We acknowledge the financial support for the project from the French grant REPLIPOX ANR-13-BSV8-0014 and by research grants from the Service de Sant&#233; des Arm&#233;es and the D&#233;l&#233;gation G&#233;n&#233;rale pour l&#39;Armement. We are thankful to the ESRF for the SAXS beam time. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). We thank Wim P. Burmeister and Fr&#233;d&#233;ric Iseni for financial and scientific support and we also thank Dr. Jesse Hopkins from BioCAT at the APS for his help and for developing BioXTAS RAW.

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The authors have nothing to disclose.

It is desired to have a monodisperse sample before starting a SAXS experiment, but in reality, many data collections do not satisfy this and must be improved by combining the measurement with inline chromatography&#8212;SEC in most cases. However, even the shortage of time between purification and data acquisition monodispersity of the sample is not guaranteed. Most commonly, this applies to experiments where components are too close in size or in their physical properties to be separated or are prone to fast dynamics. Here, we have provided a protocol combining single value decomposition with evolving factor analysis to remove the influence of DNAbound E9 exominus from its unboundform creating a monodisperse scattering profile that we were then able to analyze with the SAXS package Scatter IV.
SVD with EFA of SEC-SAXS data are very powerful methods developed to deconvolute SAXS data and improve analysis, but they do have limitations. They require that noise or drift in the buffer baseline of the SEC-SAXS is kept to a minimum. This may involve extra column equilibration (better to use more than 3 column volumes, depending on the buffer) before sample loading. However, the most critical step is the choice of the number of the singular values and the range of data used, as this will greatly affect the accuracy of the deconvolution. It is for this reason that the results should not be taken on their own but further analyzed using techniques such as analytical ultracentrifugation (AUC) or multi-angle-laser-light-scattering (MALLS) for biological interpretation.
Scatter IV is a new, software package, free for research and industrial use with an intuitive user interface that allows even non-experts to analyze their data. Scatter IV has several new features that help to improve the analysis of SEC-SAXS data, such as the heat map linked to the signal plot, enabling greater accuracy with choice of frame selection. In primary data analysis, the Guinier Peak analysis and the cross-validation plot associated with the P(r) analysis offer an integrated troubleshooting ability in the software.
It should be mentioned that many other programs can be used for primary data analysis; these contain the same basic features and are also updated regularly such as BioXTAS RAW17 ATSAS package24 and US-SOMO15 to name a few.
But regardless of which SAXS package is used for analysis, the major limitations are common: the sample preparation, before collection and analysis. In the E9 exominus example shown, it is clear to see the improvement in the signal to noise ratio and with a reduction in the Rg the dmax associated with a monodisperse sample. This will greatly aid further processing of the data such as fitting or modeling with known high-resolution structures.

