冷冻电子显微学在结构生物学研究中的现状与展望

Hans Ris – from chromatin fibres, through nuclear tracks, to nuclear pores

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

Type IV collagen forms a network that provides the major structural support of basement membranes. We have determined the nucleotide alterations and phenotypes of 17 mutant alleles of the Caenorhabditis elegans alpha 2(IV) collagen gene let-2. All 17 mutations are within the triple helical (Gly-X-Y) repeat domain of the molecule. Fifteen of the mutations are replacements of Gly-X-Y repeat glycines with aspartate, glutamate or arginine, and they cause a wide range of phenotypes. The mildest alleles are nearly wild-type at 15 and 20 degrees C but embryonic lethal at 25 degrees C, while the most severe allele is embryonic lethal at all three temperatures. Mutations resulting in severe phenotypes are generally located in areas of lower calculated thermal stability of the type IV collagen molecule. An alanine to threonine substitution at position X of a Gly-X-Y triplet immediately following an interruption results in a severe phenotype. This mutation is unusual because substitutions at positions X or Y have not generally been found to cause strong phenotypes in C. elegans or human collagens. An intron splice acceptor mutation causes a strict embryonic lethal phenotype, but does not completely abolish gene function. Pairs of independent mutations affect each of three glycines, indicating a non-random distribution of mutations in the molecule. It is suggested that this clustering results because many glycine substitutions may cause dominant lethal or sterile phenotypes.

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.

Present at the flood: How structural biology came about, by Dickerson, Richard E.. 2005. Sinauer Associates, Sunderland, MA. 307 pp. $34.95 (paper) Robert M. Stroud*, * Department of Biochemistry, University of California at San Francisco, San Francisco, CA 94143, USA Present at the Flood: How Structural Biology Came About is an essential book for every structural biologist and a must for everyone who reflects on the origins of molecular biology. Present at the Flood, authored by Richard E. Dickerson, enchantingly traces the critical path to the discovery of the underlying atomic structures of proteins and DNA. These discoveries became the foundations of modern molecular biology. Present at the Flood interprets the 30 years that revolutionized structural and molecular biology, from 1933 to 1963, and is dedicated to Francis Crick. Francis also helped Dickerson to unearth some of the unpublished materials that illustrate the true history, such as the draft of the first erroneous DNA model. The moments of critical insight and of erroneous ideas are brilliantly captured by Dickerson, who was himself an essential, key ingredient in the discovery of the first protein structure (he was indeed “present at the flood”), and at the same institution where both the myoglobin and hemoglobin protein structures and the structure of DNA were discovered. These discoveries totally revolutionized biology. They were clearly synergistic with each other in a way that depended on the interplay between the personalities just as much as between structural thinkers. Present at the Flood reflects Dickerson's own total engagement and thrill in the process of discovery. It also reflects his keen recollection of the nuance, the slight remark that triggered a cascade of thought—the essence of some of the most important discoveries in all of biology. Like a detective novel, the book draws the reader into the fabric of discovery. It became a reality after the author presented a course of the same title in 2004 at UCLA. Dickerson retreads the pathway that he and colleagues in structural biology walked, carefully reasoning from the knowledge at the time to retrace the steps and ask precisely how insight came about. The book is very different from, for example, James Watson's personal perspective in The Double Helix; rather, it is a historical analysis of the ideas in the progression to discovery and how personalities augmented or bumped the process forward, backward, or sideways. Thus, he recounts mistakes in the early interpretations of Pauling and Corey and Crick and Watson, and recalls Erwin Chargaff's indignation after the DNA structure was determined. This all makes the historical analysis a personal encounter with the players themselves and a delight to read. Each of the first seven chapters begins with a hand-crafted Dickersonian pun! “In this life one should Perutz high goals, but ultimately settle for what one Kendrew”! Then, there follows a list of key publications, some of which are reprinted at the end. In total, over 40 of the original publications are reprinted, an invaluable record in itself. The text focuses onto Dickerson's interpretation of the meaning of research results, the flash of brilliance, the chance encounter, the stumbles, and communications that became the process of scientific discovery. Dickerson reflects colloquially on personalities, and their quirks that coupled to their contributions. The book reiterates the very words of the cast, including the sometimes sarcastic or even mean-spirited responses to competitors. Thus, the reader comes away knowing a lot about the people involved and the interplay of personality in discovery. Dickerson also questions the missed discoveries that could, or even should, have been made earlier—sometimes errors in logic, and happy accidents. The appendices present an invaluable guide to “Highly Recommended Readings,” and identifies those of key contributions, each with commentary as to why they are important milestones. Appendix 3 reflects on Irving Geis, the first artist and illustrator of the meaning of molecular structure who, with Dickerson, illustrated so many of the discoveries throughout the birth of structural biology and with whom Dickerson had a long very productive interaction. One of their books, The Proteins, coauthored by Dickerson and Geis (published in 1969), represented the first picture “from within” of what structural molecular biology revealed. It was also the beginning of the field of “computer graphic” displays of molecular structures. The Flood is dedicated to Francis H.C. Crick, whose contributions, like Dickerson's, were seminal both to the first discovery of the structure of proteins and to the discovery of the structure of DNA. While Crick worked with Perutz on the hemoglobin structure, Dickerson, in 1957, developed the mathematical basis and the key process to refine the “phases” of myoglobin, and thus, to determine its structure. As Crick realized the structure of DNA with Watson and Franklin's diffraction patterns in 1953, Dickerson, in 1980, was the first to determine the atomic structure of B-form DNA at atomic resolution. He recounts Crick's response on seeing Dickerson's atomic “B-form” structure: “So that's what it looks like!” Books written by investigators who shared in the discovery process often have a special intimacy to their story, and this book certainly is rich in this. Dickerson comments on Max Perutz, “Perutz turned out to be a gifted writer and essayist, which is more than many scientists can say.” Even more could be said of Dickerson's consummate ability to engage his readership! Few writers from the inside have been able to capture and analyze the process of discovery as well as Dickerson; he is scholarly, exact, balanced, and selfless. Dickerson's keen humor and insight draws the reader into the fun and excitement at the birth of molecular biology. Present at the Flood is important for the scientific record, as perhaps the only book that focuses on the discovery of protein structure seen in three-dimensional maps of linked atoms alongside and intertwined with the deduction of the structure of DNA. As such, it is a must for all who may reflect on the impact that structural biology has on molecular and cell biology, genetics, proteomics, biochemistry, or drug discovery.

