures are then translated into an understanding of the forces and interactions governing complex systems. An example is given in Figure 2 where the modulations of supermolecular structure and interaction of neurofilament hydrogel are studied by X-ray scattering under osmotic pressure [1]. The high penetration of X-ray enables an experimental setup measuring nano-structures on macroscopic hydrogel samples. The ability to measure the sample in solution allowed for environmental modifications, of both ionic strength and osmotic pressure. In this case,

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SAXS measurements on neurofilament hydrogel at several ionic strengths and osmotic pressures. (a) Scattering curves of the hydrogel in solution at varying polyethylene glycol wt%, which induces osmotic pressure that compresses adjacent filaments. Perpendicular lines mark the average reciprocal inter-filament distance (q = 2p/l). The inter-filament distance appears in the cross-section schematic illustration in (c) and (d). (b) Phasediagram shows high-osmotic-pressure transition at 10 kPa from expanded state to condensed state which illustrated in (c) and (d). The transition is explained by inter penetrations of the disordered polypeptide brushes at high pressure.Adapted from [1]. Current Opinion in Biotechnology 2013, 24:716–723

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SAXS successfully provided structural information on a wide range of 200–800 nm, and the relations to the intermolecular interactions of the peptides. Prominent example systems are multiple-component lipid-based systems. X-ray scattering is a powerful tool to study the strong Bragg diffraction of lipid mesophases and associated bio-molecules interacting with them. Lipid systems can demonstrate the main advantages of X-ray scattering. The wide range of layer length-scales found in these systems is easily covered by X-ray measurements. For example, hydrocarbon tails ordering is on the subnanometer length-scale, while bilayer separation is typically few to tenths of nanometers. In view of possible applications, such as drug delivery, non-intrusiveness is very important [39–43,44]. In-solution measurements enable an investigation of the interactions determining the measured Bragg peaks. Therefore, X-ray scattering is a key technique to study lipid interactions, membrane elasticity and ion adsorption at various conditions [45–47]. X-ray scattering studies include numerous structural and controlled-release mechanisms for drug-delivery of lipidDNA complexes [48,49], nanoparticles composed of copolymers, anticancer drugs and cholesterol [50] and dendrimer–siRNA complexes [51], to name only a few. For example, a recent study reported the formation of liquid-crystalline phases of short double-stranded DNA confined between two-dimensional cationic lipid bilayers. The order and liquid-crystalline phase transition were found to be strongly dependent on the DNA length and the over-hanging single strands. Moreover, an increase in the DNA to lipid mole ratio in the complexes was achieved in a columnar nematic phase of end-to-end stacked short DNA [49]. Such effective packing of short DNA in cationic liposomes may have important implications on delivery systems for gene therapy [39]. X-ray scattering was optimal for this study, as it allowed for minimum intervention and in solution measurements, both crucial in achieving a more representative physiological conditions. Structural information was obtained simultaneously on the lipid bilayer spacing and the short DNA packing. Another large family of biological systems of interest consists of polymer networks and polymer brushes. This includes recent studies on protein adsorption and interaction between cytoskeleton filaments [1,52–56]. Last, a comprehensive review of ion–nucleic acid interactions studied by SAXS [23] is now available, with examples of anomalous SAXS.

Modern technological advances With many third generation synchrotron radiation sources available around the world, and more under construction (http://www.lightsources.org/), X-ray scattering has become a ubiquitous tool for the characterization of www.sciencedirect.com

materials of all kinds. The frontier lies with the possibility to produce X-rays with higher intensity and coherency, and to be able to measure with higher time resolution. These properties will experience tremendous improvement by using the next generation radiation sources based on the X-ray free electron laser (XFEL) technology [57]. One significant recent achievement in this area is the ‘self-seeding’ procedure, which promises a new level of all three mentioned properties [58]. Another recent development in XFELs is about delay lines providing a time-scale enhancement that allows ultra-fast Xray photon correlation spectroscopy measurements [59]. For state of the art in XFEL experiments, with emphasis on applications for biology, see recent reviews [57,60]. In-house scattering technology has not been left behind, with recent advancements such as scatterless slits [61] which reduce parasitic scattering, and a new type of liquid-metal jet anode design which is expected to yield a 10-fold increase in brilliance [62,63]. This new X-ray source is expected to give great benefits for medical imaging as well as for materials research [64,65]. Figure 3

Camera Lysozyme crystal

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A confocal microscope built into one of CHESS synchrotron facility X-ray beamlines. Key components of the setup are noted. This setup was used to align a microscopic lysozyme crystal for diffraction.Image adapted from [71]. Current Opinion in Biotechnology 2013, 24:716–723

