Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

Integrative Structure Modeling: Overview and Assessment.

Integrative structure modeling computationally combines data from multiple sources of information with the aim of obtaining structural insights that are not revealed by any single approach alone. In the first part of this review, we survey the commonly used sources of structural information and the computational aspects of model building. Throughout the past decade, integrative modeling was applied to various biological systems, with a focus on large protein complexes. Recent progress in the field of cryo-electron microscopy (cryo-EM) has resolved many of these complexes to near-atomic resolution. In the second part of this review, we compare a range of published integrative models with their higher-resolution counterparts with the aim of critically assessing their accuracy. This comparison gives a favorable view of integrative modeling and demonstrates its ability to yield accurate and informative results. We discuss possible roles of integrative modeling in the new era of cryo-EM and highlight future challenges and directions.

Cellular Electron Cryotomography: Toward Structural Biology In Situ.

Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.

Meet the authors: Max E. Wilkinson, Sebastian M. Fica, and Wojciech P. Galej.

We talk to the authors of "Structural basis for conformational equilibrium of the catalytic spliceosome" about their paper, advances in cryo-EM that made it possible, and the influence of their late mentor Kiyoshi Nagai, to whom the paper is dedicated.

Ups and downs in early electron cryo-microscopy.

This is a tale of two scientists who, in their younger days, had their scientific judgement clouded by the promise of a big discovery. Two years later, they found that their conclusions had been considerably exaggerated. They were lucky, though, as their later work would prove to be significant. Now, more than 30 years after those events, they met again and put in writing their understanding of what went wrong.

After the revolution: how is Cryo-EM contributing to muscle research?

The technique of electron microscopy (EM) has been fundamental to muscle research since the days of Huxley and Hanson. Direct observation of how proteins in the sarcomere are arranged and visualising the changes that occur upon activation have greatly increased our understanding of function. In the 1980s specimen preparation techniques for biological EM moved away from traditional fixing and staining. The technique known as cryo-electron microscopy (Cryo-EM) was developed, which involves rapidly freezing proteins in liquid ethane which maintains them in a near native state. Within the last 5 years there has been a step change in the achievable resolution using Cryo-EM. This 'resolution revolution' can be attributed to advances in detector technology, microscope automation and maximum likelihood image processing. In this article we look at how Cryo-EM has contributed to the field of muscle research in this post revolution era, focussing on recently published high resolution structures of sarcomeric proteins.

The 2017 Nobel Prize in Chemistry: cryo-EM comes of age.

The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution." This feature article summarizes some of the major achievements leading to the development of cryo-EM and recent technological breakthroughs that have transformed the method into a mainstream tool for structure determination.

The sleeping beauty kissed awake: new methods in electron microscopy to study cellular membranes.

Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM. The past 15-20 years are hallmarked by a tremendous interest in EM, driven by important technological advances. Cryo-EM, in particular, is now capable of revealing structures of proteins at a near-atomic resolution owing to improved sample preparation methods, microscopes and cameras. In this review, we focus on the challenges associated with the imaging of membranes by EM and give examples from the field of host-pathogen interactions, in particular of virus-infected cells. Despite the advantages of imaging membranes under native conditions in cryo-EM, conventional EM will remain an important complementary method, in particular if large volumes need to be imaged.

Cryo-EM--the first thirty years.

Thirty years ago, in December 1981, The Journal of Microscopy published a very short paper entitled 'Vitrification of pure water for electron microscopy'. It turned out to be important for the development of cryo-electron microscopy and it contributed to reverse, from foe to friend, the status of water in electron microscopists' minds. This change has brought obvious gains. The future will tell how many more are still to come.

Gaia Pigino: Inside the cell.

Gaia Pigino studies the molecular mechanisms and principles of self-organization in cilia using 3D cryo-EM.

How electron cryotomography is opening a new window onto prokaryotic ultrastructure.

Electron cryotomography is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozen-hydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. Following years of visionary technology development by a few key pioneers, several laboratories are now using the technique to produce biological results of major significance in the field of prokaryotic ultrastructure. Recent discoveries have included the surprising generality and complexity of the cytoskeleton, the connectivity of key membrane compartments, the location and architecture of large macromolecular machines such as the ribosome and flagellar motors, and the structure of some extraordinary external appendages. Through further technology development, identification of the most revealing model systems and sustained effort, electron cryotomography is poised to help resolve many fundamentally important questions about prokaryotic ultrastructure.

On cross-correlations, averages and noise in electron microscopy.

Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, averaging of multiple copies of macromolecular assembly subvolumes extracted from tomographic reconstructions can lead to a virtually noise-free high-resolution structure. Cross-correlation methods are often used in the alignment and classification steps of averaging processes for both 2D images and 3D volumes. However, the high noise level can bias alignment and certain classification results. While other approaches may be implicitly affected, sensitivity to noise is most apparent in multireference alignments, 3D reference-based projection alignments and projection-based volume alignments. Here, the influence of the image signal-to-noise ratio on the value of the cross-correlation coefficient is analyzed and a method for compensating for this effect is provided.

Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age.

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future.

Methods for preserving specimen hydration in protein crystals were pursued in the early 1970s as a prerequisite for protein crystallography using an electron microscope. Three laboratories approached this question from very different directions. One built a differentially pumped hydration chamber that could maintain the crystal in a liquid water environment, a second maintained hydration by rapidly freezing the protein crystal and examining it in a cold stage, and the third replaced the water of hydration by using glucose in the same way as one had previously used "negative stains". Each of these early efforts succeeded in preserving the structures of protein crystals at high resolution within the vacuum of the electron microscope, as demonstrated by electron diffraction patterns. The next breakthrough came in the early 1980s when a technique was devised to preserve noncrystalline specimens by freezing them within vitreous ice. Since then, with the development of high stability cold stages and transfer mechanisms compatible with many instrument platforms, and by using commercially provided low dose imaging techniques to avoiding radiation damage, there has been an explosion of applications. These now include single particles, helical filaments, 2-D arrays and even whole cells, where the most exciting recent applications involve cryoelectron tomography. These achievements and possibilities generate a new set of research opportunities associated with increasing the reliability and throughput with which specimens can be studied by cryoEM.

Structural Molecular Biology-A Personal Reflection on the Occasion of John Kendrew's 100th Birthday.

Here, I discuss the development and future of structural molecular biology, concentrating on the eukaryotic transcription machinery and reflecting on John Kendrew's legacy from a personal perspective.