The universe of cell biology is expanding. One way to sense this expansion is to attend a major meeting dedicated to the field, such as that of the American Society for Cell Biology. The poster sessions allow one to take a leisurely stroll through the exhibits and, even without stopping to look at any single poster, get a very good visceral feel for the scope of cell biology. For those who have taken such a stroll periodically over the past decade or two, the dynamic nature of cell biology and its explosive growth have been obvious—there is simply more and more about more and more. Once upon a time, poster sessions were dominated by images of cells fixed (literally) and captured in black and white by electron microscopists. While the electron microscope continues to contribute to our understanding of cell architecture, more recent poster sessions have witnessed the emergence of row upon row of television monitors where one can view computer-enhanced video images of living cells captured in the not-so-living colors of rhodamine red, fluorescein yellow, and the characteristic green that gives the now-famous fluorescent protein its name. Freeze-etching has been joined by freeze-frame. Furthermore, interspersed among the television sets and whirring VCRs are posters that document detailed molecular characterizations of a vast array of biochemical components that comprise the diverse cell types being investigated. Other posters record the latest genes to fall to the seemingly relentless onslaught of the cloners and sequencers, and still others offer functional insights obtained by knocking out these genes. Every point of the compass has its blot! Genetic studies in yeast, flies, and worms abound on the one hand, and on the other, a growing number of the presentations form bridges between basic research in cell biology and the practice of clinical medicine. Clearly, the scientists who today identify themselves as cell biologists are a diverse community, and great value resides in that diversity. Borders that once separated research disciplines have faded, and cell biologists have come to appreciate that no single approach in isolation will open the profound mysteries of the cell. New techniques and new technologies come alongside the tried-and-true as the tools of cell biology expand along with the field. It is this changing face of cell biology and its methodologies that represented the greatest challenge in pulling together Current Protocols in Cell Biology. A foundational question addressed by the editors of this work involved where to draw the boundaries around the field of cell biology. Our decision has been to refuse to draw such boundaries—they are artificial at best and counterproductive at worst. Instead, we will attempt in this effort to match the diversity of our field. We will include “classic” methods that remain valuable tools for the modern cell biologist and will also provide protocols that we believe are destined to become tomorrow's classics. There is no reason to suspect that the expanding universe of cell biology will cease expanding anytime soon. Indeed, part of the thrill of being a cell biologist is being constantly surprised by new innovations and discoveries. As a consequence, however, our community needs a reliable and user-friendly source of laboratory methods that is as expansive as the field itself. To begin to address this need, we have assembled a series of protocols that cover many aspects of cell biology. While this set of methods is incomplete, it can be considered a “starter toolbox” that includes many of the most versatile and essential instruments of our trade. This collection will expand as regular quarterly supplements are added to Current Protocols in Cell Biology. Through these supplements, the editors will endeavor to make the toolbox increasingly more useful over time. Because of the rapid pace of innovation and discovery in cell biology, we are expecting that our efforts will be met with some degree of very pleasant frustration as we strive to keep up with this fast-moving and exciting field. Subjects in this manual are organized by chapters, and protocols are contained in units. Protocol units, which constitute the bulk of the book, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Other discussion units present more general information. Page numbering reflects the modular arrangement by unit; for example, page 1.2.3 refers to Chapter 1 (Cell Culture), UNIT Unavailable (Media for Culture of Mammalian Cells), page 3 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used and recipes for common reagents are supplied in APPENDIX Unavailable. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Because this publication is first and foremost a compilation of laboratory techniques in cell biology, we have included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. One or more basic protocols are presented first in each unit and generally cover the recommended or most universally applicable approaches. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., RIPA buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are provided once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, and equipment. In some cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use Committee (IACUC) or must conform to appropriate governmental regulations regarding the care and use of laboratory animals. Written approval from the IACUC (or equivalent) committee is absolutely required prior to undertaking any live-animal studies. Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your IACUC or governmental guidelines to obtain more extensive information. Most of the protocols included in this manual are used routinely in the authors' laboratories. These protocols work for them; to make them work for you they have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. We encourage readers to send their comments to currentprotocols@wiley.com. Another valuable resource is the companion website http://www.currentprotocols.com. This site features tools, calculators, apps, troubleshooting tips, webinars, and videos to assist in the experiments presented in this manual. The individual article pages provide valuable summary information and easy access to the content. While the editorial board enlists contributors for most of the topics presented in this manual, we invite individuals to submit letters of intent describing new topics that they would like to publish in CPCB. The editorial board will carefully consider suggested topics and invite authors to submit full manuscripts in some cases. Letters of intent may be submitted by email to currentprotocols@wiley.com. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work by the Current Protocols editorial staff at John Wiley and Sons. We are extremely grateful for the critical contributions by Kathy Morgan (Series Editor) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Janet Blair, Kathy Wisch, Michael Gates, Demetra Kagdis, Alice Ro, and Scott Holmes. The extensive copyediting required to produce an accurate protocols manual was ably handled by Rebecca Barr, Allen Ranz, Elizabeth Harkins, Lisa Christenson, Connie Parks, Karen Hopkin, Monte Kendrick, and Cathy Lundmark, and electronic illustrations were prepared by Gae Xavier Studios. A basic introductory cell biology text written by the authors of Molecular Biology of the Cell. Two comprehensive and lucid textbooks that convey effectively the synergistic convergence of biochemistry, genetics, structural biology, and traditional cell biology to form modern molecular and cell biology.

