Integrating machine learning with α -SAS for enhanced structural analysis in small-angle scattering: applications in biological and artificial macromolecular complexes.

Small-Angle Scattering (SAS), encompassing both X-ray (SAXS) and Neutron (SANS) techniques, is a crucial tool for structural analysis at the nanoscale, particularly in the realm of biological macromolecules. This paper explores the intricacies of SAS, emphasizing its application in studying complex biological systems and the challenges associated with sample preparation and data analysis. We highlight the use of neutron-scattering properties of hydrogen isotopes and isotopic labeling in SANS for probing structures within multi-subunit complexes, employing techniques like contrast variation (CV) for detailed structural analysis. However, traditional SAS analysis methods, such as Guinier and Kratky plots, are limited by their partial use of available data and inability to operate without substantial a priori knowledge of the sample's chemical composition. To overcome these limitations, we introduce a novel approach integrating α -SAS, a computational method for simulating SANS with CV, with machine learning (ML). This approach enables the accurate prediction of scattering contrast in multicomponent macromolecular complexes, reducing the need for extensive sample preparation and computational resources. α -SAS, utilizing Monte Carlo methods, generates comprehensive datasets from which structural invariants can be extracted, enhancing our understanding of the macromolecular form factor in dilute systems. The paper demonstrates the effectiveness of this integrated approach through its application to two case studies: Janus particles, an artificial structure with a known SAS intensity and contrast, and a biological system involving RNA polymerase II in complex with Rtt103. These examples illustrate the method's capability to provide detailed structural insights, showcasing its potential as a powerful tool for advanced SAS analysis in structural biology.

Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Two populations of protein molecules detected by small-angle neutron and X-ray scattering (SANS and SAXS) in lyophilized protein:lyoprotector (disaccharide) systems.

Two protein interaction peaks are observed in pharmaceutically-relevant protein (serum albumin) : disaccharide 1 : 1 and 1 : 3 (w/w) freeze-dried systems for the first time. In samples with a higher disaccharide content, the protein-protein distances are longer for both populations, while the fraction of the protein population with a shorter protein-protein distance is lower. Both factors would favor better stability against aggregation for disaccharide-rich protein formulations. This study provides direct experimental support for a "dilution" hypothesis as a potential stabilization mechanism for freeze-dried protein formulations.

Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS.

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations (CL14:0, CL18:1, CL18:2) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations (C14:0 and C 18:1). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added. Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

Nonstereotypical Distribution and Effect of Ergosterol in Lipid Membranes.

Ergosterol, found in fungi and some protist membranes, is understudied compared with cholesterol from animal membranes. Generally, ergosterol is assumed to modulate membranes in the same manner as cholesterol, based on their similar chemical structures. Here we reveal some fundamental structural and dynamical differences between them. Neutron diffraction shows that ergosterol is embedded in the lipid bilayer much shallower than cholesterol. Ergosterol does not change the membrane thickness as much as cholesterol does, indicating little condensation effect. Neutron spin echo shows that ergosterol can rigidify and soften membranes at different concentrations. The lateral lipid diffusion measured by quasielastic neutron scattering indicates that ergosterol promotes a jump diffusion of the lipid, whereas cholesterol keeps the same continuous lateral diffusion as the pure lipid membrane. Our results point to quite distinct interactions of ergosterol with membranes compared with cholesterol. These insights provide a basic understanding of membranes containing ergosterol with implications for phenomena such as lipid rafts and drug interactions.

Biomolecular condensates form spatially inhomogeneous network fluids.

The functions of biomolecular condensates are thought to be influenced by their material properties, and these will be determined by the internal organization of molecules within condensates. However, structural characterizations of condensates are challenging, and rarely reported. Here, we deploy a combination of small angle neutron scattering, fluorescence recovery after photobleaching, and coarse-grained molecular dynamics simulations to provide structural descriptions of model condensates that are formed by macromolecules from nucleolar granular components (GCs). We show that these minimal facsimiles of GCs form condensates that are network fluids featuring spatial inhomogeneities across different length scales that reflect the contributions of distinct protein and peptide domains. The network-like inhomogeneous organization is characterized by a coexistence of liquid- and gas-like macromolecular densities that engenders bimodality of internal molecular dynamics. These insights suggest that condensates formed by multivalent proteins share features with network fluids formed by systems such as patchy or hairy colloids.

