Frontiers in SAS

Conference: 2020: 70th ACA Annual Meeting
08/03/2020: 12:00 PM - 3:00 PM
Oral Session 


Recent advances in light sources, experimental methods and computational algorithms have enabled exciting new discoveries using small angle scattering (SAS). This session is devoted to discussing the latest advances in methods and applications of X-ray and neutron SAS. The primary aim is to bring together cutting-edge advances utilizing SAS on both soft matter and biological systems, including time-resolved studies, contrast matching, dynamic and flexible systems, hybrid modeling, novel experimental apparatus and methods, and new computational approaches. This session will reflect the state of the art in SAS methods.


Atomistic ensembles of proteins and soft matter complexes from MD simulations and solution scattering data

Understanding the function of disordered proteins or soft-matter complexes requires understanding of their conformational ensembles. However, experimental data alone is often insufficient for defining all degrees of freedom of such systems, whereas simulations may be biased by poor sampling or force field limitations. We developed a method for coupling atomistic simulations on-the-fly to small- and wide-angle X-ray scattering (SAXS/WAXS) data, based on Jaynes' principle of maximum entropy, with the aim to obtain accurate atomistic ensembles biomolecular and soft-matter systems. As examples, we show that the method is capable of overcoming force field inaccuracies in simulations of an intrinsically disordered protein and of a detergent micelle. In addition, we critically review capabilities and limitations of widely used continuum models in deriving structures of soft-matter complexes.

[1] Hub, Curr Opin Struct Biol, 49, 18-26 (2018)
[2] Hermann and Hub, J Chem Theory Comput, 15, 95103-5115 (2019)
[3] Ivanović, Bruetzel, Lipfert, Hub, Angew Chem Int Ed, 57, 5635-5639 (2018)
[4] Ivanović, Hermann, Wójcik, Pérez, Hub, J. Phys. Chem. Lett., 11, 945-951 (2020) 

View Abstract 248


Jochen Hub

Cryogenic methods for biological small-angle x-ray scattering

Small-angle X-ray scattering (SAXS) is a key tool for probing the structure and function of proteins, nucleic acids, and macromolecular complexes. Most synchrotron sources have BioSAXS beam lines, but efforts to improve their throughput have not kept pace with user demand. Large sample volumes and low duty cycles are critical bottlenecks in the expansion of BioSAXS. The reduction in radiation damage at T = 100 K significantly reduces the amount of protein required per measurement and sample holders compatible with standard macromolecular cryocrystallography infrastructure could allow for sample preparation in the home lab immediately after purification, easy sample storage and shipping, and automated high-throughput data collection. This will enable dramatically more efficient use of both biomolecules and synchrotron beam time, and significantly expand the potential scope of BioSAXS studies.
Demonstrations of CryoSAXS have shown the potential of this technique1,2, however, the lack of a robust experimental platform has prevented CryoSAXS from becoming a routine method. The need to subtract a highly matched background scattering pattern from the macromolecule's scatter and the difficulty in vitrifying bulk-like solutions have posed serious technical challenges for the development of sample holders adequate for routine use. We have developed a new generation of CryoSAXS devices that have demonstrated encouraging results in synchrotron applications. These microfabricated devices have fixed pathlengths, low background scatter X-ray windows and small thermal mass, allowing for rapid vitrification. Each individual device is compatible with macromolecular cryocrystallography goniometer bases and pucks and allows for multiple samples per device.

1. Meisburger, S. P., et al., (2013) Biophys. J., 104, 227-236.
2. Hopkins, J. B., et al., (2015) J. Appl. Cryst. 48, 227-237. 

View Abstract 202


David Moreau, Cornell University Ithaca, NY 

Additional Author

Robert Thorne, MiTeGen, LLC Ithaca, NY 

HP-Bio: High Pressure BioSAXS for Deep Life and Extreme Biophysics

The structural biology and biophysics of biomolecules under extreme pressure have attracted an increased interest in recent years with the discovery of copious microbial life in extreme environments such as rock pores kilometers down in the deep hot subsurface, cold ocean trenches 11 km deep, and hydrothermal vents as hot as 122˚C. How proteins and other biomolecules are able to function and maintain integrity under such extreme conditions that would normally destroy surface life is a question of practical importance for food and pharmaceutical preparation. It may also shed light on the origins and limits of life, as well as the potential for life elsewhere in the universe. Biophysically speaking, virtually anything a biomolecule does changes its volume and hence is potentially influenced by pressure. Consequently, pressure offers a unique way to probe cavities and volume changes in biological systems, and to unfold or dissociate systems in a controlled way. The Cornell High Energy Synchrotron Source now hosts a facility dedicated to high-pressure structural biology. Two of our main technologies will be discussed here: "batch" high pressure BioSAXS that can reach pressures in excess of 400 MPa, and a novel high-pressure chromatography-coupled BioSAXS system that can simultaneously separate and measure biomolecules at pressures of up to 100 MPa (~10km deep in the ocean). This talk will cover design, performance, practical considerations for doing experiments, and biological applications. 