Cryo-EM in molecular and cellular biology

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein-coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling the determination of macromolecular structures at near-atomic resolution. The recent Nobel Prizes awarded to John Hopfield and Geoffrey Hinton in Physics, and to David Baker, Demis Hassabis, and John Jumper in Chemistry, highlight the growing convergence of artificial intelligence (AI) and computational innovation in advancing scientific discovery. Cryo-EM has emerged as a powerful tool for elucidating the architectures and dynamics of complex biological assemblies. Its ability to capture multiple conformational states provides mechanistic insights that are critical for understanding molecular function. However, traditional cryo-EM data processing pipelines face challenges in handling large, heterogeneous datasets. Manual particle selection, extensive computational demands, and uneven sampling of conformational states often limit resolution, especially for rare or transient conformations. Recent advances in AI-driven methods are reshaping this landscape. Deep neural networks now facilitate automated particle picking, denoising, classification, and three-dimensional reconstruction, substantially reducing human bias and computational bottlenecks. Machine learning models have also enhanced the accuracy of map interpretation and atomic modeling, accelerating the transition from raw micrographs to high-resolution structures. These developments represent a paradigm shift, positioning AI as an integral component of the “resolution revolution” in structural biology. This comprehensive article examines the state of the art in cryo-electron microscopy, highlighting how artificial intelligence is redefining every stage of the workflow-from automated particle picking and map reconstruction to structure modeling and high-resolution refinement. The integration of deep learning and neural network-based tools has transformed cryo-EM into a data-driven and highly precise discovery platform. By showcasing these innovations, we emphasize AI’s pivotal role in advancing next-generation cryo-EM research and accelerating molecular insights with profound implications for structural biology and drug discovery. Not applicable.

