Structure-based prediction and characterization of photo-crosslinking in native protein-RNA complexes.

UV-crosslinking of protein and RNA in direct contacts has been widely used to study protein-RNA complexes while our understanding of the photo-crosslinking mechanisms remains poor. This knowledge gap is due to the challenge of precisely mapping the crosslink sites in protein and RNA simultaneously in their native sequence and structural contexts. Here we systematically analyze protein-RNA interactions and photo-crosslinking by bridging crosslinked nucleotides and amino acids mapped using different assays with protein-RNA complex structures. We developed a computational method PxR3D-map which reliably predicts crosslink sites using structural information characterizing protein-RNA interaction interfaces. Analysis of the informative features revealed that photo-crosslinking is facilitated by base stacking with not only aromatic residues, but also dipeptide bonds that involve glycine, and distinct mechanisms are utilized by different RNA-binding domains. Our work suggests protein-RNA photo-crosslinking is highly selective in the cellular environment, which can guide data interpretation and further technology development for UV-crosslinking-based assays.

Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

Recovery of Conformational Continuum From Single-Particle Cryo-EM Images: Optimization of ManifoldEM Informed by Ground Truth.

This work is based on the manifold-embedding approach to study biological molecules exhibiting continuous conformational changes. Previous work established a method-now termed ManifoldEM-capable of reconstructing 3D movies and accompanying free-energy landscapes from single-particle cryo-EM images of macromolecules exercising multiple conformational degrees of freedom. While ManifoldEM has proven its viability in several experimental studies, critical limitations and uncertainties have been found throughout its extended development and use. Guided by insights from studies with cryo-EM ground-truth data, simulated from atomic structures undergoing conformational changes, we have built a novel framework, ESPER, able to retrieve the free-energy landscape and respective 3D Coulomb potential maps for all states simulated. As shown by a direct comparison of ground truth vs. recovered maps, and analysis of experimental data from the 80S ribosome and ryanodine receptor, ESPER offers substantial improvements relative to the previous work.

An immune response characterizes early Alzheimer's disease pathology and subjective cognitive impairment in hydrocephalus biopsies.

Early Alzheimer's disease (AD) pathology can be found in cortical biopsies taken during shunt placement for Normal Pressure Hydrocephalus. This represents an opportunity to study early AD pathology in living patients. Here we report RNA-seq data on 106 cortical biopsies from this patient population. A restricted set of genes correlate with AD pathology in these biopsies, and co-expression network analysis demonstrates an evolution from microglial homeostasis to a disease-associated microglial phenotype in conjunction with increasing AD pathologic burden, along with a subset of additional astrocytic and neuronal genes that accompany these changes. Further analysis demonstrates that these correlations are driven by patients that report mild cognitive symptoms, despite similar levels of biopsy ß-amyloid and tau pathology in comparison to patients who report no cognitive symptoms. Taken together, these findings highlight a restricted set of microglial and non-microglial genes that correlate with early AD pathology in the setting of subjective cognitive decline.

What is in the black box? - A perspective on software in cryoelectron microscopy.

This article bemoans the demise of truly modular open-source image processing systems, such as SPIDER, in recent years' development of tools for three-dimensional reconstruction in cryo-electron microscopy. Instead, today's users have to rely on the functionality of software systems that have little or no transparency. As a consequence, users of such packages no longer gain a conceptual understanding and intuitive grasp of the analytical routes leading from the stream of input data to the final density map. Possible remedies of this situation with free software are discussed.

A glycan gate controls opening of the SARS-CoV-2 spike protein.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded 'down' to an exposed 'up' state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the 'up' and 'down' states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways. Together with ManifoldEM analysis of cryo-electron microscopy data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408 and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein represents a landmark study for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

A glycan gate controls opening of the SARS-CoV-2 spike protein.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 µs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

Propagation of Conformational Coordinates Across Angular Space in Mapping the Continuum of States from Cryo-EM Data by Manifold Embedding.

Recent approaches to the study of biological molecules employ manifold learning to single-particle cryo-EM data sets to map the continuum of states of a molecule into a low-dimensional space spanned by eigenvectors or "conformational coordinates". This is done separately for each projection direction (PD) on an angular grid. One important step in deriving a consolidated map of occupancies, from which the free energy landscape of the molecule can be derived, is to propagate the conformational coordinates from a given choice of "anchor PD" across the entire angular space. Even when one eigenvector dominates, its sign might invert from one PD to the next. The propagation of the second eigenvector is particularly challenging when eigenvalues of the second and third eigenvector are closely matched, leading to occasional inversions in their ranking as we move across the angular grid. In the absence of a computational approach, this propagation across the angular space has been done thus far "by hand" using visual clues, thus greatly limiting the general use of the technique. In this work we have developed a method that is able to solve the propagation problem computationally, by using optical flow and a probabilistic graphical model. We demonstrate its utility by selected examples.

