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The fit of atomic models of protein structures to high-resolution cryo-electron microscopy maps can be assessed with a validation tool, EMRinger. Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger ( https://github.com/fraser-lab/EMRinger ) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3–4.5Å), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids – typical in a macromolecular assembly – is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover. Understanding how structural dynamics contribute to protein function is a longstanding challenge in structural biology. Now, time-resolved X-ray solution scattering following an infrared laser-induced temperature jump has been used to probe functional, intramolecular motions in the dynamic enzyme cyclophilin A.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps. Here, the authors quantify the effect of cryo-EM data acquisition with stage-tilt on the global resolution of reconstructions and present a tool for predicting an optimal stage-tilt angle to ameliorate the effects of preferred specimen orientation.

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we image tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing membrane modification and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virus particles bud. Naturally occurring furin site variants determine whether virus particles mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation. In this study, Dahmane et al use a method called cryo-electron tomography to uncover new details of how tick-borne flaviviruses transform cells into virus factories.

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.Shifts in temperature alter the structure and dynamics of macromolecules. Now, infra-red laser-induced temperature jump is combined with X-ray crystallography to observe protein structural dynamics in real time. Using this method, motions related to the catalytic cycle of lysozyme, a model enzyme, are visualized at atomic resolution and across broad timescales.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across amembrane. Ordered water molecules arranged in “wires” inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Despite containing their own distinct genome, mitochondria rely heavily on the nucleus to encode 99% of the proteins necessary for mitochondrial function. Most of these proteins are synthesized by cytoplasmic ribosomes before targeting and import to mitochondria. However, a subset of proteins is imported co-translationally, with their synthesis and import occurring simultaneously. Although evidence of co-translational import into the mitochondria was discovered nearly five decades ago, the molecular mechanisms mediating this elusive process have remained unclear and somewhat debated. Our lab harnessed cellular cryo-electron tomography imaging and computational tools to provide a new molecular perspective to this elusive process. We show that cytoplasmic ribosomes engaged in co-translational import make multiple contacts with the mitochondrial outer membrane. We show that ribosomes primed for import exhibit a high degree of clustering on the mitochondrial surface in an arrangement that suggests the formation of polysomes. Interestingly, these ribosomes localize at sites of local constrictions of the outer and inner mitochondrial membrane, suggesting that local membrane remodeling may facilitate efficient protein import into the distinct mitochondrial compartments. Our work sets the stage for enabling future studies to identify molecular mechanisms mediating mitochondrial co-translational import.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum. The ability of an organism to grow and reproduce, that is, it’s “fitness”, is determined by how its genes interact with the environment. Yeast is a model organism in which researchers can control the exact mutations present in the yeast’s genes (its genotype) and the conditions in which the yeast cells live (their environment). This allows researchers to measure how a yeast cell’s genotype and environment affect its fitness. Ubiquitin is a protein that many organisms depend on to manage cell stress by acting as a tag that targets other proteins for degradation. Essential proteins such as ubiquitin often remain unchanged by mutation over long periods of time. As a result, these proteins evolve very slowly. Like all proteins, ubiquitin is built from a chain of amino acid molecules linked together, and the ubiquitin proteins of yeast and humans are made of almost identical sequences of amino acids. Although ubiquitin has barely changed its sequence over evolution, previous studies have shown that – under normal growth conditions in the laboratory – most amino acids in ubiquitin can be mutated without any loss of cell fitness. This led Mavor et al. to hypothesize that treating the yeast cells with chemicals that cause cell stress might lead to amino acids in ubiquitin becoming more sensitive to mutation. To test this idea, a class of graduate students at the University of California, San Francisco grew yeast cells with different ubiquitin mutations together, and with different chemicals that induce cell stress, and measured their growth rates. Sequencing the ubiquitin gene in the thousands of tested yeast cells revealed that three of the chemicals cause a shared set of amino acids in ubiquitin to become more sensitive to mutation. This result suggests that these amino acids are important for the stress response, possibly by altering the ability of yeast cells to target certain proteins for degradation. Conversely, another chemical causes yeast to become more tolerant to changes in the ubiquitin sequence. The experiments also link changes in particular amino acids in ubiquitin to specific stress responses. Mavor et al. show that many of ubquitin’s amino acids are sensitive to mutation under different stress conditions, while others can be mutated to form different amino acids without effecting fitness. By testing the effects of other chemicals, future experiments could further characterize how the yeast’s genotype and environment interact.

Lipid bilayers form the basis of organellar architecture, structure, and compartmentalization in the cell. Decades of biophysical, biochemical, and imaging studies on purified or reconstituted liposomes have shown that variations in lipid composition influence the physical properties of membranes, such as thickness and curvature. However, similar studies characterizing these membrane properties within the native cellular context have remained technically challenging. Recent advancements in cellular cryo-electron tomography (cryo-ET) imaging enable high-resolution, three-dimensional views of native organellar membrane architecture preserved in near-native conditions. We previously developed a 'Surface Morphometrics' pipeline that generates surface mesh reconstructions to model and quantify cellular membrane ultrastructure from cryo-ET data. Here, we expand this pipeline to measure the distance between the phospholipid head groups (PHG) of the membrane bilayer as a readout of membrane thickness. Using this approach, we demonstrate thickness variations both within and between distinct organellar membranes. We also demonstrate that membrane thickness positively correlates with other features, such as membrane curvedness. Further, we show that subcompartments of the mitochondrial inner membrane exhibit varying membrane thicknesses that are independent of network morphology (i.e., fragmented versus elongated networks). Finally, we demonstrate that large membrane-associated macromolecular complexes exhibit distinct density profiles that correlate with local variations in membrane thickness. Overall, our updated Surface Morphometrics pipeline provides a framework for investigating how changes in membrane composition in various cellular and disease contexts affect organelle ultrastructure and function.