scholarly journals Concentrated protein solutions investigated using acoustic levitation and small-angle X-ray scattering

2020 ◽  
Vol 27 (2) ◽  
pp. 396-404
Author(s):  
Pernille Sønderby ◽  
Christopher Söderberg ◽  
Christian G. Frankær ◽  
Günther Peters ◽  
Jens T. Bukrinski ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Strength Parameter ◽  
Protein Solutions ◽  
Limited Sample ◽  
Saxs Data ◽  
X Ray Scattering ◽  
Levitated Droplet ◽  
Flow Through ◽  
Concentration Space ◽  
Selection Of

An acoustically levitated droplet has been used to collect synchrotron SAXS data on human serum albumin protein solutions up to a protein concentration of 400 mg ml−1. A careful selection of experiments allows for fast data collection of a large amount of data, spanning a protein concentration/solvent concentration space with limited sample consumption (down to 3 µL per experiment) and few measurements. The data analysis shows data of high quality that are reproducible and comparable with data from standard flow-through capillary-based experiments. Furthermore, using this methodology, it is possible to achieve concentrations that would not be accessible by conventional cells. The protein concentration and ionic strength parameter space diagram may be covered easily and the amount of protein sample is significantly reduced (by a factor of 100 in this work). Used in routine measurements, the benefits in terms of protein cost and time spent are very significant.

scholarly journals Smaller capillaries improve the small-angle X-ray scattering signal and sample consumption for biomacromolecular solutions

2018 ◽  
Vol 25 (4) ◽  
pp. 1113-1122 ◽  
Author(s):  
Martin A. Schroer ◽  
Clement E. Blanchet ◽  
Andrey Yu. Gruzinov ◽  
Melissa A. Gräwert ◽  
Martha E. Brennich ◽  
...  
Keyword(s):  
Small Angle ◽  
A Priori ◽  
Protein Solutions ◽  
Biological Macromolecules ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
A Cell ◽  
Flow Through ◽  
Ray Scattering

Radiation damage by intense X-ray beams at modern synchrotron facilities is one of the major complications for biological small-angle X-ray scattering (SAXS) investigations of macromolecules in solution. To limit the damage, samples are typically measured under a laminar flow through a cell (typically a capillary) such that fresh solution is continuously exposed to the beam during measurement. The diameter of the capillary that optimizes the scattering-to-absorption ratio at a given X-ray wavelength can be calculated a priori based on fundamental physical properties. However, these well established scattering and absorption principles do not take into account the radiation susceptibility of the sample or the often very limited amounts of precious biological material available for an experiment. Here it is shown that, for biological solution SAXS, capillaries with smaller diameters than those calculated from simple scattering/absorption criteria allow for a better utilization of the available volumes of radiation-sensitive samples. This is demonstrated by comparing two capillary diameters d i (d i = 1.7 mm, close to optimal for 10 keV; and d i = 0.9 mm, which is nominally sub-optimal) applied to study different protein solutions at various flow rates. The use of the smaller capillaries ultimately allows one to collect higher-quality SAXS data from the limited amounts of purified biological macromolecules.

The Oxford Handbook of the Operatic Canon

2020 ◽  
Keyword(s):  
Musical Theater ◽  
Geographical Location ◽  
The United States ◽  
Late Nineteenth Century ◽  
Limited Sample ◽  
The United Kingdom ◽  
Production And Consumption ◽  
Formation History ◽  
Artistic Directors ◽  
Selection Of

This collection examines the phenomenon of the operatic canon: its formation, history, current ontology and practical influence, and future. It does so by taking an international and interdisciplinary view: the workshops from which it was derived included the participation of critics, producers, artistic directors, stage directors, opera company CEOs, and even economists, from the United Kingdom, the United States, France, Italy, Ireland, Germany, the Netherlands, Denmark, and Canada. The volume is structured as a series of dialogues: each subtopic is addressed by two essays, introduced jointly by the authors, and followed by a jointly compiled list of further reading. These paired essays complement each other in different ways, for example by treating the same geographical location in different periods, by providing different national or regional perspectives on the same period, or by thinking through similar conceptual issues in contrasting milieus. Part I consists of a selection of surveys of operatic production and consumption contexts in France, Italy, Germany, England, Russia, and the Americas, arranged in rough order from the late seventeenth century to the late nineteenth century. Part II is a (necessarily) limited sample of subjects that illuminate the operatic canon from different—sometimes intentionally oblique—angles, ranging from the influence of singers to the contiguous genres of operetta and musical theater, and the effects of recording and broadcast over almost 150 years. The volume concludes with two essays written by prominent figures from the opera industry who give their sense of the operatic canon’s evolution and prospects.

