Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility.

RNA-binding proteins (RBPs) are crucial regulators of gene expression, often composed of defined domains interspersed with flexible, intrinsically disordered regions. Determining the structure of ribonucleoprotein (RNP) complexes involving such RBPs necessitates integrative structural modeling due to their lack of a single stable state. In this study, we integrate magnetic resonance, mass spectrometry, and small-angle scattering data to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1/hnRNP I) bound to an RNA fragment from the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV).

Guidelines for DC preparation and flow cytometry analysis of mouse lymphohematopoietic tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human DC from lymphoid organs, and various non-lymphoid tissues. Within this chapter, detailed protocols are presented that allow for the generation of single-cell suspensions from mouse lymphohematopoietic tissues including spleen, peripheral lymph nodes, and thymus, with a focus on the subsequent analysis of DC by flow cytometry. However, prepared single-cell suspensions can be subjected to other applications including sorting and cellular enrichment procedures, RNA sequencing, Western blotting, and many more.

Resolving distance variations by single-molecule FRET and EPR spectroscopy using rotamer libraries.

Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy are complementary techniques for quantifying distances in the nanometer range. Both approaches are commonly employed for probing the conformations and conformational changes of biological macromolecules based on site-directed fluorescent or paramagnetic labeling. FRET can be applied in solution at ambient temperature and thus provides direct access to dynamics, especially if used at the single-molecule level, whereas EPR requires immobilization or work at cryogenic temperatures but provides data that can be more reliably used to extract distance distributions.

Intermolecular background decay in RIDME experiments.

The relaxation-induced dipolar modulation enhancement (RIDME) technique allows the determination of distances and distance distributions in pairs containing two paramagnetic metal centers, a paramagnetic metal center and an organic radical, and, under some conditions, also in pairs of organic radicals. The strengths of the RIDME technique are its simple setup requirements, and the absence of bandwidth limitations for spin inversion which occurs through relaxation. A strong limitation of the RIDME technique is the background decay, which is often steeper than that in the double electron electron resonance experiment, and the absence of an appropriate description of the intermolecular background signal.

Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure.

Morphological and molecular characteristics determine the function of biological tissues. Attempts to combine immunofluorescence and electron microscopy invariably compromise the quality of the ultrastructure of tissue sections. We developed NATIVE, a correlated light and electron microscopy approach that preserves ultrastructure while showing the locations of multiple molecular moieties, even deep within tissues.