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Molecular Structure Determination by Electron Microscopy of Unstained Crystalline Specimens
Abstract and Figures
The projected structures of two unstained periodic biological specimens, the purple membrane and catalase, have been determined by electron microscopy to resolutions of 7 Å and 9 Å, respectively. Glucose was used to facilitate their in vacuo preservation and extremely low electron doses were applied to avoid their destruction.The information on which the projections are based was extracted from defocussed bright-field micrographs and electron diffraction patterns. Fourier analysis of the micrograph data provided the phases of the Fourier components of the structures; measurement of the electron diffraction patterns provided the amplitudes.Large regions of the micrographs (3000 to 10,000 unit cells) were required for each analysis because of the inherently low image contrast (<1%) and the statistical noise due to the low electron dose.Our methods appear to be limited in resolution only by the performance of the microscope at the unusually low magnifications which were necessary. Resolutions close to 3 Å should ultimately be possible.
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... The first 2D and 3D reconstruction algorithms were developed for regular aggregates of molecules like: helical fibres [DeRosier 1968;DeRosier 1970]; icosahedral viruses [Crowther 1971]; two-dimensional crystals [Unwin 1975]; and three-dimensional reconstruction from images recorded by tilting the sample [Hoppe 1974;Oettl 1983;Harauz 1987;Radermacher 1987]. These techniques were developed for use with the available high-contrast, negative staining specimen preparation approaches. ...
... Life arose in aqueous environments and removing water from the biological samples normally destroys the sample's structural integrity. Early landmark successes in cryogenic EM were achieved by low-dose imaging of 2D crystals like purple membrane and porins that are sometimes found naturally in cell membranes of and which samples could be embedded in sugars for structural preservation [Unwin 1975;Sass 1989;Henderson 1990]. It was especially the remarkable discovery of the "vitreous-ice" state of water which enabled the direct visualization of individual biological complexes in their hydrated state, within the EM [Fernandez Moran 1960;Taylor 1976;Chanzy 1976;Brüggeller 1980;Dubochet 1981;Jeng 1984;Chiu 1986], and of the "freeze-plunging" specimen-preparation technique [Adrian1984; Dubochet 1988] that gave rise to today's Cryo-EM routine. ...
... The raw Cryo-images are extremely noisy to minimise the electron-bombardment damage to the biological molecules in the cooled sample. Averaging procedures are necessary to extract the repetitive information present in, say, 2D crystals [Unwin 1975] which is a relatively simple operation because of the repetitive nature of a 2D-crystal sample. In single-particle analysis, however, the averaging procedure is by far more complicated since the individual molecular complexes are not held in a fixed orientation (by its crystal neighbours). ...
Electron microscopy has been an important method for visualising biological structures and processes since the 1940s. The discovery of a practical vitreous-ice specimen-preparation technique in the mid-1980s [Adrian 1984] led to modern-day Cryogenic Electron Microscopy (Cryo-EM) which in recent years has become a major technique for studying the architecture of biological macromolecules. Many further instrumental and data-analysis improvements were established in the decades after the introduction of the “vitreous-ice” state of water. Especially the advent of direct electron detectors boosted the quality of the recorded data, allowing atomic-resolution information of biological complexes to be harvested in the early 2010s, developments that truly revolutionized the use of Cryo-EM in structural biology. Single-Particle Analysis (SPA) of isolated molecules, prepared in a thin layer of vitreous water, has proven a most successful approach in structural biology and now often supplants the use of classical techniques like X-ray crystallography, especially for large biological complexes. The ever-increasing number of researchers using Cryo-EM is reflected by the growing number of depositions in the Electron Microscopy Data Bank (EMDB). Explaining this methodology to a new generation of researchers has now become a priority. In writing this review we were reminded of some persistent confusions that emerged in the early days of Cryo-EM but that continue to muddle the field. A new problem with the prolific use of Graphic User Interfaces (GUIs), is that the underlying methodology is often no longer transparent to the users of these “black boxes”. Complicated procedures, well hidden behind a GUI window, may contain methodological flaws that the user must be aware of. The conquering of markets in this booming Cryo-EM field – crucial for developing new pharmaceuticals – must not prevail over scientific integrity. We here describe and critically review the principles of single-particle Cryo-EM. We warn for procedures that have gone astray and could generate serious problems especially in the quality-control of Single-Particle Cryogenic Electron Microscopy.
