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.

Characterizing the resolution and throughput of the Apollo direct electron detector.

Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase.

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S2- oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur. SiR is a dodecameric oxidoreductase composed of an octameric flavoprotein reductase (SiRFP) and four hemoprotein metalloenzyme oxidases (SiRHPs). SiR performs the electron transfer reduction reaction to produce sulfide from sulfite through coordinated domain movements and subunit interactions without release of partially reduced intermediates. Efforts to understand the electron transfer mechanism responsible for SiR's efficiency are confounded by structural heterogeneity arising from intrinsically disordered regions throughout its complex, including the flexible linker joining SiRFP's flavin-binding domains. As a result, high-resolution structures of SiR dodecamer and its subcomplexes are unknown, leaving a gap in the fundamental understanding of how SiR performs this uniquely large-volume electron transfer reaction. Here, we use deuterium labeling, in vitro reconstitution, analytical ultracentrifugation (AUC), small-angle neutron scattering (SANS), and neutron contrast variation (NCV) to observe the relative subunit positions within SiR's higher-order assembly. AUC and SANS reveal SiR to be a flexible dodecamer and confirm the mismatched SiRFP and SiRHP subunit stoichiometry. NCV shows that the complex is asymmetric, with SiRHP on the periphery of the complex and the centers of mass between SiRFP and SiRHP components over 100 Å apart. SiRFP undergoes compaction upon assembly into SiR's dodecamer and SiRHP adopts multiple positions in the complex. The resulting map of SiR's higher-order structure supports a cis/trans mechanism for electron transfer between domains of reductase subunits as well as between tightly bound or transiently interacting reductase and oxidase subunits.

Reconstruction of Average Subtracted Tubular Regions (RASTR) enables structure determination of tubular filaments by cryo-EM.

As the field of electron microscopy advances, the increasing complexity of samples being produced demand more involved processing methods. In this study, we have developed a new processing method for generating 3D reconstructions of tubular structures. Tubular biomolecules are common throughout many cellular processes and are appealing targets for biophysical research. Processing of tubules with helical symmetry is relatively straightforward for electron microscopy if the helical parameters are known, but tubular structures that deviate from helical symmetry (asymmetrical components, local but no global order, etc) present myriad issues. Here we present a new processing technique called Reconstruction of Average Subtracted Tubular Regions (RASTR), which was developed to reconstruct tubular structures without applying symmetry. We explain the RASTR approach and quantify its performance using three examples: a simulated symmetrical tubular filament, a symmetrical tubular filament from cryo-EM data, and a membrane tubule coated with locally ordered but not globally ordered proteins.

Out-of-Register Parallel ß-Sheets and Antiparallel ß-Sheets Coexist in 150-kDa Oligomers Formed by Amyloid-ß(1-42).

We present solid-state NMR measurements of ß-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-ß peptide (Aß(1-42)). We build upon our previous report of a ß-strand spanned by residues 30-42, which arranges into an antiparallel ß-sheet. New results presented here indicate that there is a second ß-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second ß-strand is organized into a parallel ß-sheet despite the co-existence of an antiparallel ß-sheet in the same structure. In addition, the in-register parallel ß-sheet commonly observed for amyloid fibril structure does not apply to residues 11-24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the ß-sheet. We corroborated this unexpected scheme for ß-strand organization using multiple two-dimensional NMR and 13C-13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P1 and P6, both to 1.5 Šresolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

Archaeal and eukaryotic homologs of Hfq: A structural and evolutionary perspective on Sm function.

Hfq and other Sm proteins are central in RNA metabolism, forming an evolutionarily conserved family that plays key roles in RNA processing in organisms ranging from archaea to bacteria to human. Sm-based cellular pathways vary in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in each of these pathways being mediated by an RNA-associated molecular assembly built upon Sm proteins. Though the first structures of Sm assemblies were from archaeal systems, the functions of Sm-like archaeal proteins (SmAPs) remain murky. Our ignorance about SmAP biology, particularly vis-à-vis the eukaryotic and bacterial Sm homologs, can be partly reduced by leveraging the homology between these lineages to make phylogenetic inferences about Sm functions in archaea. Nevertheless, whether SmAPs are more eukaryotic (RNP scaffold) or bacterial (RNA chaperone) in character remains unclear. Thus, the archaeal domain of life is a missing link, and an opportunity, in Sm-based RNA biology.