View Abstract 335


Richard Gillilan, CHESS, Cornell Univ Ithaca, NY 

Additional Author(s)

Durgesh K Rai, Cornell High Energy Synchrotron Source Ithaca, NY 
Robert Miller, Cornell High Energy Synchrotron Source Ithaca, NY 
Sol Gruner, Physics Dept. & CHESS, Cornell Univ
Nozomi Ando, Cornell University Ithaca, NY 
Qingqiu Huang, Cornell University Ithaca, NY 

Coffee Break

Application of neural networks for small angle scattering data analysis

Small angle X-ray scattering (SAXS) is a powerful technique for analyzing dilute solutions of proteins, nucleic acids and other macromolecules in solution under a wide range of conditions. The primary SAXS data analysis steps include estimation of the overall particle parameters (the radius of gyration Rg, maximum intraparticle distance Dmax, molecular weight MW) and computation of the pair distance distribution function p(r). The latter represents the histogram of distances between pairs of points in the particle, weighted by the product of their scattering contrasts. Mathematically, the p(r) function is a Fourier transform of the scattering intensities versus the momentum transfer (i.e. scattering angle). The limited angular range of recorded experimental data, as well as the presence of experimental noise, makes the evaluation of p(r) an ill-posed problem. This problem is typically solved by an indirect Fourier transformation (IFT) approach, e.g. Glatter (1977), Svergun (1992), Hansen (2000). We propose a new approach for computing p(r) from the experimental data based on the state-of-the-art machine learning principles. We employed neural networks trained on a manifold of simulated and experimental SAXS data to perform primary data analysis. For a given experimental data set from protein, RNA or DNA our stack of networks evaluates Rg, Dmax, MW, p(r) and produces a denoised scattering curve. This approach has proved to be robust against experimental errors, applicable to data from particles of various nature, size and shape and does not require human input. The implementation of this approach as a publicly available web service with a graphical interface was developed, providing the possibility to inspect and download the results.
This work is supported by the SAS-BSOFT grant: "Curation and development of databases for X-ray and neutron small angle control in biology and soft matter". 

View Abstract 420


Al Kikhney, EMBL Hamburg 22607

Additional Author(s)

Dmitry Molodenskiy, EMBL Hamburg Hamburg, NON-US 
Dmitri Svergun, EMBL Hamburg Hamburg

Applications of Microfluidic Mixers for Time-Resolved SAXS and Crystallography Experiments

Both SAXS and X-ray Crystallography (MX) have paved the way for rapid determination of macromolecular structures, including proteins, DNA, and RNA. However, structural information alone cannot fully explain the biological functions that these macromolecules perform. Relatively recent developments in microfluidic mixers allow for the rapid diffusion of a ligand, such as an ion or small molecule, into a co-flowing stream that contains a sample, like a protein or RNA. This triggers millisecond scale interactions that can be visualized by various structural techniques. The combination of these mixers with either SAXS or MX creates an exciting frontier for time-resolved experiments. The microfluidic mixers are constructed from concentric capillaries and are comprised of a flow-focused mixing region and a delay region (Calvey et al., 2016; Calvey et al., 2019). The mixing region creates a narrow stream of sample surrounded by ligand, which supports fast diffusion for uniform mixing of the ligand and sample. Next, there is a delay region, which simply controls how long the sample and ligand interact before eventually being probed by the X-ray beam. The mixers have a seemingly endless range of applications as they can be situated upstream of standard sample delivery hardware and are compatible with samples free in solution or in crystal form.

A recent Time-Resolved SAXS experiment (Plumridge et al., 2018) incorporated these mixers with a standard SAXS sample cell to visualize the effect of added Mg2+ ions on a small segment of RNA, exploring reaction timescales from 10ms to 3000ms. This wide range of detection times was accessed using a combination of flow rates and mixers with the appropriately sized mixing and delay regions. An additional application for these mixers links them with Gas-Dynamic Virtual Nozzles (GDVNs) for Mix-and-Inject Femtosecond Serial Crystallography (Olmos et al., 2018). Key to this technique is the use of microcrystals and the rapid diffusion of small molecules to access proteins within them. Snapshots of the protein-ligand interactions can then be captured on the millisecond scale and beyond. These mixers are also currently being adapted for time-resolved spectroscopy. Overall, the mixers can be applied widely to probe different biological systems and designed for compatibility with multiple structural techniques. This technology has the potential to elucidate the details of many essential biological processes.