Structural refinement by restrained molecular-dynamics algorithm with small-angle X-ray scattering constraints for a biomolecule

2004 ◽  
Vol 37 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Masaki Kojima ◽  
Alexander A. Timchenko ◽  
Junichi Higo ◽  
Kazuki Ito ◽  
Hiroshi Kihara ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Small Angle ◽  
Protein Structures ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Saxs Curve ◽  
Restrained Molecular Dynamics ◽  
Ray Scattering

A new algorithm to refine protein structures in solution from small-angle X-ray scattering (SAXS) data was developed based on restrained molecular dynamics (MD). In the method, the sum of squared differences between calculated and observed SAXS intensities was used as a constraint energy function, and the calculation was started from given atomic coordinates, such as those of the crystal. In order to reduce the contribution of the hydration effect to the deviation from the experimental (objective) curve during the dynamics, and purely as an estimate of the efficiency of the algorithm, the calculation was first performed assuming the SAXS curve corresponding to the crystal structure as the objective curve. Next, the calculation was carried out with `real' experimental data, which yielded a structure that satisfied the experimental SAXS curve well. The SAXS data for ribonuclease T1, a single-chain globular protein, were used for the calculation, along with its crystal structure. The results showed that the present algorithm was very effective in the refinement and adjustment of the initial structure so that it could satisfy the objective SAXS data.

scholarly journals Structural Analysis of the Partially Disordered Protein EspK from Mycobacterium tuberculosis

Crystals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 18
Author(s):  
Abril Gijsbers ◽  
Nuria Sánchez-Puig ◽  
Ye Gao ◽  
Peter J. Peters ◽  
Raimond B. G. Ravelli ◽  
...  
Keyword(s):  
Host Response ◽  
Limited Proteolysis ◽  
Maximal Dimension ◽  
Globular Domain ◽  
Saxs Data ◽  
X Ray ◽  
Disordered Protein ◽  
X Ray Scattering ◽  
C Terminus ◽  
New Treatments

For centuries, tuberculosis has been a worldwide burden for human health, and gaps in our understanding of its pathogenesis have hampered the development of new treatments. ESX-1 is a complex machinery responsible for the secretion of virulence factors that manipulate the host response. Despite the importance of these secreted proteins for pathogenicity, only a few of them have been structurally and functionally characterised. Here, we describe a structural study of the ESX-secretion associated protein K (EspK), a 74 kDa protein known to be essential for the secretion of other substrates and the cytolytic effects of ESX-1. Small-Angle X-ray Scattering (SAXS) data show that EspK is a long molecule with a maximal dimension of 228 Å. It consists of two independent folded regions at each end of the protein connected by a flexible unstructured region driving the protein to coexist as an ensemble of conformations. Limited proteolysis identified a 26 kDa globular domain at the C-terminus of the protein consisting of a mixture of α-helices and β-strands, as shown by circular dichroism (CD) and SAXS. In contrast, the N-terminal portion is mainly helical with an elongated shape. Sequence conservation suggests that this architecture is preserved amongst the different mycobacteria species, proposing specific roles for the N- and C-terminal domains assisted by the middle flexible linker.

scholarly journals Universal nature of collapsibility in the context of protein folding and evolution

2018 ◽  
Author(s):  
D. Thirumalai ◽  
Himadri S. Samanta ◽  
Hiranmay Maity ◽  
Govardhan Reddy
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Saxs Data ◽  
X Ray ◽  
Model Free ◽  
X Ray Scattering ◽  
Theoretical Predictions ◽  
Helical Proteins ◽  
Universal Nature

AbstractTheory and simulations predicted sometime ago that the sizes of unfolded states of globular proteins should decrease continuously as the denaturant concentration is shifted from a high to a low value. However, small angle X-ray scattering (SAXS) data were used to assert the opposite, while interpretation of single molecule Forster resonance energy transfer experiments (FRET) supported the theoretical predictions. The disagreement between the two experiments is the SAXS-FRET controversy. By harnessing recent advances in SAXS and FRET experiments and setting these findings in the context of a general theory and simulations, we establish that compaction of unfolded states is universal. The theory also predicts that proteins rich in β-sheets are more collapsible than α-helical proteins. Because the extent of compaction is small, experiments have to be accurate and their interpretations should be as model free as possible. Theory also suggests that collapsibility itself could be a physical restriction on the evolution of foldable sequences, and provides a physical basis for the origin of multi-domain proteins.

scholarly journals Plasmodium falciparum Kelch13 and its artemisinin-resistant mutants assemble as hexamers in solution: a SAXS data driven shape restoration study

2021 ◽  
Author(s):  
Nainy Goel ◽  
Kanika Dhiman ◽  
Nidhi Kalidas ◽  
Anwesha Mukhopadhyay ◽  
Ashish ◽  
...  
Keyword(s):  
Artemisinin Resistance ◽  
Resistant Mutant ◽  
Data Driven ◽  
Wild Type ◽  
Saxs Data ◽  
Mutant Proteins ◽  
X Ray ◽  
X Ray Scattering ◽  
Shape Restoration ◽  
Spatial Positioning

