Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Carbon footprint impact of the choice of inhalers for asthma and COPD.

In the 1990s, metered dose inhalers (MDIs) containing chlorofluorocarbons were replaced with dry-powder inhalers (DPIs) and MDIs containing hydrofluorocarbons (HFCs). While HFCs are not ozone depleting, they are potent greenhouse gases. Annual carbon footprint (CO2e), per patient were 17 kg for Relvar-Ellipta/Ventolin-Accuhaler; and 439 kg for Seretide-Evohaler/Ventolin-Evohaler. In 2017, 70% of all inhalers sold in England were MDI, versus 13% in Sweden. Applying the Swedish DPI and MDI distribution to England would result in an annual reduction of 550 kt CO2e. The lower carbon footprint of DPIs should be considered alongside other factors when choosing inhalation devices.

Single particle electron cryomicroscopy: trends, issues and future perspective.

There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2- to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

From Electron Crystallography to Single Particle CryoEM (Nobel Lecture).

Pictures are a key to knowledge: The development of electron microscopy from its beginnings to modern single particle cryo-EM is described by R. Henderson in his Nobel lecture. Shown is the first projection structure at 7 Šresolution of the purple membrane from October 1974.

Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference.

With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.

Avoiding the pitfalls of single particle cryo-electron microscopy: Einstein from noise.

Single particle cryo-electron microscopy is currently poised to produce high-resolution structures of many biological assemblies, but several pitfalls can trap the unwary. This critique highlights one problem that is particularly relevant when smaller structures are being studied. It is known as "Einstein from noise," in which the experimenter honestly believes they have recorded images of their particles, whereas in reality, most if not all of their data consist of pure noise. Selection of particles using cross-correlation methods can then lead to 3D maps that resemble the model used in the initial selection and provide the illusion of progress. Suggestions are given about how to circumvent the problem.

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography.

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.

Overview and future of single particle electron cryomicroscopy.

Electron cryomicroscopy (cryoEM) has experienced a quantum leap in its capability in recent years, due to improved microscopes, better detectors and better software. It is now possible to obtain near-atomic resolution 3D density maps of macromolecular assemblies using single particle cryoEM without the need for crystals. Although this recent progress has produced some outstanding achievements, we have still only partly realised the full potential of single particle cryoEM. If one or two remaining problems can be solved, it will become an even more powerful method in structural biology that should closely approach the limit of what is theoretically possible.

Longitudinal assessment of bone growth and development in a facility-based population of young adults with cerebral palsy.

AIM: Osteoporosis is a significant clinical problem in persons with moderate to severe cerebral palsy (CP), causing fractures with minimal trauma. Over the past decade, most studies examining osteoporosis and CP have been cross-sectional in nature, focused exclusively on children and adolescents and only involving one evaluation of bone mineral density (BMD). The purpose of this study was to assess BMD in a group including adults with CP, and changes in each individual's BMD over a 5- to 6-year period. METHOD: The study group included 40 residents of a long-term care facility aged 6 to 26 years at the time of their initial evaluation. Twenty-one patients (52.5%) were male, 35 (88%) were white, and 38 (95%) were in Gross Motor Function Classification System level V. BMD was assessed by dual-energy X-ray absorptiometry on the right and left distal femurs for three distinct regions of interest. RESULTS: Five residents had a fracture that occurred during the study period; this represented a fracture rate of 2.1% per year in the study group. Longitudinally, annualized change in the median BMD was 0.7% to 1.0% per year in the different regions of the distal femur, but ranged widely among the study group, with both increases and decreases in BMD. Increase in BMD over time was negatively correlated with age and positively correlated with change in weight. INTERPRETATION: Changes in BMD over time in profoundly involved persons with CP can range widely, which is important to recognize when evaluating potential interventions to improve BMD. Age and changes in body weight appear the most relevant factors.

Adaptation of the lateral distal femur DXA scan technique to adults with disabilities.

The technique that best addresses the challenges of assessing bone mineral density in children with neuromuscular impairments is a dual-energy X-ray absorptiometry (DXA) scan of the lateral distal femur. The purpose of this study was to adapt this technique to adults with neuromuscular impairments and to assess the reproducibility of these measurements. Thirty-one adults with cerebral palsy had both distal femurs scanned twice, with the subject removed and then repositioned between each scan (62 distal femurs, 124 scans). Each scan was independently analyzed twice by 3 different technologists of varying experience with DXA (744 analyses). Precision of duplicate analyses of the same scan was good (range: 0.4%-2.3%) and depended on both the specific region of interest and the experience of the technologist. Precision was reduced when comparing duplicate scans, ranging from 7% in the metaphyseal (cancellous) region to 2.5% in the diaphyseal (cortical) region. The least significant change was determined as recommended by the International Society for Clinical Densitometry for each technologist and each region of interest. Obtaining reliable, reproducible, and clinically relevant assessments of bone mineral density in adults with neuromuscular impairments can be challenging. The technique of obtaining DXA scans of the lateral distal femur can be successfully applied to this population but requires a commitment to developing the necessary expertise.

Structure of a beta1-adrenergic G-protein-coupled receptor.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

`Cryo-EM': electron cryomicroscopy, cryo electron microscopy or something else?

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. During the last ten years, cryoEM has become widely available and has provided a major boost to structure determination of membrane proteins and large multi-protein complexes. Many of the structures have now been made available at resolutions around 2 Å, where fundamental questions regarding enzyme mechanisms can be addressed. Over the years, the abbreviation cryoEM has been understood to stand for different things. We wish the wider community to engage and clarify the definition of cryoEM so that the expanding literature involving cryoEM is unified.

Structures of membrane proteins.

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class.

Beam-induced motion of vitrified specimen on holey carbon film.

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 µm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.

Curcumin encapsulation in submicrometer spray-dried chitosan/Tween 20 particles.

Optimal curcumin delivery for medicinal applications requires a drug delivery system that both solubilizes curcumin and prevents degradation. To achieve this, curcumin has been encapsulated in submicrometer chitosan/Tween 20 particles via a benchtop spray-drying process. Spray-drying parameters have been optimized using a Taguchi statistical approach to minimize particle size and to favor spheroid particles with smooth surfaces, as evaluated with scanning electron microscopy (SEM) imaging. Nearly spherical particles with 285 ± 30 nm diameter and 1.21 axial ratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within the chitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 h period.