A simple flash and freeze system for cryogenic time-resolved electron microscopy

Practical Guide for Implementing Cryogenic Electron Microscopy Structure Determination in Dermatology Research.

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.

Structural flexibility and/or dynamic interactions with other molecules is a critical aspect of protein function. CryoEM provides direct visualization of individual macromolecules sampling different conformational and compositional states. While numerous methods are available for computational classification of discrete states, characterization of continuous conformational changes or large numbers of discrete state without human supervision remains challenging. Here we present e2gmm, a machine learning algorithm to determine a conformational landscape for proteins or complexes using a 3-D Gaussian mixture model mapped onto 2-D particle images in known orientations. Using a deep neural network architecture, e2gmm can automatically resolve the structural heterogeneity within the protein complex and map particles onto a small latent space describing conformational and compositional changes. This system presents a more intuitive and flexible representation than other manifold methods currently in use. We demonstrate this method on both simulated data as well as three biological systems, to explore compositional and conformational changes at a range of scales. The software is distributed as part of EMAN2.

Free volume pockets inside a cell membrane play a prominent role in a variety of dynamic processes such as the permeability of small molecules across membranes and the diffusion of, e.g., lipids, drugs, and electron carriers in the plane of the membrane. Nonetheless, by now the chances for characterizing free volume voids in a nonperturbative manner through experiments have been very limited. Here we use lipid membranes as an example to show how positron annihilation spectroscopy (PALS) together with atomistic simulations can be employed to gauge changes in free volume pockets in biological macromolecular complexes. The measurements show that PALS is a viable technique to probe free volume in biomolecular systems. As examples, we consider the gel-to-fluid transition and the role of increasing cholesterol concentration in a lipid membrane. Further applications proposed in this work for PALS are likely to provide a great deal of insight into the understanding of the role of free volume in the dynamics of biomolecular complexes.

Digital holographic imaging has emerged as a label-free, non-invasive, and powerful tool, offering a transformative method for the real-time investigation of morphology, density, and other physical and mechanical features of non-biological and biological samples. The rapid development of these portable, economical imaging systems for clinical/non-clinical setups, especially in remote areas, is of great interest. These systems are early point-of-care applications in diagnosis, research, education, and training. This article reports a general-purpose, fully automated portable imaging tool, ‘HoloLume,’ that offers high-resolution 3D imaging that facilitates the comprehensive understanding and analysis of different media. The proposed microscopic system HoloLume realizes the interferograms generation of samples using common-path optics configuration based on a Fresnel biprism, and its motorized stage precisely scans the whole sample with a step of 3.87 μm for quick identification of the region of interest, which makes sure a time-efficient and robust analysis. The prototype, assembled from off-the-shelf components, costs approximately USD ∼776 and weighs around 3 kg. To validate and verify the performance of the HoloLume microscope, we have conducted experiments on both non-biological and biological samples. Thus, its non-invasive nature, high temporal stability, and economical lightweight prototyping make it an ideal choice for hands-on learning and quantitative analysis, reducing the need for time-consuming sample preparation and conventionally expensive microscopic systems. Furthermore, the adaptability of the proposed imaging tool to remote environments ensures wider accessibility and its easy integration with other imaging modalities enables a compact and cost-effective solution for advanced research and in field measurements.