AbstractArtemisinin-resistant mutations in PfKelch13 identified worldwide are mostly confined to its BTB/POZ and KRP domains. To date, only two crystal structures of the BTB/POZ-KRP domains as tight dimers are available, which limits structure-based interpretations of its functionality. Our solution Small-Angle X-ray Scattering (SAXS) data driven shape restoration of larger length of protein brought forth that: i) PfKelch13 forms a stable hexamer in P6 symmetry, ii) interactions of the N-termini drive the hexameric assembly, and iii) the six KRP domains project independently in space, forming a cauldron-like architecture. While artemisinin-sensitive mutant A578S packed like the wild-type, hexameric assemblies of dominant artemisinin-resistant mutant proteins R539T and C580Y displayed detectable differences in spatial positioning of their BTB/POZ-KRP domains. Lastly, mapping of mutations known to enable artemisinin resistance explained that most mutations exist mainly in these domains because they are non-detrimental to assembly of mutant PfKelch13 and yet can alter the flux of downstream events essential for susceptibility to artemisinin.

scholarly journals Not the story you want? Assessing the fit of a conceptual framework characterising mental health recovery narratives

2019 ◽  
Vol 55 (3) ◽  
pp. 295-308 ◽  
Author(s):  
Joy Llewellyn-Beardsley ◽  
Stefan Rennick-Egglestone ◽  
Simon Bradstreet ◽  
Larry Davidson ◽  
Donna Franklin ◽  
...  
Keyword(s):  
Mental Health ◽  
Conceptual Framework ◽  
Intervention Development ◽  
Mental Health Recovery ◽  
Research Use ◽  
Narrative Construction ◽  
Limited Sample ◽  
Recovery Narratives ◽  
Recovery Approach ◽  
Selection Of

Abstract Purpose Narratives of recovery have been central to the development of the recovery approach in mental health. However, there has been a lack of clarity around definitions. A recent conceptual framework characterised recovery narratives based on a systematic review and narrative synthesis of existing literature, but was based on a limited sample. The aims of this study were to assess the relevance of the framework to the narratives of more diverse populations, and to develop a refined typology intended to inform narrative-based research, practice and intervention development. Method 77 narrative interviews were conducted with respondents from four under-researched mental health sub-populations across England. Deductive and inductive analysis was used to assess the relevance of the dimensions and types of the preliminary typology to the interview narratives. Results Five or more dimensions were identifiable within 97% of narratives. The preliminary typology was refined to include new definitions and types. The typology was found not to be relevant to two narratives, whose narrators expressed a preference for non-verbal communication. These are presented as case studies to define the limits of the typology. Conclusion The refined typology, based on the largest study to date of recovery narratives, provides a defensible theoretical base for clinical and research use with a range of clinical populations. Implications for practice include ensuring a heterogeneous selection of narratives as resources to support recovery, and developing new approaches to supporting non-verbal narrative construction.

Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering

2010 ◽  
Vol 43 (2) ◽  
pp. 237-243 ◽  
Author(s):  
Shuji Akiyama
Keyword(s):  
Quality Control ◽  
Molecular Mass ◽  
Small Angle ◽  
Biological Macromolecules ◽  
Average Deviation ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Standard Quality ◽  
Ray Scattering

Small-angle X-ray scattering (SAXS) is a powerful technique with which to evaluate the size and shape of biological macromolecules in solution. Forward scattering intensity normalized relative to the particle concentration,I(0)/c, is useful as a good measure of molecular mass. A general method for deducing the molecular mass from SAXS data is to determine the ratio ofI(0)/cof a target protein to that of a standard protein with known molecular mass. The accuracy of this interprotein calibration is affected considerably by the monodispersity of the prepared standard, as well as by the precision in estimating its concentration. In the present study, chromatographic fractionation followed by hydrodynamic characterization is proposed as an effective procedure by which to prepare a series of monodispersed protein standards. The estimation of molecular mass within an average deviation of 8% is demonstrated using monodispersed bovine serum albumin as a standard. The present results demonstrate the importance of protein standard quality control in order to take full advantage of interprotein calibration.

scholarly journals Structural Study of the Photo-Mediated Growth of Silver Nanoprisms

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5413
Author(s):  
Matti Knaapila ◽  
Ulla Vainio ◽  
Sophie E. Canton ◽  
Gunnel Karlsson
Keyword(s):  
Particle Size ◽  
Small Angle ◽  
Volume Ratio ◽  
Direct Methods ◽  
Saxs Data ◽  
Aqueous Dispersions ◽  
X Ray ◽  
Silver Nanoprisms ◽  
X Ray Scattering ◽  
Ag Particles

We present a small-angle X-ray scattering (SAXS) study of the anisotropic photoinduced growth of silver (Ag) nanoprisms in aqueous dispersions. The growth of nearly spherical (<10 nm) Ag particles into large (>40 nm) and thin (<10 nm) triangular nanoprisms induced by 550 nm laser is followed in terms of particle size using indirect and direct methods for irradiation times up to 150 min. During the process, the surface-to-volume ratio of the particles decreased. The SAXS data of the initial solution fit well to the model of polydisperse spheres with pronounced average diameters around 7.4 nm and 10 nm. The data after 45 min irradiation fit well to the model containing approximately the same amount of the initial particles and the end product, the nanoprisms.

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