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Small-Angle X-Ray Scattering for Macromolecular Complexes
Adv Exp Med Biol. 2024;3234:163-172. doi: 10.1007/978-3-031-52193-5_11.ABSTRACTSmall angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including s...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Stephanie Hutin Mark D Tully Martha Brennich Source Type: research

Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines
Adv Exp Med Biol. 2024;3234:173-190. doi: 10.1007/978-3-031-52193-5_12.ABSTRACTHigh-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buff...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Aur élien Deniaud Burak V Kabasakal Joshua C Bufton Christiane Schaffitzel Source Type: research

Characterization of Complexes and Supramolecular Structures by Electron Microscopy
Adv Exp Med Biol. 2024;3234:191-205. doi: 10.1007/978-3-031-52193-5_13.ABSTRACTRecent advancements in cryo-electron microscopy (cryo-TEM) have enabled the determination of structures of macromolecular complexes at near-atomic resolution, establishing it as a pivotal tool in Structural Biology. This high resolution allows for the detection of ligands and substrates under physiological conditions. Enhancements in detectors and imaging devices, like phase plates, improve signal quality, facilitating the reconstruction of even smaller macromolecular complexes. The 100-kDa barrier has been surpassed, presenting new opportunitie...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Jos é L Carrascosa Source Type: research

Immunoprecipitation Methods to Isolate Messenger Ribonucleoprotein Complexes (mRNP)
Adv Exp Med Biol. 2024;3234:1-15. doi: 10.1007/978-3-031-52193-5_1.ABSTRACTThroughout their life cycle, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Each mRNA is part of multiple successive mRNP complexes that participate in their biogenesis, cellular localization, translation and decay. The dynamic composition of mRNP complexes and their structural remodelling play crucial roles in the control of gene expression. Studying the endogenous composition of different mRNP complexes is a major challenge. In this chapter, we describe the variety of protein-centric immunoprecipitation methods ...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Hassan Hayek Lauriane Gross Fatima Alghoul Franck Martin Gilbert Eriani Christine Allmang Source Type: research

Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods
Adv Exp Med Biol. 2024;3234:17-29. doi: 10.1007/978-3-031-52193-5_2.ABSTRACTThroughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Aur élie Janvier Hassan Hayek Fatima Alghoul Lauriane Gross Christine Allmang Franck Martin Gilbert Eriani Source Type: research

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes
Adv Exp Med Biol. 2024;3234:31-40. doi: 10.1007/978-3-031-52193-5_3.ABSTRACTIn the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample pr...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Gianluca Degliesposti Source Type: research

Probing Protein Complexes Composition, Stoichiometry, and Interactions by Peptide-Based Mass Spectrometry
Adv Exp Med Biol. 2024;3234:41-57. doi: 10.1007/978-3-031-52193-5_4.ABSTRACTThe characterization of a protein complex by mass spectrometry can be conducted at different levels. Initial steps regard the qualitative composition of the complex and subunit identification. After that, quantitative information such as stoichiometric ratios and copy numbers for each subunit in a complex or super-complex is acquired. Peptide-based LC-MS/MS offers a wide number of methods and protocols for the characterization of protein complexes. This chapter concentrates on the applications of peptide-based LC-MS/MS for the qualitative, quantita...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Gianluca Degliesposti Source Type: research

Discovery and Characterization of Linear Motif Mediated Protein-Protein Complexes
Adv Exp Med Biol. 2024;3234:59-71. doi: 10.1007/978-3-031-52193-5_5.ABSTRACTThere are myriads of protein-protein complexes that form within the cell. In addition to classical binding events between globular domains, many protein-protein interactions involve short disordered protein regions. The latter contain so-called linear motifs binding specifically to ordered protein domain surfaces. Linear binding motifs are classified based on their consensus sequence, where only a few amino acids are conserved. In this chapter we will review experimental and in silico techniques that can be used for the discovery and characterizati...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Andr ás Zeke Anita Alexa Attila Rem ényi Source Type: research

Protein-Protein Binding Kinetics by Biolayer Interferometry
Adv Exp Med Biol. 2024;3234:73-88. doi: 10.1007/978-3-031-52193-5_6.ABSTRACTThe specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, i...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Jorge Santos-L ópez Sara G ómez Francisco J Fern ández M Cristina Vega Source Type: research

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods
Adv Exp Med Biol. 2024;3234:89-107. doi: 10.1007/978-3-031-52193-5_7.ABSTRACTCellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and the...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Carlos Alfonso Marta Sobrinos-Sanguino Juan Rom án Luque-Ortega Silvia Zorrilla Bego ña Monterroso Oscar M Nuero Germ án Rivas Source Type: research

The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions
Adv Exp Med Biol. 2024;3234:109-123. doi: 10.1007/978-3-031-52193-5_8.ABSTRACTNuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in ...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Elisabetta Boeri Erba Annalisa Pastore Source Type: research

X-Ray Crystallography for Macromolecular Complexes
Adv Exp Med Biol. 2024;3234:125-140. doi: 10.1007/978-3-031-52193-5_9.ABSTRACTX-ray crystallography has for most of the last century been the standard technique to determine the high-resolution structure of biological macromolecules, including multi-subunit protein-protein and protein-nucleic acids as large as the ribosome and viruses. As such, the successful application of X-ray crystallography to many biological problems revolutionized biology and biomedicine by solving the structures of small molecules and vitamins, peptides and proteins, DNA and RNA molecules, and many complexes-affording a detailed knowledge of the st...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Francisco J Fern ández Javier Querol-Garc ía Sergio Navas-Yuste Fabrizio Martino M Cristina Vega Source Type: research

Characterization of Biological Samples Using Ultra-Short and Ultra-Bright XFEL Pulses
Adv Exp Med Biol. 2024;3234:141-162. doi: 10.1007/978-3-031-52193-5_10.ABSTRACTThe advent of X-ray Free Electron Lasers (XFELs) has ushered in a transformative era in the field of structural biology, materials science, and ultrafast physics. These state-of-the-art facilities generate ultra-bright, femtosecond-long X-ray pulses, allowing researchers to delve into the structure and dynamics of molecular systems with unprecedented temporal and spatial resolutions. The unique properties of XFEL pulses have opened new avenues for scientific exploration that were previously considered unattainable. One of the most notable applic...
Source: Advances in Experimental Medicine and Biology - March 20, 2024 Category: Research Authors: Adam Round E Jungcheng Carsten Fortmann-Grote Klaus Giewekemeyer Rita Graceffa Chan Kim Henry Kirkwood Grant Mills Ekaterina Round Tokushi Sato Sakura Pascarelli Adrian Mancuso Source Type: research