Small-angle X-ray and neutron scattering applied to lipid-based nanoparticles: Recent advancements across different length scales.

Lipid-based nanoparticles (LNPs), ranging from nanovesicles to non-lamellar assemblies, have gained significant attention in recent years, as versatile carriers for delivering drugs, vaccines, and nutrients. Small-angle scattering methods, employing X-rays (SAXS) or neutrons (SANS), represent unique tools to unveil structure, dynamics, and interactions of such particles on different length scales, spanning from the nano to the molecular scale. This review explores the state-of-the-art on scattering methods applied to unveil the structure of lipid-based nanoparticles and their interactions with drugs and bioactive molecules, to inform their rational design and formulation for medical applications. We will focus on complementary information accessible with X-rays or neutrons, ranging from insights on the structure and colloidal processes at a nanoscale level (SAXS) to details on the lipid organization and molecular interactions of LNPs (SANS). In addition, we will review new opportunities offered by Time-resolved (TR)-SAXS and -SANS for the investigation of dynamic processes involving LNPs. These span from real-time monitoring of LNPs structural evolution in response to endogenous or external stimuli (TR-SANS), to the investigation of the kinetics of lipid diffusion and exchange upon interaction with biomolecules (TR-SANS). Finally, we will spotlight novel combinations of SAXS and SANS with complementary on-line techniques, recently enabled at Large Scale Facilities for X-rays and neutrons. This emerging technology enables synchronized multi-method investigation, offering exciting opportunities for the simultaneous characterization of the structure and chemical or mechanical properties of LNPs.

Alcohol-Induced Denaturation of Hen Egg White Lysozyme Studied by Infrared, Circular Dichroism, and Small-Angle Neutron Scattering.

In aqueous binary solvents with fluorinated alcohols, 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), and aliphatic alcohols, ethanol (EtOH) and 2-propanol (2-PrOH), the denaturation of hen egg white lysozyme (HEWL) with increasing alcohol mole fraction xA has been investigated in a wide view from the molecular vibration to the secondary and ternary structures. Circular dichroism (CD) measurement showed that the secondary structure of α-helix content of HEWL increases on adding a small amount of the fluorinated alcohol to the aqueous solution, while the ß-sheet content decreases. On the contrary, the secondary structure does not significantly change by the addition of the aliphatic alcohols. Correspondingly, the infrared (IR) spectroscopic measurements revealed that the amide I band red-shifts on the addition of the fluorinated alcohol. However, the band remains unchanged in the aliphatic alcohol systems with increasing alcohol content. To observe the ternary structure of HEWL, small-angle neutron scattering (SANS) experiments with H/D substitution technique have been applied to the HEWL solutions. The SANS experiments were successful in revealing the details of how the geometry of the HEWL changes as a function of xA. The SANS profiles indicated the spherical structure of HEWL in all of the alcohol systems in the xA range examined. The mean radius of HEWL in the two fluorinated alcohol systems increases from ∼16 to ∼18 Å during the change in the secondary structure against the increase in the fluorinated alcohol content. On contrast, the radius does not significantly change in both aliphatic alcohol systems below xA = 0.3 but expands to ∼19 Å as the alcohol content is close to the limitation of the HEWL solubility. According to the present results, together with our knowledge of the alcohol cluster formation and the interaction of the trifluoromethyl (CF3) groups with the hydrophobic moieties of biomolecules, the effects of alcohols on the denaturation of the protein have been discussed on a molecular scale.

Computation of X-ray and Neutron Scattering Patterns to Benchmark Atomistic Simulations against Experiments.

