Resolution Structure Research Articles

Scaffold-enabled high-resolution cryo-EM structure determination of RNA

Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.

Engineering dimer mutants of human geranylgeranyl pyrophosphate synthase.

Geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme in protein prenylation, plays a critical role in cellular signal transduction and is a promising target for cancer therapy. However, the enzyme's native hexameric quaternary structure presents challenges for crystallographic studies. The primary objective of this study was to engineer dimeric forms of human GGPPS to facilitate high-resolution crystallographic analysis of its ligand binding interactions. Through site-directed mutagenesis, we disrupted the inter-dimer interactions required for hexamer assembly, generating three stable double-site mutants: Y246D/C247L, Y246D/C205A, and Y246K/C247L. Enzyme assays confirmed that all mutants retained wild-type catalytic activity under both saturating and subsaturating substrate conditions. Differential scanning fluorimetry showed that the mutant proteins had a ~10°C lower melting temperature than the wild-type enzyme but exhibited similar shifts in melting temperature in the presence of the known inhibitors risedronate and zoledronate. Crystallographic analysis of the Y246D/C247L mutant yielded a 2.1 Å resolution structure, providing detailed insights into the binding of isopentenyl pyrophosphate. Closer inspection also revealed the unexpected formation of intermolecular disulfide bonds connecting neighboring dimers, which may explain the enhanced crystallizability of the Y246D/C247L mutant compared to the wild-type and other mutants. These findings highlight the potential of the dimeric mutants as substitutes for wild-type GGPPS in future studies. Optimized dimeric mutants could serve as valuable molecular tools to further our understanding of the enzyme's structural and functional properties and aid in the rational design of novel therapeutic agents targeting GGPPS.

The Dynamism of the Structure and Mechanism of the Alternative Dispute Resolution (ADR) System in Bangladesh for Dispute Resolution Outside the Formal Courts

This study aimed to investigate the dynamic nature of this hybrid system to serve society in contemporary Bangladesh's changing circumstances effectively. Through a content analysis of documents and literature, it was found that the system is quite dynamic and effective in terms of structure and mechanism. Over the years, it has expanded in terms of scope and institutions to cover resolutions of conflicts relating to not only family affairs but civil disputes and even selected criminal cases. It is now compulsory for civil disputes to be addressed first through ADR. The system is gaining popularity in the country. The various ADR institutions entertain a large and increasing number of disputes of both natures every year. In most cases, the rate of success in resolving the cases submitted to these institutions is as high as 88%. However, handling civil disputes through ADR has yet to become popular. The record shows that in four districts, only 2.2% of civil suits were disposed of through ADR during 2012 and 2014. It suggests that adequate efforts need to be made to make ADR popular in this respect as well.

High resolution structures of Myosin-IC reveal a unique actin-binding orientation, ADP release pathway, and power stroke trajectory.

Myosin-IC (myo1c) uses an ATP-driven 'power-stroke' to support slow intracellular membrane and vesicle transport. We used cryo-electron microscopy to understand adaptations of myo1c to perform its unique roles. We discovered an altered interface between myo1c and actin compared with the closely related myo1b, which repositions the motor domain and alters the trajectory of its lever arm swing compared to other myosins. This explains why myo1c propels actin filaments in a leftward circular path. We also discovered a unique role in force sensing for a structural element called the N-terminal extension and built a full-length atomic model for the myo1c power-stroke. Our findings highlight how myosins can tune their power-stroke geometries and force-sensing properties to adapt to diverse cellular functions.

Exploring the reaction dynamics of alanine racemase using serial femtosecond crystallography

Alanine racemase (Alr) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent racemization between l- and d-alanine in bacteria. Owing to the potential interest in targeting Alr for antibacterial drug development, several studies have determined the structures of Alr from different species, proposing models for the reaction mechanism. Insights into its reaction dynamics may be conducive to a better understanding of the Alr reaction mechanism. In this study, we determined the structures of the apo and reaction states of Bacillus subtilis Alr (BsAlr) at room temperature using a fixed-target based X-ray free-electron laser. The 2.3 Å resolution structures revealed the alanine substrate or intermediate in various positions at the active site. Conformational change between the N- and C-terminal domains of BsAlr expanded the entryway for substrate binding. In the reaction state of BsAlr, two main alanine binding states were observed: one alanine molecule is positioned away from PLP, whereas the other alanine molecule is covalently bonded to PLP. These structures might represent the dynamic states of the substrate for entrance into, reaction with, or exit from the active site. Our approach provides a simple and rapid method for elucidating the intermediate structure of Alr, which can be expanded to other enzymes.

Impact of data structure types and spatial resolution on landslide volumetric change measurements

Terrain is a dynamic component of the landscape, subject to rapid changes, particularly in scenarios such as landslides. This study investigates how the spatial resolution and data structure of digital terrain models (DTMs) influence the estimation of landslide volume changes. We selected a landslide formed by the undercutting action of the Belá River in Slovakia as our research site. Our findings indicate that raster data structures, across various spatial resolutions, generally yield more consistent volume estimates compared to 3D mesh data structures. Nonetheless, at higher spatial resolutions (0.1 m and 0.25 m), the 3D mesh data structure demonstrates superior capability in capturing detailed terrain features, resulting in more precise volume estimations of the landslide.

Specificity and diversity of Klebsiella pneumoniae phage-encoded capsule depolymerases.

