SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface.

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with ß-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

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.

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.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Mass spectrometers as cryoEM grid preparation instruments.

Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air-water interface often leads to sample damage and preferential orientation of the particles. Using native mass spectrometry to create cryoEM samples, potentially avoids these problems and allows the use of mass spectrometry sample isolation techniques during EM grid creation. We review the recent publications that have demonstrated protein complexes can be ionized, flown through the mass spectrometer, gently landed onto EM grids, imaged, and reconstructed in 3D. Although many uncertainties and challenges remain, the combination of cryoEM and MS has great potential.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Replication fork binding triggers structural changes in the PriA helicase that govern DNA replication restart in E. coli.

Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (PriA-PriB) selectively acts on replication forks, we determined the cryogenic-electron microscopy structure of a PriA/PriB/replication fork complex. Replication fork specificity arises from extensive PriA interactions with each arm of the branched DNA. These interactions reshape the PriA protein to create a pore encircling single-stranded lagging-strand DNA while also exposing a surface of PriA onto which PriB docks. Together with supporting biochemical and genetic studies, the structure reveals a switch-like mechanism for replication restart initiation in which restructuring of PriA directly couples replication fork recognition to PriA/PriB complex formation to ensure robust and high-fidelity replication re-initiation.

From primordial clocks to circadian oscillators.

Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator1. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock2. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood3-6, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism7-9. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.

Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching.

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.

Onto Grid Purification and 3D Reconstruction of Protein Complexes Using Matrix-Landing Native Mass Spectrometry.

Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.

Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes.

Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexes─GroEL, the 20S proteasome core particle, and ß-galactosidase─and obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (∼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.

Structural basis of transposon end recognition explains central features of Tn7 transposition systems.

Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.

Three-dimensional structure determination of protein complexes using matrix-landing mass spectrometry.

Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe the use of a chemical landing matrix that preserves the structural integrity of the deposited particles. With this system we obtain a three-dimensional reconstruction of the 800 kDa protein complex GroEL from gas-phase deposited GroEL ions. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

Locating macromolecular assemblies in cells by 2D template matching with cisTEM.

For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently limiting throughput. Additionally, the high-resolution signal preserved in the raw tomograms is currently limited by a number of technical difficulties, leading to an increased false-positive detection rate when using 3D template matching to find molecular complexes in tomograms. We have recently described a 2D template matching approach that addresses these issues by including high-resolution signal preserved in single-tilt images. A current limitation of this approach is the high computational cost that limits throughput. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells with high precision and sensitivity, demonstrating that this is a versatile tool for in situ visual proteomics and in situ structure determination. We benchmark the results with 3D template matching of tomograms acquired on identical sample locations and identify strengths and weaknesses of both techniques, which offer complementary information about target localization and identity.

Assessing the water scarcity footprint of food crops by growing season available water remaining (AWARE) characterization factors in Thailand.

Water scarcity problems are a national agenda that must be prioritized. Thailand is an agricultural country with agriculture consuming a large amount of water. Sustainable water management for the agricultural sector is urgently needed. This study assessed the impacts of water consumption by agricultural products using a water scarcity footprint (WSF) assessment. This study presents the use of the growing season available water remaining characterization factors (AWARE CFs) of specific crops, based on growth months of that crop, instead of the annual AWARE CFs of agricultural products to assess the value of this approach for an appropriate assessment of water resource and the planning of crop production priorities. The original marginal AWARE CF was considered for determining the growing season AWARE CFs of major rice, second rice, sugarcane, maize, and cassava in 25 Thai watersheds. The growing season AWARE CFs of the food crops were used to determine their WSFs for the 25 Thai watersheds. The growing season AWARE CFs of major rice, second rice, sugarcane, maize, and cassava for Thailand were 1.74, 11.5, 6.01, 3.28, and 7.96 m3 world-eq. per m3 consumed, respectively. There were statistical differences between the means of annual AWARE CFs and growing season AWARE CFs for all Thai food crops in almost all watersheds. The WSFs using growing season AWARE CFs of major rice, second rice, sugarcane, maize, and cassava were 0.05-3.66, 1.10-193, 0.51-7.99, 1.09-8.28, and 1.65-30.3 m3 world-eq. per kilogram, respectively. This work identified suitable watersheds for growing food crops and compiled them as databases for the use of zoning food crop cultivation by the Thai government. Regarding WSF values, the least suitable watershed for growing major rice, second rice, sugarcane, and cassava was the Petchaburi watershed. The least suitable watershed for growing maize was the Chao Phraya watershed.

A Survey of Contact Lens Wearers and Eye Care Professionals on Satisfaction with a New Smart-Surface Silicone Hydrogel Daily Disposable Contact Lens.

PURPOSE: To assess the patient and ECP preference of verofilcon A lenses over approximately a one-week period when worn on a daily wear, daily disposable (DD) basis by current DD contact lens wearers (CLWs) and new CLWs. ECPs assessed the lens fit, performance on eye and lens preference (to habitual DD lens) for current wearers. METHODS: A total of 218 current DD CLWs and 129 new CLWs aged >18 years participated in the survey. Baseline demographics and one-week data were obtained by 31 ECPs after fitting up to 10 current and 10 new wearers each. The survey was conducted online for both the participants and the ECPs. The survey took place in Australia and New Zealand from April to May 2019. RESULTS: For new wearers, the surveyed results demonstrated strong wearer preference for the verofilcon A lens with quality of life as a main driver. For current wearers, comfort was the main driver for verofilcon A preference. Post survey, 73% of ECPs stated they would most likely continue to fit verofilcon A lenses.