Calvey G. D. et al. (2016) Struct. Dyn. 3, 054301
Calvey G. D. et al. (2019) Anal. Chem. 91(11), 7139-7144
Olmos J. L. et al. (2018) BMC Biol. 16(59)
Plumridge A. et al. (2018) Nucleic Acids Res. 14(21), 7354-7365 

View Abstract 181


Kara Zielinski, Cornell University Ithaca, NY 

Additional Author

Lois Pollack, Cornell University Ithaca, NY 

Real-time pressure-temperature reaction studies of biological systems using small-angle neutron scattering technique.

Scientific experiments that require the need to use extreme sample environments are ideally suited for neutron experiments due to the high penetration and non-destructive nature of biomass. The Bio-SANS instrument at Oak Ridge National Laboratory has developed a pressure cell that allows to heat the cell up to 300 °C as well as apply a maximum pressure of 1 kbar. Initial motivation for these capabilities were to track, in real-time, chemical reactions that span multiple hours and more recently expanded these capabilities to perform solution scattering of thermophilic and mesophilic proteins under pressure and/or temperature. Synergistically, the dynamic q-range for the detector system of the Bio-SANS instrument was improved from a factor of 20 to ~300 by the installation of an additional detector array in the high scattering angle region. This talk will present the technical developments addressed to achieve these capabilities and two scientifically themed experiments will be presented. The first class of experiments will cover chemical reaction of plant biosystems and plant biopolymers. This will include dilute acid (acidic), sodium hydroxide (basic), co-solvents (acidic water/THF) as well as organic solvents (catalytic super-critical methanol) reactions required to breakdown plant biosystems. The presentation will cover new in-sights gained by these experiments that were not possible by the ex-situ based techniques. In fact, these measurements produced a paradigm shift in the thinking of biotechnologists pursuing biomass deconstruction strategies. The second set of experiments that is a relatively new capability, will cover solution scattering of thermophilic and mesophilic proteins. These measurements were carried out by varying the temperature, pressure and a combination of temperature/pressure of the protein solution system. 

View Abstract 332


Sai Venkatesh Pingali, Oak Ridge National Laboratory Oak Ridge, TN 

Additional Author(s)

Loukas Petridis, Oak Ridge National Laboratory Oak Ridge, TN 
Utsab Shrestha, Oak Ridge National Laboratory Oak Ridge, TN 
SHUO Qian, Oak Ridge National Laboratory KNOXVILLE, TN 
Volker Urban, Oak Ridge National Laboratory Oak Ridge, TN 
Hugh O'Neill, Oak Ridge National Laboratory Oak Ridge, TN 

REGALS: data analysis at the SAS frontier

SAS experiments at the frontier often seek to measure macromolecular components whose concentrations vary, as in ligand titrations, time-resolved methods, or chromatographic separations. These experiments produce large datasets that must be processed to extract the individual component scattering curves. Mathematically, the problem is related to matrix factorization and can be solved by singular value decomposition (SVD). However, the basis vectors from SVD are usually non-physical, and there is the additional problem of finding linear combinations that satisfy physically motivated restraints. Previously, we used evolving factor analysis (EFA) to analyze size-exclusion chromatography (SEC) SAXS experiments with overlapping peaks [1]. The method was integrated into a well-known software package [2] and has been used by many research groups. However, EFA cannot handle severe peak overlap or changing background. To address a wider class of problems, we developed a computational method called REGALS (for REGularized Alternating Least Squares) that performs matrix factorization such that "physically-meaningful" solutions are enforced by regularization. Regularization functions chosen by the user are applied separately to each component and can include smoothness and extent of the concentration peak, smoothness of the SAXS profile, or smoothness and maximum dimension of the real-space P(r) function. We recently applied REGALS to ion-exchange (IEX) SAXS data, which cannot be analyzed by EFA because the salt gradient produces a changing background [3]. In my presentation I will describe the further development of REGALS and its performance on challenging chromatography datasets. In addition, I will explore the method's potential for other types of experiments, including equilibrium titrations and time-resolved SAXS. Finally, I will describe the REGALS software package, which is free and open-source and includes both Python and MATLAB implementations.

[1] Meisburger SP, Taylor AB, Khan CA, Zhang S, Fitzpatrick PF, Ando N. (2016). Domain movements upon activation of phenylalanine hydroxylase characterized by crystallography and chromatography-coupled small-angle X-ray scattering. JACS 138(20): 6506–16.
[2] Hopkins, J. B., Gillilan, R. E., & Skou, S. (2017). BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis. J. Appl. Crystallogr. 50(5): 1545–53.
[3] Parker MJ, Maggiolo AO, Thomas WC, Kim A, Meisburger SP, Ando N, Boal AK, Stubbe J. (2018). An endogenous dAMP ligand in Bacillus subtilis class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP. PNAS 115(20): E4594-E4603. 

View Abstract 344


Steve Meisburger, Cornell University Ithaca

Additional Author(s)

Darren Xu, Cornell University Ithaca, NY 
Nozomi Ando, Cornell University Ithaca, NY