... The first 2D and 3D reconstruction algorithms were developed for regular aggregates of molecules like: helical fibres [DeRosier 1968;DeRosier 1970]; icosahedral viruses [Crowther 1971]; two-dimensional crystals [Unwin 1975]; and three-dimensional reconstruction from images recorded by tilting the sample [Hoppe 1974;Oettl 1983;Harauz 1987;Radermacher 1987]. These techniques were developed for use with the available high-contrast, negative staining specimen preparation approaches. ...
... Life arose in aqueous environments and removing water from the biological samples normally destroys the sample's structural integrity. Early landmark successes in cryogenic EM were achieved by low-dose imaging of 2D crystals like purple membrane and porins that are sometimes found naturally in cell membranes of and which samples could be embedded in sugars for structural preservation [Unwin 1975;Sass 1989;Henderson 1990]. It was especially the remarkable discovery of the "vitreous-ice" state of water which enabled the direct visualization of individual biological complexes in their hydrated state, within the EM [Fernandez Moran 1960;Taylor 1976;Chanzy 1976;Brüggeller 1980;Dubochet 1981;Jeng 1984;Chiu 1986], and of the "freeze-plunging" specimen-preparation technique [Adrian1984; Dubochet 1988] that gave rise to today's Cryo-EM routine. ...
... The raw Cryo-images are extremely noisy to minimise the electron-bombardment damage to the biological molecules in the cooled sample. Averaging procedures are necessary to extract the repetitive information present in, say, 2D crystals [Unwin 1975] which is a relatively simple operation because of the repetitive nature of a 2D-crystal sample. In single-particle analysis, however, the averaging procedure is by far more complicated since the individual molecular complexes are not held in a fixed orientation (by its crystal neighbours). ...
Electron microscopy has been an important method for visualising biological structures and processes since the 1940s. The discovery of a practical vitreous-ice specimen-preparation technique in the mid-1980s [Adrian 1984] led to modern-day Cryogenic Electron Microscopy (Cryo-EM) which in recent years has become a major technique for studying the architecture of biological macromolecules. Many further instrumental and data-analysis improvements were established in the decades after the introduction of the “vitreous-ice” state of water. Especially the advent of direct electron detectors boosted the quality of the recorded data, allowing atomic-resolution information of biological complexes to be harvested in the early 2010s, developments that truly revolutionized the use of Cryo-EM in structural biology. Single-Particle Analysis (SPA) of isolated molecules, prepared in a thin layer of vitreous water, has proven a most successful approach in structural biology and now often supplants the use of classical techniques like X-ray crystallography, especially for large biological complexes. The ever-increasing number of researchers using Cryo-EM is reflected by the growing number of depositions in the Electron Microscopy Data Bank (EMDB). Explaining this methodology to a new generation of researchers has now become a priority. In writing this review we were reminded of some persistent confusions that emerged in the early days of Cryo-EM but that continue to muddle the field. A new problem with the prolific use of Graphic User Interfaces (GUIs), is that the underlying methodology is often no longer transparent to the users of these “black boxes”. Complicated procedures, well hidden behind a GUI window, may contain methodological flaws that the user must be aware of. The conquering of markets in this booming Cryo-EM field – crucial for developing new pharmaceuticals – must not prevail over scientific integrity. We here describe and critically review the principles of single-particle Cryo-EM. We warn for procedures that have gone astray and could generate serious problems especially in the quality-control of Single-Particle Cryogenic Electron Microscopy.
