ABSTRACT
Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.
Subject(s)
Cell Biology , Cells , Cryoelectron Microscopy , Electron Microscope Tomography , Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trends , Electron Microscope Tomography/methods , Electron Microscope Tomography/trends , Macromolecular Substances/analysis , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Macromolecular Substances/ultrastructure , Cell Biology/instrumentation , Cells/chemistry , Cells/cytology , Cells/metabolism , Cells/ultrastructure , HumansSubject(s)
Analytic Sample Preparation Methods , Cryoelectron Microscopy , Mass Spectrometry , Proteins , Molecular Biology/methods , Molecular Biology/trends , Cryoelectron Microscopy/methods , Cryoelectron Microscopy/standards , Cryoelectron Microscopy/trends , Mass Spectrometry/methods , Mass Spectrometry/standards , Mass Spectrometry/trends , Proteins/chemistry , Proteins/ultrastructure , Analytic Sample Preparation Methods/methods , Analytic Sample Preparation Methods/standardsABSTRACT
Mammalian gametes-the sperm and the egg-represent opposite extremes of cellular organization and scale. Studying the ultrastructure of gametes is crucial to understanding their interactions, and how to manipulate them in order to either encourage or prevent their union. Here, we survey the prominent electron microscopy (EM) techniques, with an emphasis on considerations for applying them to study mammalian gametes. We review how conventional EM has provided significant insight into gamete ultrastructure, but also how the harsh sample preparation methods required preclude understanding at a truly molecular level. We present recent advancements in cryo-electron tomography that provide an opportunity to image cells in a near-native state and at unprecedented levels of detail. New and emerging cellular EM techniques are poised to rekindle exploration of fundamental questions in mammalian reproduction, especially phenomena that involve complex membrane remodelling and protein reorganization. These methods will also allow novel lines of enquiry into problems of practical significance, such as investigating unexplained causes of human infertility and improving assisted reproductive technologies for biodiversity conservation.
Subject(s)
Cell Biology/trends , Cytological Techniques , Germ Cells/ultrastructure , Microscopy, Electron/trends , Animals , Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trends , Fertilization/physiology , Germ Cells/physiology , Humans , Mammals , Microscopy, Electron/methodsSubject(s)
Apoferritins/chemistry , Apoferritins/ultrastructure , Cryoelectron Microscopy/methods , Electrons , Cryoelectron Microscopy/trends , Crystallography, X-Ray/trends , Drug Design , Histamine/chemistry , Histamine/metabolism , Models, Molecular , Receptors, GABA-A/chemistry , Receptors, GABA-A/ultrastructure , Time Factors , Water/chemistry , Water/metabolismABSTRACT
The number of new X-ray crystallography-based submissions to the Protein Data Bank appears to be at the beginning of a decline, perhaps signalling an end to the era of the dominance of X-ray crystallography within structural biology. This letter, from the viewpoint of a young structural biologist, applies the Copernican method to the life expectancy of crystallography and asks whether the technique is still the mainstay of structural biology. A study of the rate of Protein Data Bank depositions allows a more nuanced analysis of the fortunes of macromolecular X-ray crystallography and shows that cryo-electron microscopy might now be outcompeting crystallography for new labour and talent, perhaps heralding a change in the landscape of the field.
Subject(s)
Cryoelectron Microscopy/trends , Crystallography, X-Ray/trends , Proteins/chemistry , Databases, Protein/trends , Multiprotein Complexes/chemistry , Protein ConformationSubject(s)
Cryoelectron Microscopy/statistics & numerical data , Cryoelectron Microscopy/trends , Databases, Chemical , Proteins/ultrastructure , Cryoelectron Microscopy/instrumentation , Cryoelectron Microscopy/methods , Crystallization , Crystallography, X-Ray/statistics & numerical data , Nobel Prize , Protein Stability , Proteins/chemistry , SoftwareSubject(s)
Computer Simulation , Cryoelectron Microscopy/trends , Genetic Therapy/trends , Genome/physiology , Microbiota/physiology , Sequence Analysis, RNA/trends , Single-Cell Analysis/trends , Aptamers, Nucleotide/analysis , Disease Progression , Enhancer Elements, Genetic/genetics , Genome/genetics , Humans , Microbiota/genetics , Neoplasms/genetics , Neoplasms/pathologyABSTRACT
With the rapid improvement of cryo-electron microscopy (cryo-EM) resolution, new computational tools are needed to assist and improve upon atomic model building and refinement options. This communication demonstrates that microscopists can now collaborate with the players of the computer game Foldit to generate high-quality de novo structural models. This development could greatly speed the generation of excellent cryo-EM structures when used in addition to current methods.
Subject(s)
Citizen Science , Cryoelectron Microscopy/trends , Video Games , Algorithms , Biochemistry , Cryoelectron Microscopy/methods , Models, MolecularABSTRACT
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 â¼2 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.
Subject(s)
Cryoelectron Microscopy/trends , Macromolecular Substances/chemistry , Macromolecular Substances/ultrastructure , Animals , Biomedical Engineering , Electron Microscope Tomography/trends , Electron Transport Chain Complex Proteins/chemistry , Electron Transport Chain Complex Proteins/ultrastructure , Humans , Imaging, Three-Dimensional , Ion Channels/chemistry , Ion Channels/ultrastructure , Macromolecular Substances/isolation & purification , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/ultrastructure , Models, Molecular , Spliceosomes/chemistry , Spliceosomes/ultrastructureABSTRACT
Eva Nogales introduces cryo-electron microscopy used to visualize macromolecular structures.
Subject(s)
Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trends , Macromolecular SubstancesABSTRACT
Cryo-electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.
Subject(s)
Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trends , Single Molecule Imaging/methods , Single Molecule Imaging/trends , Animals , Catalase/chemistry , Catalase/ultrastructure , Crystallography, X-Ray , Humans , Transient Receptor Potential Channels/chemistryABSTRACT
Bacterial secretion systems are responsible for releasing macromolecules to the extracellular milieu or directly into other cells. These membrane complexes are associated with pathogenicity and bacterial fitness. Understanding of these large assemblies has exponentially increased in the last few years thanks to electron microscopy. In fact, a revolution in this field has led to breakthroughs in characterizing the structures of secretion systems and other macromolecular machineries so as to obtain high-resolution images of complexes that could not be crystallized. In this review, we give a brief overview of structural advancements in the understanding of secretion systems, focusing in particular on cryo-electron microscopy, whether tomography or single-particle analysis. We describe how such techniques have contributed to knowledge of the mechanism of macromolecule secretion in bacteria and the impact they will have in the future.
Subject(s)
Bacteria/enzymology , Bacterial Secretion Systems/ultrastructure , Cryoelectron Microscopy/methods , Cryoelectron Microscopy/trendsABSTRACT
The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (â¼3.0â¯Å) and size (â¼310.0â¯Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508â¯Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9â¯Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.