Dataset Information

Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

ABSTRACT: Proteins and nucleic acids, alone and in complex are among the essential building blocks of living organisms. Obtaining a molecular level understanding of their structures, and the changes that occur as they interact, is critical for expanding our knowledge of life processes or disease progression. Here, we motivate and describe an application of solution small angle X-ray scattering (SAXS) which provides valuable information about the structures, ensembles, compositions and dynamics of protein-nucleic acid complexes in solution, in equilibrium and time-resolved studies. Contrast variation (CV-) SAXS permits the visualization of the distinct molecular constituents (protein and/or nucleic acid) within a complex. CV-SAXS can be implemented in two modes. In the simplest, the protein within the complex is effectively rendered invisible by the addition of an inert contrast agent at an appropriate concentration. Under these conditions, the structure, or structural changes of only the nucleic acid component of the complex can be studied in detail. The second mode permits observation of both components of the complex: the protein and the nucleic acid. This approach requires the acquisition of SAXS profiles on the complex at different concentrations of a contrast agent. Here, we review CV-SAXS as applied to protein-nucleic acid complexes in both modes. We provide some theoretical framework for CV-SAXS but focus primarily on providing the necessary information required to implement a successful experiment including experimental design, sample quality assessment, and data analysis.

SUBMITTER: San Emeterio J

PROVIDER: S-EPMC10015503 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

San Emeterio Josue J Pabit Suzette A SA Pollack Lois L

Methods in enzymology 20220922

Proteins and nucleic acids, alone and in complex are among the essential building blocks of living organisms. Obtaining a molecular level understanding of their structures, and the changes that occur as they interact, is critical for expanding our knowledge of life processes or disease progression. Here, we motivate and describe an application of solution small angle X-ray scattering (SAXS) which provides valuable information about the structures, ensembles, compositions and dynamics of protein- ...[more]

PMID: 36410957

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Project description:Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is an increasingly used digital complement, supplement, or alternative to traditional dissection-based anatomical research. The diceCT protocol, which has evolved and expanded over the past decade, employs passive diffusion of Lugol's iodine (KI3) to increase soft tissue radiodensity and improve structure contrast in the CT or microCT imaging of specimens. The development and application of diceCT has focused largely on specimens under 1 kg, and the varying reporting of methods on studies of both small and large specimens has initiated, but not yet established, an effective diceCT protocol for larger specimens based on monitored experiments of several fundamental variables (e.g., Lugol's iodine concentration, duration, and impacts of Lugol's iodine on tissues). In this study, we have experimentally assessed the efficacy of diceCT protocols for imaging whole-body specimens of the 1-4.5 kg Australian brushtail possum (Trichosurus vulpecula) using sequential CT imaging assessment across experimental conditions. We assessed the impact of varying Lugol's iodine concentration, the presence/absence of skin, solution volume and agitation on tissue radiodensity changes through weekly CT-based monitoring of tissue radiodensities over an 8-week experimental period. We have also quantified tissue volumetric changes across our experiment to assess the impact of diceCT applications on subsequent analyses of imaging datasets. Our results indicate that substantial changes in both soft-tissue radiodensity and soft-tissue volume occur within the first 28 days of Lugol's iodine treatment, followed by a slower rate of progressive soft-tissue radiodensity and volume changes across the experiment duration. Our results demonstrate the negligible benefit of skinning larger specimens to improve solution diffusion, and document significant soft-tissue volumetric changes with high concentration solutions (e.g., 10%) and long-duration exposure (e.g., beyond 5 weeks) that should guide individual diceCT protocol design and/or quantification and analysis for mammal specimens above 1 kg.

Dataset Information

Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

Publications

Contrast variation SAXS: Sample preparation protocols, experimental procedures, and data analysis.

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