Biological macromolecules are too small to be seen even with the best light microscopes. Current methods to determine their structures generally involve crystallizing the protein or measurements on vast numbers of identical molecules at the same time. While crystallography provides information on the atomic level, it represents an artificial sample environment, given that most macromolecules are not presented in a crystalline form in the cell. During the last couple of years cryo-electron microscopy delivered similar high-resolution structures of large macromolecules / macromolecular complexes, but although the samples are closer to physiological condition, they are still frozen, hence immobile and static. Bio-small angle X-ray scattering (BioSAXS) provides a structural measurement of the macromolecule, in conditions that are relevant to biology. This state can be visualized as a low resolution 3-D shape determined on nanometer scale and captures the entire conformational space of the macromolecule in solution. BioSAXS experiments efficiently assess oligomeric state, domain and complex arrangements as well as flexibility between domains1,2,3. The method is accurate, mostly non-destructive and usually requires only a minimum of sample preparation and time. However, for the best interpretation of the data, the samples need to be monodisperse. This is challenging; biological molecules are often susceptible to contaminations, poor purification and aggregation, for example from freeze thawing4. The development of inline chromatography followed by immediate SAXS measurement helps mitigate these effects. Size-exclusion chromatography separates the samples by size thus excluding most contaminants and aggregations5,6,7,8,9,10. However, in some cases even SEC-SAXS is not sufficient to produce a monodisperse sample, because the mixture may consist of components that are too close in size or their physical properties or their fast dynamics lead to overlapping peaks in the SEC UV trace. In these cases, a software-based deconvolution step of the obtained SAXS data might lead to an idealized SAXS curve of the individual component5,11,12. As an example, in protocol section 2, we show the standard SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant (E9 exominus) in complex with DNA. Vaccinia represents the model organism of the Poxviridae, a family containing several pathogens, for example the human smallpox virus. The polymerase was shown to bind tightly to DNA in biochemical approaches, with the structure of the complex recently solved by X-ray crystallography13.
Most synchrotron facilities will provide an automated data processing pipeline that will perform data normalization and integration producing a set of unsubtracted frames. But the approach described in this manuscript could also be use with a lab source provided SEC-SAXS is performed. Furthermore, additional automation may be available that will reject radiation-damaged frames and perform the buffer subtraction14. We will show how to perform primary data analysis on pre-processed data and make the most of the available data in section 2.
In section 3, we show how to deconvolute SEC-SAXS data and analyze the curves efficiently. While there are several deconvolution methods such as the Gaussian peak deconvolution, implemented in US-SOMO15 and the Guinier optimized maximum likelihood method, implemented in the DELA software16, these generally require a model for the peak shape12. The finite size of individual peaks we are investigating allows the use of evolving factor analysis (EFA), as an enhanced form of singular value decomposition (SVD) to deconvolute overlapping peaks, without relying on the peak shape or scattering profile5,11. A SAXS-specific implementation can be found in BioXTAS RAW17. EFA was first used on chromatography data when 2D diode array data allowed matrices to be formed from absorbance against retention time and wavelength data18. Where EFA excels is that it focuses on the evolving character of singular values, how they change with the appearance of new components, with the caveat that there is an inherent order in the acquisition10. Fortunately, SEC-SAXS data provides all the necessary ordered acquisition data in organized 2D data arrays, lending itself nicely to the EFA technique.
In section 4, we will demonstrate the basics of model-independent SAXS analysis from the buffer-background subtracted SAXS curve. Model-independent analysis determines the particle&#8217;s radius-of-gyration (Rg), volume-of-correlation (Vc), Porod Volume (Vp), and Porod-Debye Exponent (PE). The analysis provides a semi-quantitative assessment of the particle&#8217;s thermodynamic state in terms of compactness or flexibility via the dimensionless Kratky plot2,4,19.
Finally, SAXS data are measured in reciprocal space units and we will show how to transform the SAXS data to real-space to recover the pair-distance, P(r), distribution function. The P(r)-distribution is the set of all distances found within the particle and includes the particle&#8217;s maximum dimension, dmax. Since this is a thermodynamic measurement, the P(r)-distribution represents the physical space occupied by the particles&#8217; conformational space. Proper analysis of a SAXS dataset can provide solution-state insights that complement high-resolution information from crystallography and cryo-EM.

<table><tbody><tr><td>Beamline control software BsXCuBE</td><td>ESRF</td><td>Pernot et al. (2013), J. Synchrotron Rad. 20, 660-664</td><td>local development</td></tr><tr><td>BioXTAS Raw 1.2.3.</td><td>MacCHESS</td><td>http://bioxtas-raw.readthedocs.io/en/latest/index.html</td><td>First developed in 2008 by Soren Skou as part of the biological x-ray total analysis system (BioXTAS) project. Since then it has been extensively developed, with recent work being done by Jesse B. Hopkins</td></tr><tr><td>HPLC program LabSolutions</td><td>Shimadzu</td><td>n.a.</td><td></td></tr><tr><td>ISPyB</td><td>ESRF</td><td>De Maria Antolinos et al. (2015). Acta Cryst. D71, 76-85.</td><td>local development</td></tr><tr><td>NaCl</td><td>VWR Chemicals (BDH Prolabo)</td><td>27808.297</td><td></td></tr><tr><td>Scatter</td><td>Diamond Light Source Ltd</td><td>http://www.bioisis.net/tutorial/9</td><td>Supported by SIBYLS beamline (ALS berkeley, Ca) and Bruker Cororation (Karlsruhe, Germany)</td></tr><tr><td>Superdex 200 Increase 5/150 GL column</td><td>GE Healthcare</td><td>28990945</td><td>SEC-SAXS column used</td></tr><tr><td>Tris base</td><td>Euromedex</td><td>26-128-3094-B</td><td></td></tr></tbody></table>

analysis of sec-saxs data via efa deconvolution and scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