... Taylor and Glaeser showed that high-resolution diffraction data could be obtained by freezing catalase crystals as they were embedded in sugar, thus preserving the native hydrated state. 7 In 1975, Henderson and Unwin published the first 3D structural model of glucose-embedded 2D crystals of the purple membrane protein bacteriorhodopsin and bovine liver catalase at 7 and 9Å resolutions, respectively, 8,9 by combining the intensities from electron diffraction with the phases from electron microscopy images. Later, with the development of the plunge freezing technique in 1984 by Dubochet and co-workers, it became possible to rapidly freeze and vitrify biological samples in their native hydrated state. ...
Cryo-electron microscopy (cryo-EM) is a significant driver of recent advances in structural biology. Cryo-EM is comprised of several distinct and complementary methods, which include single particle analysis, cryo-electron tomography, and microcrystal electron diffraction. In this Perspective, we will briefly discuss the different branches of cryo-EM in structural biology and the current challenges in these areas.
... A fundamental limitation of cryo-EM is the fact that inelastic scattering deposits energy in the specimen, causing irreversible sample damage and rapid deterioration of highresolution details, a process termed radiation damage (R. M. Glaeser 1971;Henderson 1995). The deleterious effects of radiation damage on the imaged sample have been well documented by measuring the reduction of intensities of diffraction spots in exposure series obtained from 2D and thin 3D crystals (Unwin and Henderson 1975;Hayward and Glaeser 1979;Stark, Zemlin, and Boettcher 1996;Baker et al. 2010), as well as more recent measurements in noncrystalline, biological material (Grant and Grigorieff 2015). ...
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these voltages, specifically a ~4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope. We also achieved a ~3.2 Å resolution reconstruction for a 115 kDa asymmetric protein complex, proving its effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine’s capability for high-resolution data acquisition and screening on lower-end systems by obtaining ~3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, possibly bringing smaller sized particles within the scope of cryo-EM.
... Given the dose regime used to collect the data 16 , it is likely that the passivating organic ligands that cover the surface of the nanocrystals are damaged in the first few frames and that nanocrystals with a damaged passivation layer are imaged for the remainder of the time-series. To what degree radiation damage of the organic material affects the configuration of the metallic surface atoms would have to be addressed with low-dose TEM [26][27][28] . This is beyond the scope of this study. ...
In situ structures of Platinum (Pt) nanoparticles (NPs) can be determined with graphene liquid cell transmission electron microscopy. Atomic-scale three-dimensional structural information about their physiochemical properties in solution is critical for understanding their chemical function. We here analyze eight atomic-resolution maps of small (<3 nm) colloidal Pt NPs. Their structures are composed of an ordered crystalline core surrounded by surface atoms with comparatively high mobility. 3D reconstructions calculated from cumulative doses of 8500 and 17,000 electrons/pixel, respectively, are characterized in terms of loss of atomic densities and atomic displacements. Less than 5% of the total number of atoms are lost due to dissolution or knock-on damage in five of the structures analyzed, whereas 10–16% are lost in the remaining three. Less than 5% of the atomic positions are displaced due to the increased electron irradiation in all structures. The surface dynamics will play a critical role in the diverse catalytic function of Pt NPs and must be considered in efforts to model Pt NP function computationally.
Type 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic β cell. However, genetic and physiologic data indicate that defects in β cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the β cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X‐ray tomography, have been utilized to investigate the structure‐function relationship within the β cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach. © 2024 American Physiological Society. Compr Physiol 14:5243‐5267, 2024.
Microcrystal electron diffraction (MicroED) has advanced structural methods across a range of sample types, from small molecules to proteins. This cryogenic electron microscopy (cryo-EM) technique involves the continuous rotation of small 3D crystals in the electron beam, while a high-speed camera captures diffraction data in the form of a movie. The crystal structure is subsequently determined by using established X-ray crystallographic software. MicroED is a technique still under development, and hands-on expertise in sample preparation, data acquisition and processing is not always readily accessible. This comprehensive guide on MicroED sample preparation addresses commonly used methods for various sample categories, including room temperature solid-state small molecules and soluble and membrane protein crystals. Beyond detailing the steps of sample preparation for new users, and because every crystal requires unique growth and sample-preparation conditions, this resource provides instructions and optimization strategies for MicroED sample preparation. The protocol is suitable for users with expertise in biochemistry, crystallography, general cryo-EM and crystallography data processing. MicroED experiments, from sample vitrification to final structure, can take anywhere from one workday to multiple weeks, especially when cryogenic focused ion beam milling is involved.