Molecular Dynamics simulations study material structure and dynamics at the atomic level. X-ray and neutron scattering experiments probe exactly the same time- and length scales as the simulations. In order to benchmark simulations against measured scattering data, a program is required that computes scattering patterns from simulations with good single-core performance and support for parallelization. In this work, the existing program Sassena is used as a potent solution to this requirement for a range of scattering methods, covering pico- to nanosecond dynamics, as well as the structure from some Ångströms to hundreds of nanometers. In the case of nanometer-level structures, the finite size of the simulation box, which is referred to as the finite size effect, has to be factored into the computations for which a method is described and implemented into Sassena. Additionally, the single-core and parallelization performance of Sassena is investigated, and several improvements are introduced.

Characterization of the In Vivo Deuteration of Native Phospholipids by Mass Spectrometry Yields Guidelines for Their Regiospecific Customization.

Customization of deuterated biomolecules is vital for many advanced biological experiments including neutron scattering. However, because it is challenging to control the proportion and regiospecificity of deuterium incorporation in live systems, often only two or three synthetic lipids are mixed together to form simplistic model membranes. This limits the applicability and biological accuracy of the results generated with these synthetic membranes. Despite some limited prior examination of deuterating Escherichia coli lipids in vivo, this approach has not been widely implemented. Here, an extensive mass spectrometry-based profiling of E. coli phospholipid deuteration states with several different growth media was performed, and a computational method to describe deuterium distributions with a one-number summary is introduced. The deuteration states of 36 lipid species were quantitatively profiled in 15 different growth conditions, and tandem mass spectrometry was used to reveal deuterium localization. Regressions were employed to enable the prediction of lipid deuteration for untested conditions. Small-angle neutron scattering was performed on select deuterated lipid samples, which validated the deuteration states calculated from the mass spectral data. Based on these experiments, guidelines for the design of specifically deuterated phospholipids are described. This unlocks even greater capabilities from neutron-based techniques, enabling experiments that were formerly impossible.

Improved joint X-ray and neutron refinement procedure in Phenix.

Neutron diffraction is one of the three crystallographic techniques (X-ray, neutron and electron diffraction) used to determine the atomic structures of molecules. Its particular strengths derive from the fact that H (and D) atoms are strong neutron scatterers, meaning that their positions, and thus protonation states, can be derived from crystallographic maps. However, because of technical limitations and experimental obstacles, the quality of neutron diffraction data is typically much poorer (completeness, resolution and signal to noise) than that of X-ray diffraction data for the same sample. Further, refinement is more complex as it usually requires additional parameters to describe the H (and D) atoms. The increase in the number of parameters may be mitigated by using the `riding hydrogen' refinement strategy, in which the positions of H atoms without a rotational degree of freedom are inferred from their neighboring heavy atoms. However, this does not address the issues related to poor data quality. Therefore, neutron structure determination often relies on the presence of an X-ray data set for joint X-ray and neutron (XN) refinement. In this approach, the X-ray data serve to compensate for the deficiencies of the neutron diffraction data by refining one model simultaneously against the X-ray and neutron data sets. To be applicable, it is assumed that both data sets are highly isomorphous, and preferably collected from the same crystals and at the same temperature. However, the approach has a number of limitations that are discussed in this work by comparing four separately re-refined neutron models. To address the limitations, a new method for joint XN refinement is introduced that optimizes two different models against the different data sets. This approach is tested using neutron models and data deposited in the Protein Data Bank. The efficacy of refining models with H atoms as riding or as individual atoms is also investigated.

Cellular dynamics as a marker of normal-to-cancer transition in human cells.

Normal-to-cancer (NTC) transition is known to be closely associated to cell´s biomechanical properties which are dependent on the dynamics of the intracellular medium. This study probes different human cancer cells (breast, prostate and lung), concomitantly to their healthy counterparts, aiming at characterising the dynamical profile of water in distinct cellular locations, for each type of cell, and how it changes between normal and cancer states. An increased plasticity of the cytomatrix is observed upon normal-to-malignant transformation, the lung carcinoma cells displaying the highest flexibility followed by prostate and breast cancers. Also, lung cells show a distinct behaviour relative to breast and prostate, with a higher influence from hydration water motions and localised fast rotations upon NTC transformation. Quasielastic neutron scattering techniques allowed to accurately distinguish the different dynamical processes taking place within these highly heterogeneous cellular systems. The results thus obtained suggest that intracellular water dynamics may be regarded as a specific reporter of the cellular conditions-either healthy or malignant.

