Large macromolecular protein complexes play an important role in the quest of un- derstanding life at a molecular level. For this endeavor, the highly-resolved 3D structure of a large complex and its changes, which are called conformational dy- namics, are of high interest due to their close relation to biological function. A pop- ular method to gain access to this information is cryo-electron microscopy. Here, biological samples are vitrified in their native buffer conditions and then im- aged in a transmission electron microscope. The large amount of recorded noisy images, called microgra...
Cryo-EM Part A: Sample Preparation and Data Collection is dedicated to a description of the instruments, samples, protocols, and analyses that belong to cryo-EM. It emphasizes the relatedness of the ideas, instrumentation, and methods underlying all cryo-EM approaches, which allow practitioners to easily move between them. Within each section, the articles are ordered according to the most common symmetry of the sample to which their methods are applied. Includes time-tested core methods and new innovations applicable to any researcher Methods included are useful to both established researchers and newcomers to the field Relevant background and reference information given for procedures can be used as a guide
The book reproduces 55 of more than 300 articles written by the author, representing milestones in methods development of single-particle cryo-EM as well as important results obtained by this technique in the study of biological macromolecules and their interactions. Importantly, neither symmetries nor ordered arrangements (as in two-dimensional crystals, helical assemblies, icosahedral viruses) are required. Although the biological applications are mainly in the area of ribosome structure and function, the elucidation of membrane channel structures and their activation and gating mechanisms are represented, as well. The book is introduced by a commentary that explains the original development of concepts, describes the contributions of the author's colleagues and students, and shows how challenges were overcome as the technique matured. Along the way, the ribosome served as an example for a macromolecule with intricate structure and conformational dynamics that pose challenges for three-dimensional visualization. Toward the end of the book -- bringing us to the present time -- molecular structures with near-atomic resolution are presented, and a novel type of computational analysis, manifold embedding, is introduced. Single-particle cryo-EM is currently revolutionizing structural biology, presenting a powerful alternative to X-ray crystallography as a means to solve the structure of biological macromolecules. The book presents in one place a number of articles containing key advances in mathematical and computational methods leading up to the present time. Secondly, the development of the technique over the years is reflected by ever-expanding discoveries in the field of ribosome structure and function. Thirdly, as all histories of ideas, the history of concepts pertaining to this new method of visualization is fascinating all in itself.
Cryo-electron Tomography: A Journey from Sample Preparation to Data Mining offers a comprehensive guide to the latest state-of-the-art methods and techniques available to researchers. The book begins with a section on sample preparation, considering a range of different sample types including virus-like particles, viruses, bacteria, organoids and tissues, with some chapters dedicated to specific tools like in situ cellular tomography and correlative techniques. The book then moves on to a section on data mining and specific steps and methods, delivering a complete overview of this advancing technique and providing researchers with the knowledge and tools required for their own investigations. Provides a detailed overview of cryo-ET sample preparation and data mining protocols and approaches in a single resource Includes foundational knowledge underpinning the field Explores the latest methods, techniques and recent developments in cryo-ET Offers step-by-step guidance to researchers for key procedures Written by an international range of experts
Cryo-electron microscopy, in combination with tomography, has emerged as a new technology for visualizing molecular structures at a resolution beyond even 1 Å. Using this technology has revealed the native molecular details of viruses, membranes, enzymes, ribosomes, and cells. This comprehensive volume brings together authoritative overviews of these methods from structural and biological perspectives. It is a must-have for researchers and graduate students, as well as those working in industry, primarily in the areas of biophysics, structural biology, crystallography, and genomics. Key Features • Focuses on the applications of cryo-EM to structural biology • Documents the importance of cryo-EM/ET approaches in studying the structural determinants of cellular organelle and membrane protein biochemistry • Reviews the applications of high-resolution structures of viruses • Emphasizes structural insights of nuclear and gene machineries • Includes a section focused entirely on the applications of cryo-EM/ET in drug discovery and therapeutic development
Advances in Protein Molecular and Structural Biology Methods offers a complete overview of the latest tools and methods applicable to the study of proteins at the molecular and structural level. The book begins with sections exploring tools to optimize recombinant protein expression and biophysical techniques such as fluorescence spectroscopy, NMR, mass spectrometry, cryo-electron microscopy, and X-ray crystallography. It then moves towards computational approaches, considering structural bioinformatics, molecular dynamics simulations, and deep machine learning technologies. The book also covers methods applied to intrinsically disordered proteins (IDPs)followed by chapters on protein interaction networks, protein function, and protein design and engineering. It provides researchers with an extensive toolkit of methods and techniques to draw from when conducting their own experimental work, taking them from foundational concepts to practical application. Presents a thorough overview of the latest and emerging methods and technologies for protein study Explores biophysical techniques, including nuclear magnetic resonance, X-ray crystallography, and cryo-electron microscopy Includes computational and machine learning methods Features a section dedicated to tools and techniques specific to studying intrinsically disordered proteins
cryoEM, a new volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods and new developments in recording images, the creation, evaluation and validation of 3D maps from the images, model building into maps and refinement of the resulting atomic structures, and applications of essentially single particle methods to helical structures and to sub-tomogram averaging. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods that determine the structures of biological molecules, a vital step for understanding their function Contains the technical developments underpinning the advances of cryoEM and captures the exciting insights that have resulted
Biophysical Approaches for the Study of Membrane Structure, Part A, Volume 700 explores lipid membrane asymmetry and lateral heterogeneity. A burst of recent research has shown that bilayers whose leaflets differ in their physical properties—such as composition, phase state, or lateral stress—exhibit many fascinating new characteristics, but also pose a host of new challenges related to their creation, characterization, simulation, and theoretical description. Chapters in this new release include Evaluation of functional transbilayer coupling in live cells by controlled lipid exchange and imaging FCS, Effects of lateral and hydrostatic pressure on membrane structure and properties, and much more. Other sections cover Using the yeast vacuole as a system to test the lipid drivers of membrane heterogeneity in living cells, Direct quantification of cellular membrane lipids using ratiometric fluorescence sensors, The spectral phasor approach to resolving membrane order with environmentally sensitive dyes, The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains, Advanced microscopy methods to study membrane pores, Use of cryo-EM to study membrane phase separation, and much more. Explore the state-of-the-art of lipid membrane asymmetry Covers experimental, theoretical, and computational techniques to create and characterize asymmetric lipid membranes Teaches how these kinds of approaches create and characterize laterally inhomogeneous membranes