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ~ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ~ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine’s effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine’s capability for high-resolution data acquisition and screening on lower-end systems by obtaining ~ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.
Type 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic β cell. However, genetic and physiologic data indicate that defects in β cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the β cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X‐ray tomography, have been utilized to investigate the structure‐function relationship within the β cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach. © 2024 American Physiological Society. Compr Physiol 14:5243‐5267, 2024.
This chapter discusses the determination of liver catalase. Next to urease, beef liver catalase is possibly the easiest enzyme to obtain in crystalline condition. The reasons for this are the unusual stability of this enzyme, its insolubility in water at its isoelectric point, and its relatively high concentration in beef liver. Since the preparation of crystalline catalase from beef liver by Sumner and Dounce crystalline catalases have been obtained from a number of other sources. These are: lamb liver, horse liver, beef erythrocytes, horse kidney and human liver, guinea pig liver, Micrococcus lysodeikticus, and pig liver. Catalase can be purified by being adsorbed on tricalcium phosphate in a Tswett column at pH 5.7 and later by eluting with phosphate buffer of pH 8.0. However, since most preparations of tricalcium phosphate are rather impermeable to water, it is probably better to adsorb on Celite. The chapter also describes a method for the determination of catalase activity which in all probability is the best yet devised for solutions of pure or partially purified catalase.
The radial electron-density distribution of tomato bushy stunt virus has been determined at low resolution by X-ray methods. Three experimental techniques have been used jointly to measure the spherically averaged Fourier transform of the particle in media of different electron density: (1) small-angle X-ray scattering from the virus in solution (counter recording); (2) small- and moderate- angle X-ray scattering from gels of the virus (photographic recording); and (3) single-crystal X-ray diffraction. The continuous transform corresponding to five different background densities has been obtained from the measurements with solutions and gels. The electron densities range from 0.343 to 0.408 . The single-crystal diffraction experiments show that no structural change or change in solvent binding occurs on transfer of the virus particle from one of these media to another, and that ammonium sulfate, sodium sulfate and sucrose can be used interchangeably to alter the solvent density. The single-crystal data extend to sufficiently wide angles that intensities from crystals in different media can be scaled to each other, since the background density has little effect on the diffraction pattern at spacings smaller than 20 Å. The continuous transforms can therefore be compared by scaling them to the corresponding crystal diffraction patterns.
The scattering of electrons by three-dimensional potential fields, and, in particular, the potential fields associated with a crystal lattice, is considered in terms of the new approach to physical optics proposed by Cowley & Moodie.
Since specimens are normally viewed and photographed in the electron microscope at high levels of electron beam radiation damage, it becomes important to choose a recording medium for a particular operating voltage which has the maximum sensitivity to electrons, consistent with the required recording resolution and contrast. It is shown that commonly used emulsions show a wide range of sensitivity. There is a need for manufacturere to specify the electron sensitivity, resolution and contrast of available emulsions over accelerating voltages of 40 to 1000kV and also to specify the X-ray fluorescence and back scatter characteristics for each kind of emulsion backing and cassette material. In addition, the fluorescent light and X-ray sensitivity of each emulsion should be specified. New sensitivity measurements on films and plates are reported. The extra high sensitivity of some X-ray films is emphasized and their usefulness in conventional and high voltage microscopy discussed. Suggestions are made for the manufacture of improved emulsions for high voltage work and for their protection from fogging during use. In addition, high voltage electron microscopy of biological materials requires the availability of high gain image intensifier/TV system for scanning specimens at low radiation levels and for the recording of dynamic images.