Journal of Structural Biology: X最新文献

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.

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.

RNases are varied in the RNA structures and sequences they target for cleavage and are an important type of enzyme in cells. Despite the numerous examples of RNases known, and of those with determined three-dimensional structures, relatively few examples exist with the RNase bound to intact cognate RNA substrate prior to cleavage. To better understand RNase structure and sequence specificity for RNA targets, in vitro methods used to assemble these enzyme complexes trapped in a pre-cleaved state have been developed for a number of different RNases. We have surveyed the Protein Data Bank for such structures and in this review detail methodologies that have successfully been used and relate them to the corresponding structures. We also offer ideas and suggestions for future method development. Many strategies within this review can be used in combination with X-ray crystallography, as well as cryo-EM, and other structure-solving techniques. Our hope is that this review will be used as a guide to resolve future yet-to-be-determined RNase–substrate complex structures.

Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein’s expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils’ segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of ‘intermediate motion selection’ (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter’s detection with specific dyes and positron emission tomography tracers.