Many functions in living cells are governed by macromolecular complexes. To fully describe the underlying mechanisms, they have to be understood at atomic level. The present study combines data obtained by X-ray crystallography and cryo-electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Two functions of macromolecular complexes, the downregulation of neurotransmitter release by the SNARE protein complex under oxidative stress and the translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis, were investigated. First, the hypothesis that oxidation of two cysteines on linker of the SNARE protein SNAP-25B and consequent disulfide bond formation shortens this linker sufficiently to hinder complex formation was tested. For this purpose, MD simulations of the SNARE complex with and without the disulfide bond were compared. Disulfide bond formation lead to conformational changes of the linker and of three central hydrophobic layers necessary to form the SNARE complex. Previously, mutations of residues contributing to these layers have been shown to reduce neurotransmitter release, suggesting that the stability of these layers is crucial for complex formation. The results from the simulations agree with the hypothesis that disulfide bond formation leads to a destabilization of the SNARE complex thus rendering it dysfunctional. This mechanism is interpreted as a chemomechanical regulation to shut down neurotransmitter release under oxidative stress, which has been linked to neurodegenerative diseases. In a second part I investigated the ribosome, where after peptide bond formation, bound tRNAs translocate by more than 7 nm to adjacent binding sites (A and P to P and E), accompanied by large-scale conformational motions (L1-stalk, intersubunit rotation) of the ribosome. By combining existing cryo-EM reconstructions of translocation intermediates with high resolution crystal structures, we obtained 13 near-atomic resolution structures. Subsequently, MD simulations of were carried out for each intermediate state. The obtained dynamics within these states allowed to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. Rapid motions of the L1-stalk and the small (30S) subunit on sub-microsecond timescales were revealed, whereas tRNA motions were seen to be rate-limiting for most transitions. By calculating the interaction free energy between L1-stalk and tRNA, molecular forces were derived showing that the L1-stalk is actively pulling the tRNA from P to E binding site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving key protein-tRNA contacts. This explains how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements. Translocation is accompanied by rotations of the 30S ribosomal subunit of more than 20 degrees relative to the large (50S) subunit. For each translocation intermediate, the affinity of the two subunits with each other must be finely tuned enabling such conformational flexibility while maintaining integrity of the ribosomal complex. Analyzing the trajectories at residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and primarily located close to the axis of rotation. ii) contacts that are formed and ruptured depending on the rotation angle, seen mainly on the periphery. Strikingly, also these rotation specific contacts substantially contribute to the overall stability of the ribosomal assembly and are expected to maintain a constant interaction energy with low barriers for rotation. The simulations reveal that upon removal of tRNAs peripheral contacts are weakened and, in turn, intersubunit rotation angles decrease, in agreement with cryo-EM analysis of tRNA depleted ribosomes. The identified mechanisms for lowering free energy barriers and for fine-tuning affinities might have developed similarly in other macromolecular complexes.

BackgroundProteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood.MethodsLast generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle.ResultsUsing these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation.ConclusionsRecent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the in vitro determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes in situ, without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts. This forward-looking review covers recent developments in cryoEM/ET and related technologies such as cryogenic focused ion beam milling scanning electron microscopy and correlative light microscopy, increasingly enhanced and supported by artificial intelligence algorithms. Their potential application to emerging concepts is discussed, primarily the prospect of complementing medical histopathology analysis. Machine learning solutions are poised to address current challenges posed by "big data" in cryoET of tissues, cells, and macromolecules, offering the promise of enabling novel, quantitative insights into disease processes, which may translate into the clinic and lead to improved diagnostics and targeted therapeutics.

Introduction: Understanding changes in PPI maps from a healthy and diseased state can illuminate our understanding of biological changes and disease processes. PPI maps enable a higher order of information than a simple listing of components by providing functional context, yet existing maps grossly underrepresent the total biological information potential of PPIs. Herein, we describe Proteograph, a novel platform that leverages the nano-bio interactions of nanoparticles (NPs) for deep and unbiased proteomic sampling that can provide insights on PPI across biological samples. Proteograph leverages the protein corona that forms on the surface of NPs as a function of their distinct biophysicochemical properties. NPs reproducibly bind subsets of proteins from biofluids as a function of protein concentration, protein-NP affinity, and protein-protein interactions to form a corona on the NP surface. We have employed Proteograph to quantify known PPIs using a panel of 3 distinct NPs to capture plasma proteins and derive maps of NSCLC and control subjects in order to identify biological changes in interactions, potentially indicative of health and disease. Method and Results: We collected plasma samples from 288 subjects: healthy (n=82), comorbid (n=81) and NSCLC stages I-IV (n=125). In this initial study, we used three NPs with distinct properties and evaluated the protein corona of plasma samples by mass spectrometry (MS) to quantify 1,235 protein groups (1% FDR). A fully automated assay workflow enabled preparation of 3 NPs' corona for MS analysis across 288 subjects in approximately 6 days. We mapped the protein groups to a PPI map derived from the STRING database. Partitioning the network into clusters identified 9 interaction clusters with greater than 10 protein members. These clusters enabled us to investigate differences in the PPI networks between NSCLC patients vs. controls. Evaluating the expression of proteins in these groups, we identified interaction clusters that had significant differences between cancer vs. control (t-test, p < 0.01 Bonferroni corrected). Six of the clusters show differential behavior between NSCLC vs. healthy controls (p < 0.01). Two of these clusters show differential behavior between NSCLC vs. healthy and comorbid (p < 0.01). Investigation of these differentially expressed clusters reveals links to known cancer biology with proteins related to the immune system and endocytosis pathways. Discussion: We have used the Proteograph platform to identify PPI clusters that are differentiated between NSCLC and control individuals. We believe the efficiency of the Proteograph platform applied to sufficiently powered studies may enable comprehensive understanding of known PPIs, and potentially infer and confirm new PPIs, in health and disease. Citation Format: Asim Siddiqui, John E. Blume, William C. Manning, Gregory Troiano, Philip Ma, Robert Langer, Vivek Farias, Omid C. Farokhzad. Plasma protein-protein interactome (PPI) maps derived from the protein corona captured at the nano-bio interface of nanoparticles reveal differential networks for non-small cell lung cancer (NSCLC) and control subjects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6571.