The advantage of using deconvolution over classical frame selection13 is to remove the influence of species on one another, producing a monodisperse scattering signal. This is also often followed with a better signal to noise ratio. When E9 exominus is bound to DNA and run using SEC-SAXS, two peaks are observed (Figure 1). The first, large peak (approximately frames 420&#8210;475) is the E9 exominus-DNA complex the second (approximately frames 475&#8210;540), the unbound state (see Supplementary Data: Figure 2). While the classical approach of selecting frames provides a stable Rg of the complex in the first peak (see Supplementary Data: Figure 3), the second peak is clearly merged and the Rg across the plot shows that the second peak of interest does not have a stable Rg, due to cross-peak contamination. Only 5 frames could be used that showed a semi-stable Rg, when subtracted they gave an Rg = 36.3&#160;&#197; (Figure 2, green). When the peaks were deconvoluted using EFA the corresponding curve for the second peak (Figure 2, blue) was overlaid with the original and showed a clear decrease in signal to noise, and a lower Rg, 34.1 &#197; was recorded. The Kratky plot (Figure 3) shows the complex with the deconvoluted peak (blue) is more globular. This is confirmed by the P(r) curve (Figure 4) which gives a dmax 108.5&#160;&#197; for the deconvoluted curve (blue) while the non-deconvoluted is more elongated with a dmax 120&#160;&#197; (green), this is most likely due to heterogeneity arising from the unbound E9 exominus.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61578/61578fig01.jpg" /> 
Figure 1: Signal plot of E9 exominus alone and with DNA in complex. 
The top panel shows a plot of the integral ratio to the background for each frame of a SEC-SAXS run (light blue). The red points show the Rg at each frame over the peak. The bottom panel shows the corresponding heat map showing the residuals for each frame colored according to the Durbin-Watson auto-correlation analysis, regions of high similarity are colored cyan while dissimilar frames follow darker blues to pinks and finally to red depending on the severity of the dissimilarity. <a href="https://www.jove.com/files/ftp_upload/61578/61578fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61578/61578fig02.jpg" /> 
Figure 2: Plot of intensity versus scattering vector. 
An overlay of the subtracted SAXS data form the E9 exominus . In green 5 frames (frame 517&#8210;522) averaged and subtracted from an area of semi-stable Rg and in blue the representative scattering curve derived from the EFA deconvolution of the SEC-SAXS peak. <a href="https://www.jove.com/files/ftp_upload/61578/61578fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61578/61578fig03.jpg" /> 
Figure 3: Dimensionless Kratky curve. 
Overlay of the deconvoluted (blue) and non-deconvoluted (green) Kratky curve showing E9 exominus is globular. <a href="https://www.jove.com/files/ftp_upload/61578/61578fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61578/61578fig04.jpg" /> 
Figure 4: P(r) curve. 
The overlay of the deconvoluted (blue) and non-deconvoluted (green) curves for the E9 exominus. <a href="https://www.jove.com/files/ftp_upload/61578/61578fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Data.&#160;<a href="https://www.jove.com/files/ftp_upload/61578/Supplementary data.docx" target="_blank">Please click here to download this file</a>

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Analysis of SEC-SAXS data via EFA deconvolution and Scatter

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

analysis-of-sec-saxs-data-via-efa-deconvolution-and-scatter

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Biochemistry

8.9K Views.  Structural Biology Group. Biologische kleine hoek x-ray verstrooiing biedt structurele metingen van macromolecule en macromoleculaire complexen. Idealiter moet het te meten monster monodispersed zijn. Hoewel in sommige gevallen de grootte uitsluiting chromatografie SAXS is niet voldoende om monodispersity te produceren, een software-gebaseerde deconvolutie van de SAXS-gegevens kan worden uitgevoerd om een geïdealiseerde SAXS curve te produceren.Volgens dit protocol kunnen een deconvolutieprogramma en het gebru...

Video: Analysis of SEC-SAXS data via EFA deconvolution and Scatter

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Bioengineering

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

Engineering

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

Chemistry

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

ARL Spectral Fitting as an Application to Augment Spectral Data via Franck-Condon Lineshape Analysis and Color Analysis

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis (FCLSA) on spectral data, in addition to CIE color coordinate determination and basic spectral processing. While some of the features may be found in commercial software or in programs made by&#160;academic research groups, we believe that ARL Spectral Fitting is the only application that possesses all three of the aforementioned attributes.
This program is intended as a standalone, GUI-based application for use by an average laboratory researcher without requiring any coding knowledge or proprietary software. In addition to the standalone executable hosted on ARL GitHub, the associated MATLAB files are available for use and further development.
FCLSA augments the information found in luminescence spectra, providing meaningful insight into the relationship between the ground and excited states of a photoluminescent species. This insight is achieved by modeling spectra with two versions (modes) of an equation that are characterized by either four or six parameters, depending on which mode is used. Once optimized, the value of each of these parameters can be used to gain insight into the molecule, as well as to perform further analysis (for example, the free energy content of the excited-state molecule). This application provides tools for easy by-hand fitting of imported data, as well as two methods for optimizing this fit-damped least-squares fitting, powered by the Levenberg-Marquardt algorithm, and derivative-free fitting utilizing the Nelder-Mead simplex algorithm. Furthermore, estimations of sample color can be performed and reported in CIE and RGB coordinates.

This protocol introduces Franck-Condon Lineshape Analyses (FCLSA) of emission spectra and serves as a tutorial for the use of ARL Spectral Fitting software. The open-source software provides an easy and intuitive way to perform advanced analysis of emission spectra including excited state energy calculations, CIE color coordinate determination, and FCLSA.

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

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