The Resolution Revolution in CryoEM Requires New Sample Preparation Procedures: a Rapid Pipeline to High Resolution Maps of Yeast FAS PDF Download

Author: Mirko Joppe
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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Book Description
Single-particle electron cryo-microscopy (cryoEM) has undergone a “resolution revolution” that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

Author: Mirko Joppe
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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Book Description
Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6%MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1%Å resolution map of yeast FAS from 15%000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.

Author:
Publisher: Academic Press
ISBN: 0128054352
Category : Science
Languages : en
Pages : 488

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Book Description
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

Author: David S. Goodsell
Publisher: Springer
ISBN: 3319325108
Category : Science
Languages : en
Pages : 179

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Book Description
This book will take an evidence-based approach to current knowledge about biomolecules and their place in our lives, inviting readers to explore how we know what we know, and how current gaps in knowledge may influence the way we approach the information. Biomolecular science is increasingly important in our everyday life, influencing the choices we make about our diet, our health, and our wellness. Often, however, information about biomolecular science is presented as a list of immutable facts, discouraging critical thought. The book will introduce the basic tools of structural biology, supply real-life examples, and encourage critical thought about aspects of biology that are still not fully understood.

Author: Joachim Frank
Publisher: World Scientific Publishing Company
ISBN: 9789813234857
Category : Electron microscopy
Languages : en
Pages : 0

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Book Description
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.

Author: Amitabha Chattopadhyay
Publisher: Springer
ISBN: 3319666010
Category : Science
Languages : en
Pages : 387

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Book Description
This volume brings together information on membrane organization and dynamics from a variety of spectroscopic, microscopic and simulation approaches, spanning a broad range of time scales. The implication of such dynamic information on membrane function in health and disease is a topic of contemporary interest. The chapters cover various aspects of membrane lipid and protein dynamics, explored using a battery of experimental and theoretical approaches. The synthesis of information and knowledge gained by utilizing multiple approaches will provide the reader with a comprehensive understanding of the underlying membrane dynamics and function, which will help to develop robust dynamic models for the understanding of membrane function in healthy and diseased states. In the last few years, crystal structures of an impressive number of membrane proteins have been reported, thanks to tremendous advances in membrane protein crystallization techniques. Some of these recently solved structures belong to the G protein-coupled receptor (GPCR) family, which are particularly difficult to crystallize due to their intrinsic flexibility. Nonetheless, these static structures do not provide the necessary information to understand the function of membrane proteins in the complex membrane milieu. This volume will address the dynamic nature of membrane proteins within the membrane and will provide the reader with an up-to date overview of the theory and practical approaches that can be used. This volume will be invaluable to researchers working in a wide range of scientific areas, from biochemistry and molecular biology to biophysics and protein science. Students of these fields will also find this volume very useful. This book will also be of great use to those who are interested in the dynamic nature of biological processes.

Author: Jean-Paul Renaud
Publisher: John Wiley & Sons
ISBN: 1118900502
Category : Medical
Languages : en
Pages : 1367

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Book Description
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

Author: James E. Barrett
Publisher: Springer Nature
ISBN: 3030353621
Category : Medical
Languages : en
Pages : 466

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Book Description
Celebrating 100 years of HEP, this volume will discuss key pharmacological discoveries and concepts of the past 100 years. These discoveries have dramatically changed the medical treatment paradigms of many diseases and these concepts have and will continue to shape discovery of new medicinies. Newly evolving technologies will similarly be discussed as they will shape the future of the pharmacology and, accordingly, medical therapy.

Author:
Publisher: Academic Press
ISBN: 0128170905
Category : Science
Languages : en
Pages : 326

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Book Description
Enzyme Activity in Single Cells, Volume 628, the latest release in the Methods of Enzymology series, discusses groundbreaking cellular physiology research that is taking place in the biological sciences. Chapters in this new release cover Spatial and temporal resolution of caspase waves in single Xenopus eggs during apoptosis, Spatial and temporal organization of metabolic complexes in cells, Measuring cellular efflux and biomolecular delivery: synthetic approaches to imaging and engineering cells, Slide-based, single-cell enzyme assays, Single-cell assays using integrated continuous-flow microfluidics, High-throughput screening of single-cell lysates, Microfluidic capture of single cells for drug resistance assays, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Includes the latest information on Enzyme Activity in Single Cells

Author: Joachim Frank
Publisher: Oxford University Press
ISBN: 0195150961
Category : Computers
Languages : en
Pages : 427

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Book Description
Cryoelectron microscopy of biological molecules is among the hottest growth areas in biophysics and structural biology at present, and Frank is arguably the most distinguished practitioner of this art. CryoEM is likely over the next few years to take over much of the structural approaches currently requiring X-ray crystallography, because one can now get good and finely detailed images of single molecules down to as little as 200,000 MW, covering a substantial share of the molecules of greatest biomedical research interest. This book, the successor to an earlier work published in 1996 with Academic Press, is a natural companion work to our forthcoming book on electron crystallography by Robert Glaeser, with contributions by six others, including Frank. A growing number of workers will employ CryoEM for structural studies in their own research, and a large proportion of biomedical researchers will have a growing interest in understanding what the capabilities and limits of this approach are.