Neutron crystallographic refinement with REFMAC5 from the CCP4 suite.

Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein-protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Šor higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium-low resolution.

Signature of functional enzyme dynamics in quasielastic neutron scattering spectra: The case of phosphoglycerate kinase.

We present an analysis of high-resolution quasi-elastic neutron scattering spectra of phosphoglycerate kinase which elucidates the influence of the enzymatic activity on the dynamics of the protein. We show that in the active state the inter-domain motions are amplified and the intra-domain asymptotic power-law relaxation ∝t-α is accelerated, with a reduced coefficient α. Employing an energy landscape picture of protein dynamics, this observation can be translated into a widening of the distribution of energy barriers separating conformational substates of the protein.

Exploring the effects of high pressure on hydrogen bonding in pharmaceutical cocrystals: A systematic study of pyridine dicarboxylic acid systems using synchrotron and neutron diffraction.

Pharmaceutical cocrystals use common robust hydrogen bonding synthons to create novel materials with different physicochemical properties. In this systematic study of a series of cocrystals, we explore the effect of high pressure on one of these commonly used motifs, the acid-pyridine motif, to assess the commonality of behaviour under extreme conditions. We have surveyed five pyridine dicarboxylic acid systems using both synchrotron and neutron diffraction methods to elucidate the changes in structure. We observe that the hydrogen bonding in these systems compress at a similar rate despite the changes to the molecular make-up of the solids and that on compression the changes in structure are indicative that the layers move along the major slip planes in the structure. We have observed two phase transitions to new forms of the pyrazine:malonic acid system, one for each stoichiometric ratio. This study demonstrates that the combination of two complementary diffraction approaches is key to understanding polymorphic behaviour at high pressure.

Site-specific relaxation of peptide bond planarity induced by electrically attracted proton/deuteron observed by neutron crystallography.

In structural biology, peptide bonds, fundamental linkages between hundreds of amino acids, of which a protein molecule is composed, have been commonly treated as a plane structure just as Linus Pauling et al. proposed. In this paper, a site-specific peptide bond relaxation mechanism by deuterons whose localization has been suggested by neutron crystallography is proposed. Such deuteron was observed as an arm of neutron scattering length density protruding from the carbonyl oxygen atoms in the main chain in the omit map drawn by neutron crystallography of human lysozyme. Our comprehensive study using x-ray and neutron diffraction and 15 N chemical shifts of individual amide nitrogen atoms within the same peptide bond strongly suggests the relaxation of the electronic resonance structure because of site-specific modulation by protons/deuterons localized on the electron orbital of the carbonyl oxygen. All experimental data used in this examination were obtained at room temperature, which is preferable for enzymatic activity. Such a close interaction between the electron resonance structure of a peptide bond and the exchangeable protons/deuterons well agreed with that observed in an intermediate state in an amide hydrolytic reaction simulated by the ab-initio calculation including water molecules.

Development of an automatic sample changer with variable temperature for small-angle neutron scattering at China Spallation Neutron Source.

A small-angle neutron scattering (SANS) instrument at the China Spallation Neutron Source (CSNS) is an operating instrument for studying structures and inhomogeneities with dimensions ranging from 1 to 100 nm. Preparing multiple samples at once and measuring them sequentially is a common approach in SANS experiments to reduce neutron beamline wastes and increase experimental efficiency. We present the development of an automatic sample changer for the SANS instrument, including system design, thermal simulation, optimization analysis, structure design details, and temperature control test results. It features a two-row construction that can hold 18 samples on each row. The controllable temperature range is -30 to 300 °C. Furthermore, neutron scattering experiments on SANS at CSNS proved that this instrument has good temperature control performance and low background. This automatic sample changer is optimized for usage at SANS and will be offered to other researchers through the user program.