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factors (IFs), which must dissociate before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2–5 and 70S4,6–8 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a 70S EC structure obtained directly from a 70S IC formed via an IF-catalyzed initiation reaction has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)9 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

RNAs play critical roles in most biological processes. Although the three-dimensional (3D) structures of RNAs primarily determine their functions, it remains challenging to experimentally determine these 3D structures due to their conformational heterogeneity and intrinsic dynamics. Cryogenic electron microscopy (cryo-EM) has recently played an emerging role in resolving dynamic conformational changes and understanding structure-function relationships of RNAs including ribozymes, riboswitches and bacterial and viral noncoding RNAs. A variety of methods and pipelines have been developed to facilitate cryo-EM structure determination of challenging RNA targets with small molecular weights at subnanometer to near-atomic resolutions. While a wide range of conditions have been used to prepare RNAs for cryo-EM analysis, correlations between the variables in these conditions and cryo-EM visualizations and reconstructions remain underexplored, which continue to hinder optimizations of RNA samples for high-resolution cryo-EM structure determination. Here we present a protocol that describes rigorous screenings and iterative optimizations of RNA preparation conditions that facilitate cryo-EM structure determination, supplemented by cryo-EM data processing pipelines that resolve RNA dynamics and conformational changes and RNA modeling algorithms that generate atomic coordinates based on moderate- to high-resolution cryo-EM density maps. The current protocol is designed for users with basic skills and experience in RNA biochemistry, cryo-EM and RNA modeling. The expected time to carry out this protocol may range from 3 days to more than 3 weeks, depending on the many variables described in the protocol. For particularly challenging RNA targets, this protocol could also serve as a starting point for further optimizations.

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

The need for improved separation power in Liquid Chromatography (LC), particularly for complex multi-constituent biological samples, is becoming increasingly critical. Recent advancements have led to a growing interest in Two-Dimensional Liquid Chromatography (2DLC), which offers significant advantages over traditional One-Dimensional Liquid Chromatography (1DLC) by enabling higher peak capacities and resolution. This review explores the potential of 2DLC, highlighting its ability to enhance separation by combining orthogonal separations and offering peak capacity that, under ideal conditions, is the product of the peak capacities of both dimensions. Despite its impressive separation power, comprehensive 2DLC is hindered by long analysis times, often extending several hours. Recent innovations in high-temperature LC techniques have addressed this challenge by drastically reducing the time required for the Second Dimension of 2DLC to approximately 20 seconds, enabling rapid separations with improved peak capacities exceeding 2000. These developments have led to significant reductions in analysis time, allowing complex biological samples with over 200 detectable peaks to be analyzed in under 30 minutes. This shift toward fast, high-temperature LC is transforming the capabilities of 2DLC, making it an invaluable tool for the analysis of biological samples. Additionally, the review touches on complementary techniques like NMR spin relaxation spectroscopy, which aids in characterizing protein dynamics, offering further insights into protein conformational changes on the picosecond-nanosecond timescale.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

SKELETAL MUSCLES adjust their contractile output over an exceptionally wide dynamic range, as needed to produce movements ranging from sitting and standing, to athletics and dance. At the single fiber level, the force generated by skeletal muscles is determined, ultimately, by the formation of cycling cross bridges between actin and myosin. This interaction requires Ca 2 and is governed by the simple relationship: force is proportional to intracellular ionized Ca 2 . Cross-bridge formation is inhibited in resting muscle when Ca 2 is sequestered within the sarcoplasmic reticulum (SR); it is activated when stored Ca 2 is released into the cytosol, allowing actin-myosin interactions to proceed. Contraction stops as Ca 2 is actively resequestered by the Ca 2 -ATPase of the SR (SERCA1). Thus Ca 2 is the central messenger in muscle activation, and the amplitude and time course of the intracellular Ca 2 change is the major determinant of contractile output. The key mechanisms that activate Ca 2 release from the SR are now well defined. This sequence, termed excitation-contraction coupling, begins when an excitatory input from the motor nerve triggers a propagating action potential on the muscle membrane. The action potential rapidly depolarizes the entire sarcolemma, including transverse tubules, which invaginate from the surface membrane and form specialized junctions (triads) with the SR along much of their length. The triad junction serves as a platform for assembling sarcolemmal calcium