Journal of Structural Biology: X最新文献

It is well known that insulating samples can accumulate electric charges from exposure to an electron beam. How the accumulation of charge affects imaging parameters and sample stability in transmission electron microscopy is poorly understood. To quantify these effects, it is important to know how the charge is distributed within the sample and how it builds up over time. In the present study, we determine the spatial distribution and temporal dynamics of charge accumulation on vitreous ice samples with embedded proteins through a combination of modeling and Fresnel diffraction experiments. Our data reveal a rapid evolution of the charge state on ice upon initial exposure to the electron beam accompanied by charge gradients at the interfaces between ice and carbon films. We demonstrate that ice film movement and charge state variations occur upon electron beam exposure and are dose-rate dependent. Both affect the image defocus through a combination of sample height changes and lensing effects. Our results may be used as a guide to improve sample preparation, data collection, and data processing for imaging of dose-sensitive samples.

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Heterotopic mineralization entails pathological mineral formation inside soft tissues. In human tendons mineralization is often associated with tendinopathies, tendon weakness and pain. In Achilles tendons, mineralization is considered to occur through heterotopic ossification (HO) primarily in response to tendon pathologies. However, refined details regarding HO deposition and microstructure are unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomography. Furthermore, we test the potential of studying local tissue injury by needling intact Achilles tendons and the relation between tissue microdamage and HO. The results show that HO occurs in all intact Achilles tendons at 16 weeks of age. HO deposits are characterized by an elongated ellipsoidal shape and by a fiber-like internal structure which suggests that some collagen fibers have mineralized. The data indicates that deposition along fibers initiates in the pericellular area, and propagates into the intercellular area. Within HO deposits cells are larger and more rounded compared to tenocytes between unmineralized fibers, which are fewer and elongated. The results also indicate that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption may also occur. Additionally, phase contrast synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level in response to needling. The needle injury protocol could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition.

In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2–3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the “simple insertion method” is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation.

Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50–150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.

Atomic force microscopy (AFM) and high-speed scanning have significantly advanced real time observation of biomolecular dynamics, with applications ranging from single molecules to the cellular level. To facilitate the interpretation of resolution-limited imaging, post-experimental computational analysis plays an increasingly important role to understand AFM measurements. Data-driven simulation of AFM, computationally emulating experimental scanning, and automatized fitting has recently elevated the understanding of measured AFM topographies by inferring the underlying full 3D atomistic structures. Providing an interactive user-friendly interface for simulation AFM, the BioAFMviewer software has become an established tool within the Bio-AFM community, with a plethora of applications demonstrating how the obtained full atomistic information advances molecular understanding beyond topographic imaging. This graphical review illustrates the BioAFMviewer capacities and further emphasizes the importance of simulation AFM to complement experimental observations.

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.

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six α-subunits and six β-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC β-subunits form stable homohexamers, to which different numbers of α-subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulae α₀β₆, α₁β₆, α₂β₆, α₃β₆, α₄β₆, α₅β₆, α₆β₆) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein’s hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine ¹H-¹³C/²H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10–20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record H^N- and H^C-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of ¹H–¹H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate H^N-Η^C, H^C-H^N and H^C-H^C restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[¹³C,¹⁵N] labeled GB1 sample at 95–100 kHz MAS.