Heterogeneous Aggregation of Humic Acids Studied by Small-Angle Neutron and X-ray Scattering.

Aggregation of humic acids (HAs) was studied by small-angle neutron and X-ray scattering techniques. The combination of these techniques enables us to examine the aggregation structures of HA particles. Two HAs with distinctive compositions were examined: a commercial HA (PAHA) and a HA extracted from deep sedimentary groundwater (HHA). While macroscopic coagulation tests showed that these HAs were stable in solutions except for HHA at pH < 6, small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) revealed that they formed aggregates with sizes exceeding the sub-micrometer length scale. The SAXS curves of PAHA remarkably varied with pD = log aD+, where aD+ stands for the activity of deuterium ions, whereas the SANS curves did not. With the help of theoretical fittings, it was revealed that PAHA aggregates consisted of two domains: poorly hydrated cores and well-hydrated proton-rich shells. The cores were (dis)aggregated with pD inside the aggregates of the shells. The SANS and SAXS curves of HHA resembled each other, and their intensities at low q, where q stands for the scattering vector, increased with a decrease of pD, indicating the formation of homogeneous aggregates within the spatial resolutions of SANS and SAXS. This study revealed that distinctive aggregation behaviors exist in humic substances with nm-scale heterogeneous structures like PAHA, which is important for their roles in the fate of contaminants or nutrients in aqueous environments.

Small Angle Neutron Scattering in Drug Discovery Research: A Novel Tool for Advanced Study of Structures of Biological Macromolecules.

Small Angle Neutron Scattering (SANS) is a powerful and novel tool for the study of soft condensed matter, including the microscopic and nanomaterials used for drug discovery and delivery. The sample is exposed to a neutron beam, and neutron scattering occurs, which is studied as a function of the scattering angle to deduce a variety of information about the dynamics and structure of the material. The technique is becoming very popular in biomedical research to investigate the various aspects of structural biology. The low-resolution information on large heterogeneous, solubilized biomacromolecular complexes in solution is obtained with the use of deuterium labelling and solvent contrast variation. The article reviews the basics of the SANS technique, its applications in drug delivery research, and its current status in biomedical research. The article covers and overviews the precise characterization of biological structures (membranes, vesicles, proteins in solution), mesoporous structures, colloids, and surfactants, as well as cyclodextrin complexes, lipid complexes, polymeric nanoparticles, etc., with the help of neutron scattering. SANS is continuously evolving as a medium for exploring the complex world of biomolecules, providing information regarding the structure, composition, and arrangement of various constituents. With improving modelling software automation in data reduction and the development of new neutron research facilities, SANS can be expected to remain mainstream for biomedical research.

Application of small angle scattering (SAS) in structural characterisation of casein and casein-based products during digestion.

In recent years, small and ultra-small angle scattering techniques, collectively known as small angle scattering (SAS) have been used to study various food structures during the digestion process. These techniques play an important role in structural characterisation due to the non-destructive nature (especially when using neutrons), various in situ capabilities and a large length scale (of 1 nm to ∼20 µm) they cover. The application of these techniques in the structural characterisation of dairy products has expanded significantly in recent years. Casein, a major dairy protein, forms the basis of a wide range of gel structures at different length scales. These gel structures have been extensively researched utilising scattering techniques to obtain structural information at the nano and micron scale that complements electron and confocal microscopy. Especially, neutrons have provided opportunity to study these gels in their natural environment by using various in situ options. One such example is understanding changes in casein gel structures during digestion in the gastrointestinal tract, which is essential for designing personalised food structures for a wide range of food-related diseases and improve health outcomes. In this review, we present an overview of casein gels investigated using small angle and ultra-small angle scattering techniques. We also reviewed their digestion using newly built setups recently employed in various research. To gain a greater understanding of micro and nano-scale structural changes during digestion, such as the effect of digestive juices and mechanical breakdown on structure, new setups for semi-solid food materials are needed to be optimised.