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A complementary evolution has been seen in 2D X-ray detectors, with new specialized large area detectors developed for increasing acquisition rate (as high as 22 kHz), combined with spatial resolution of 75 mm and virtually no dead-time [66]. Such high acquisition rates reduce sampling bottlenecks for XFELs and also allow the measurement of short-lived time correlations within the sample of lower than 1 ms [66]. Fast time evolution can also be measured by flow-cell techniques using mixing chambers which allow scattering measurement of reactions with resolution as high as a few milliseconds for stopped-flow experiments and submillisecond resolution for continuous-flow experiments [67].

electron microscopy to resolve self-assembly [70]. This integrative modeling is further discussed in [15]. A related trend is in device integration. For example, many synchrotron X-ray beam-lines incorporate microbiology tools, such as simultaneous use of optical microscopy (see Figure 3) [71,72] and measurement of just-purified protein using a high-performance liquid chromatography machine [73]. Other methods such as microfluidic devices allow researchers to design complicated experiments, with closed feedback loops, being constantly measured and analyzed by an X-ray scattering apparatus [74].

Coherent scattering Recent breakthroughs in coherent X-ray experiments are opening new horizons in biological, materials and medical research. Different setups enable real-space, reciprocal space, and synergetic methods. Examples of real-space experiments include transmission X-ray microscopy, scanning transmission X-ray microscopy,

One of the promising trends in X-ray scattering is the integration of instruments and data-analysis with other complementary methods. Such recent examples include the combination of SAXS with NMR [68] or FRET [69] to resolve protein complex solution structure, and with

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Reconstruction of a whole yeast cell using coherent X-ray diffraction microscopy. (a) An example of a single 2D diffraction image measured. The inset shows the missing center is confined within the centro-speckle. (b) The corresponding real space reconstructed projection. The arrow points at the spore wall. (c) A 3D image, obtained by tomographic method from the 2D projections. The dotted square marks a possible spore line protruding from the spore surface. (d) SEM image of a similar yeast spore (Scale bar: 500 nm).Adapted from [75]. Current Opinion in Biotechnology 2013, 24:716–723

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X-ray differential interference contrast, and forwardscattering phase-contrast imaging [76]. Coherent X-ray diffraction imaging (CDI), first experimentally demonstrated in 1999, has revolutionized X-ray experiments and opportunities. CDI can solve the aforementioned phase problem of a diffraction image by an iterative algorithm and oversampling [77,78]. Therefore, much more information on the sample is obtained than in conventional diffraction experiments, and the structure can be determined with few and simple a priori constraints such as the scattering area of the specimen. The method can be applied to micrometer-sized samples including whole cells [75], exploiting the long penetration of hard X-rays compared to electrons.

2. Als-Nielsen J, McMorrow D: Elements of Modern X-ray Physics.  New York: Wiley; 2001. A comprehensive and yet a most elucidate book, including most applications of X-ray physics and fine explanations of X-ray fundamentals. It is the best starting point for those who wish for an introduction to almost any topic in the X-ray sciences, including those mentioned in this review

The first reconstructed image of a biological specimen was performed in 2003, and reached 30 nm resolution [79]. A 11–13 nm resolution was achieved for goldlabeled cryogenically cooled yeast cells [80]. 3D reconstructions are also possible by tilting the sample and collecting 2D diffraction images. Unstained yeast spore cells were imaged with 50–60 nm resolution [75] (see Figure 4), along with their cellular organelles. Moreover, a protrusion was noticed on the reconstructed yeast spore, suggesting the spore germination process. Another method, called Ptychographic CDI, combines scanning X-ray microscopy with CDI [77,78]. It does not require an isolated object, and thus can produce images of extended objects, such as a cortical bone specimen taken from the mid-diaphysis of a mouse femur [81]. Last, the newly emerging technology of XFEL may allow for an in-solution, ab initio structure measurement of biological samples by overcoming the radiation damage problem. It was theorized that a sufficiently short femtosecond pulse can produce coherent diffract-and-destroy single particle measurement, without measurable radiation damage [82]. A preliminary experiment already demonstrated a coherent single-shot imaging of a minivirus [83]. The upcoming opportunities of XFEL including serial crystallography [84,85] and ankylography [86,87] were recently reviewed in [57,60].

Acknowledgments The authors would like to acknowledge the generous support of the Israeli Science Foundation (Individual Research Grant 571/11), the European Community’s 7th Framework Programme (293402) and the Sackler Institute for Biophysics at Tel Aviv University.

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