This volume 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
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.
Contents References 11 II. A short historical survey 13 References 16 III. Ribosomes within the cell 20 1. Proportion of ribosomes in tissues 21 2. Free and membrane-bound ribo- mes 22 3. Ribosomes in nucleoli 26 4. Structure of polysomes in the cell 26 5. Ribosomal crystals 33 6. References 33 IV. Some general properties of ribosomes 41 1. Physical characteristics 41 2. Chemical characteristics 43 3. References 44 V. Morphology of ribosomes and poly somes 47 1. Electron microscopy 47 a) The small ribosomal subunit 48 b) The large ribosomal subunit 48 c) The monomeric ribosome 52 d) Three-dimensional models 54 e) Polysomes 55 2. Small-angle X-ray scattering 55 a) The monomeric ribosome 56 b) Polysomes 57 3. References 57 VI. Chemical components 61 1. Ribosomal proteins 61 a) Electrophoretic separation and num ber 61 b) Preparation of single ribosomal pro teins 67 c) Molecular weights 68 d) Amino acid composition and amino acid sequences 71 e) Stoichiometry 71 f) Posttranslational modifications 72 g) Comparison of ribosomal proteins of different tissues 73 h) Species specificities and evolution 74 i) Ribosomal proteins of mitochondria and chloroplasts 75 k) Ribosomal mutants with altered pro teins 75 5 3. Formation of preribosomal 2. Phosphorylation of ribosomal prote particles 139 ins 76 4. References 141 a) In vitro phosphorylation and de phosphorylation 76 VIII. Dissociation - reassociation processes b) In vivo phosphorylation 77 of ribosomal particles 151 c) Phosphorylation of ribosomal pro tein S 6 78 1.
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
With the most comprehensive and up-to-date overview of structure-based drug discovery covering both experimental and computational approaches, Structural Biology in Drug Discovery: Methods, Techniques, and Practices describes principles, methods, applications, and emerging paradigms of structural biology as a tool for more efficient drug development. Coverage includes successful examples, academic and industry insights, novel concepts, and advances in a rapidly evolving field. The combined chapters, by authors writing from the frontlines of structural biology and drug discovery, give readers a valuable reference and resource that: Presents the benefits, limitations, and potentiality of major techniques in the field such as X-ray crystallography, NMR, neutron crystallography, cryo-EM, mass spectrometry and other biophysical techniques, and computational structural biology Includes detailed chapters on druggability, allostery, complementary use of thermodynamic and kinetic information, and powerful approaches such as structural chemogenomics and fragment-based drug design Emphasizes the need for the in-depth biophysical characterization of protein targets as well as of therapeutic proteins, and for a thorough quality assessment of experimental structures Illustrates advances in the field of established therapeutic targets like kinases, serine proteinases, GPCRs, and epigenetic proteins, and of more challenging ones like protein-protein interactions and intrinsically disordered proteins
This book highlights important techniques for cellular imaging and covers the basics and applications of electron tomography and related techniques. In addition, it considers practical aspects and broadens the technological focus by incorporating techniques that are only now becoming accessible (e.g. block face imaging). The first part of the book describes the electron microscopy 3D technique available to scientists around the world, allowing them to characterize organelles, cells and tissues. The major emphasis is on new technologies like scanning transmission electron microscopy (STEM) tomography, though the book also reviews some of the more proven technologies like electron tomography. In turn, the second part is dedicated to the reconstruction of data sets, signal improvement and interpretation
A guide to modern scanning electron microscopy instrumentation, methodology and techniques, highlighting novel applications to cell and molecular biology.
Three-Dimensional Electron Microscopy of Macromolecular Assemblies is the first systematic introduction to single-particle methods of reconstruction. It covers correlation alignment, classification, 3D reconstruction, restoration, and interpretation of the resulting 3D images in macromolecular assemblies. It will be an indispensable resource for newcomers to the field and for all using or adopting these methods.Key Features* Presents methods that offer an alternative to crystallographic techniques for molecules that cannot be crystallized* Describes methods that have been instrumental in exploring the three-dimensional structure of* the nuclear pore complex* the calcium release channel;* the ribosome* chaperonins