Structure and dynamics of the insulin receptor: implications for receptor activation and drug discovery.

Recently, major progress has been made in uncovering the mechanisms of how insulin engages its receptor and modulates downstream signal transduction. Here, we present in detail the current structural knowledge surrounding the individual components of the complex, binding sites, and dynamics during the activation process. A novel kinase triggering mechanism, the 'bow-arrow model', is proposed based on current knowledge and computational simulations of this system, in which insulin, after its initial interaction with binding site 1, engages with site 2 between the fibronectin type III (FnIII)-1 and -2 domains, which changes the conformation of FnIII-3 and eventually translates into structural changes across the membrane. This model provides a new perspective on the process of insulin binding to its receptor and, thus, could lead to future novel drug discovery efforts.

Quantitative Characterization of Domain Motions in Molecular Machines.

The work of molecular machines such as the ribosome is accompanied by conformational changes, often characterized by relative motions of their domains. The method we have developed seeks to quantify these motions in a general way, facilitating comparisons of results obtained by different researchers. Typically there are multiple snapshots of a structure in the form of pdb coordinates resulting from flexible fitting of low-resolution density maps, from X-ray crystallography, or from molecular dynamics simulation trajectories. Our objective is to characterize the motion of each domain as a coordinate transformation using moments of inertia tensor, a method we developed earlier. What has been missing until now are ancillary tools that make this task practical, general, and biologically informative. We have provided a comprehensive solution to this task with a set of tools implemented on the VMD platform. These tools address the need for reproducible segmentation of domains, and provide a generalized description of their motions using principal axes of inertia. Although this methodology has been specifically developed for studying ribosome motion, it is applicable to any molecular machine.

Multiplexed modular genetic targeting of quantum dots.

While DNA-directed nanotechnology is now a well-established platform for bioinspired nanoscale assembly in vitro, the direct targeting of various nanomaterials in living biological systems remains a significant challenge. Hybrid biological systems with integrated and targeted nanomaterials may have interesting and exploitable properties, so methods for targeting various nanomaterials to precise biological locations are required. Fluorescence imaging has benefited from the use of nanoparticles with superior optical properties compared to fluorescent organic dyes or fluorescent proteins. While single-particle tracking (SPT) in living cells with genetically encoded proteins is limited to very short trajectories, the high photon output of genetically targeted and multiplexed quantum dots (QDs) would enable long-trajectory analysis of multiple proteins. However, challenges with genetic targeting of QDs limit their application in these experiments. In this report, we establish a modular method for targeting QD nanoparticles selectively to multiple genetically encoded tags by precomplexing QD-streptavidin conjugates with cognate biotinylated hapten molecules. This approach enables labeling and SPT of multiple genetically encoded proteins on living cells at high speed and can label expressed proteins in the cytosol upon microinjection into living cells. While we demonstrate labeling with three distinct QD conjugates, the approach can be extended to other specific hapten-affinity molecule interactions and alternative nanoparticles, enabling precise directed targeting of nanoparticles in living biological systems.

Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins.

The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.

Inferring biological structures from super-resolution single molecule images using generative models.

Localization-based super resolution imaging is presently limited by sampling requirements for dynamic measurements of biological structures. Generating an image requires serial acquisition of individual molecular positions at sufficient density to define a biological structure, increasing the acquisition time. Efficient analysis of biological structures from sparse localization data could substantially improve the dynamic imaging capabilities of these methods. Using a feature extraction technique called the Hough Transform simple biological structures are identified from both simulated and real localization data. We demonstrate that these generative models can efficiently infer biological structures in the data from far fewer localizations than are required for complete spatial sampling. Analysis at partial data densities revealed efficient recovery of clathrin vesicle size distributions and microtubule orientation angles with as little as 10% of the localization data. This approach significantly increases the temporal resolution for dynamic imaging and provides quantitatively useful biological information.

Evaluation of sCMOS cameras for detection and localization of single Cy5 molecules.

The ability to detect single molecules over the electronic noise requires high performance detector systems. Electron Multiplying Charge-Coupled Device (EMCCD) cameras have been employed successfully to image single molecules. Recently, scientific Complementary Metal Oxide Semiconductor (sCMOS) based cameras have been introduced with very low read noise at faster read out rates, smaller pixel sizes and a lower price compared to EMCCD cameras. In this study, we have compared the two technologies using two EMCCD and three sCMOS cameras to detect single Cy5 molecules. Our findings indicate that the sCMOS cameras perform similar to EMCCD cameras for detecting and localizing single Cy5 molecules.