Mass spectrometry (MS) has become one of the key technologies of structural biology. In this review, the contributions of chemical cross-linking combined with mass spectrometry (XL-MS) for studying three-dimensional structures of proteins and for investigating protein-protein interactions are outlined. We summarize the most important cross-linking reagents, software tools, and XL-MS workflows and highlight prominent examples for characterizing proteins, their assemblies, and interaction networks in vitro and in vivo. Computational modeling plays a crucial role in deriving 3D-structural information from XL-MS data. Integrating XL-MS with other techniques of structural biology, such as cryo-electron microscopy, has been successful in addressing biological questions that to date could not be answered. XL-MS is therefore expected to play an increasingly important role in structural biology in the future.

In 2021, the release of AlphaFold2 - the DeepMind’s machine-learning protein structure prediction program - revolutionized structural biology. Results of the CASP14 contest were an immense surprise as AlphaFold2 successfully predicted 3D structures of nearly all submitted protein sequences. The AlphaFold2 craze has rapidly spread the life science community since structural biologists as well as untrained biologists have now the possibility to obtain high-confidence protein structures. This revolution is opening new avenues to address challenging biological questions. Moreover, AlphaFold2 is imposing itself as an essential step of any structural biology project, and requires us to revisit our structural biology workflows. On one hand, AlphaFold2 synergizes with experimental methods including X-ray crystallography and cryo-electron microscopy. On the other hand, it is, to date, the only method enabling structural analyses of large and flexible assemblies resistant to experimental approaches. We illustrate this valuable application of AlphaFold2 with the structure prediction of the whole host adhesion device from the Lactobacillus casei bacteriophage J-1. With the ongoing improvement of AlphaFold2 algorithms and notebooks, there is no doubt that AlphaFold2-driven biological stories will increasingly be reported, which questions the future directions of experimental structural biology.

In modern structural biology, there are three major methods for structural biologists to obtain structural information of macromolecules: cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR), and X-ray crystallography. Cryo-EM, in comparison with the other two methods, allows structural biologists to obtain the structures of various macromolecules in a more native and less perturbed system. Over the past decade, cryo-EM has enabled scientists to determine the structures of protein complexes at atomic resolution and made a profound impact in molecular bioscience and pharmaceutical sectors. Along with cryo-EM, another emerging technique called cryo-electron tomography (cryo-ET) has gained increasing importance in structural biology. It has the potential to visualize macromolecular complexes and assemblies in their native environments at high resolution, but there are still some challenges for small, sparse subjects and in approaching atomic resolution in situ. This chapter summarizes the major steps involved in structure determination using cryo-EM and cryo-ET and highlights the major challenges for in situ cryo-ET. We also present a few examples of near-atomic resolution structure determination of macromolecular assemblies both in purified systems in vitro and in native contexts in situ. Future perspectives are discussed as well.

A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM)). Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7-2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.

CellBio-X: celebrating the interface between Cell Biology and other disciplines

ABSTRACTAn original instrument intended to integrate the advantages of scanning probe, near and far field optical microscopy is presented. The device named Apertureless Head was specially designed to be incorporated to Ntegra scanning probe laboratory, the powerful analytical instrumentation in the nanotechnology field produced by NT-MDT Company, Russia. The device is supposed to be a powerful tool for high resolution analysis in different fields of investigations such as material sciences (optical and optoelectronic, magnetic, semi- and superconducting materials), polymers and biological sciences (structural biology, molecular and cell biology, microbiology, etc.). Probe tip plane light distributions are calculated and different light propagation schemes are discussed.