Klebsiella pneumoniae is an opportunistic pathogen with significant clinical relevance. K. pneumoniae-targeting bacteriophages encode specific polysaccharide depolymerases with the ability to selectively degrade the highly varied protective capsules, allowing for access to the bacterial cell wall. Bacteriophage depolymerases have been proposed as novel antimicrobials to combat the rise of multidrug-resistant K. pneumoniae strains. These enzymes display extraordinary diversity, and are key determinants of phage host range, however with limited data available our current knowledge of their mechanisms and ability to predict their efficacy is limited. Insight into the resolved structures of Klebsiella-specific capsule depolymerases reveals varied catalytic mechanisms, with the intra-chain cleavage mechanism providing opportunities for recombinant protein engineering. A detailed comparison of the 58 characterised depolymerases hints at structural and mechanistic patterns, such as the conservation of key domains for substrate recognition and phage tethering, as well as diversity within groups of depolymerases that target the same substrate. Another way to understand depolymerase specificity is by analyzing the targeted capsule structures, as these may share similarities recognizable by bacteriophage depolymerases, leading to broader substrate specificities. Although we have only begun to explore the complexity of Klebsiella capsule depolymerases, further research is essential to thoroughly characterise these enzymes. This will be crucial for understanding their mechanisms, predicting their efficacy, and engineering optimized enzymes for therapeutic applications.

A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure.

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

NCAP: Noncanonical Amino Acid Parameterization Software for CHARMM Potentials.

Noncanonical amino acids (ncAAs) provide numerous avenues for the introduction of novel functionality to peptides and proteins. ncAAs can be incorporated through solid-phase synthesis or genetic code expansion in conjugation with heterologous expression of the encoded protein modification. Due to the difficulty of synthesis or overexpression, wide chemical space, and lack of empirically resolved structures, modeling the effects of ncAA mutation is critical for rational protein design. To evaluate the structural and functional perturbations ncAAs introduce, we utilize molecular potentials that describe the forces in the protein structure. Most potentials such as CHARMM are designed to model canonical residues but can be parametrized to include novel ncAAs. In this work, we introduce NCAP, a software package to generate CHARMM-compatible parameters from quantum chemical calculations. Unlike currently available tools, NCAP is designed to recognize the ncAA structure and automatically bridge the gap between quantum chemical calculations and CHARMM potential parameters. For our software, we discuss the workflow, validation against canonical parameter sets, and comparison with published ncAA-protein structures.

Mapping the FF domain folding pathway via structures of transiently populated folding intermediates

Despite the tremendous accomplishments of AlpaFold2/3 in predicting biomolecular structure, the protein folding problem remains unsolved in the sense that accurate atomistic models of how protein molecules fold into their native conformations from an unfolded ensemble are still elusive. Here, using chemical exchange saturation transfer (CEST) NMR experiments and a comprehensive four-state kinetic model of the folding trajectory of a 71 residue four-helix bundle FF domain from human HYPA/FBP11 we present an atomic resolution structure of a transiently formed intermediate, I2, that along with the structure of a second intermediate, I1, provides a description of the FF domain folding trajectory. By recording CEST profiles as a function of urea concentration the extent of compaction along the folding pathway is evaluated. Our data establish that unlike the partially disordered I1 state, the I2 intermediate that is also formed before the rate-limiting folding barrier is well ordered and compact like the native conformer, while retaining nonnative interactions similar to those found in I1. The slow-interconversion from I2 to F, involving changes in secondary structure and the breaking of nonnative interactions, proceeds via a compact transition-state. Interestingly, the native state of the FF1 domain from human p190-A Rho GAP resembles the I2 conformation, suggesting that well-ordered folding intermediates can be repurposed by nature in structurally related proteins to assume functional roles. It is anticipated that the strategy for elucidation of sparsely populated and transiently formed structures of intermediates along kinetic pathways described here will be of use in other studies of protein dynamics.

Crucial role and conservation of the three [2Fe-2S] clusters in the human mitochondrial ribosome.

Mitochondria synthesize only a small set of their proteins on endogenous mitoribosomes. These particles differ in structure and composition from both their bacterial 70S ancestors and cytosolic 80S ribosomes. Recently published high resolution structures of the human mitoribosome revealed the presence of three [2Fe-2S] clusters in the small and large subunits. Each of these clusters is coordinated in a bridging fashion by cysteine residues from two different mitoribosomal proteins. Here, we investigated the cell biological and biochemical roles of all three Fe/S clusters in mitochondrial function and assembly. First, we found a requirement of both early and late factors of the mitochondrial iron-sulfur cluster assembly machinery for protein translation indicating that not only the mitoribosome [2Fe-2S] clusters but also the [4Fe-4S] cluster of the mitoribosome assembly factor METTL17 are required for mitochondrial translation. Second, siRNA-mediated depletion of the cluster-coordinating ribosomal proteins bS18m, mS25 or mL66 and complementation with either the respective wild-type or cysteine-exchange proteins unveiled the importance of the clusters for assembly, stability, and function of the human mitoribosome. As a consequence, the lack of cluster binding to mitoribosomes impaired the activity of the mitochondrial respiratory chain complexes and led to altered mitochondrial morphology with a loss of cristae membranes. Finally, in silico investigation of the phylogenetic distribution of the cluster-coordinating cysteine motifs indicated their presence in most metazoan mitoribosomes, with exception of ray-finned fish. Collectively, our study highlights the functional need of mitochondrial Fe/S protein biogenesis for both protein translation and respiratory energy supply in most metazoan mitochondria.

Cost-benefit analysis of cryogenic electron tomography subtomogram averaging of chaperonin MmCpn at near atomic resolution.

Cryogenic electron microscopy single particle analysis (cryoEM-SPA) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) toward a similar resolution, in contrast, is still under active development. Here, we use the archeal chaperonin MmCpn as a model macromolecule to quantitatively investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers, and tilt angle ranges. By delineating the feasibility and experimental factors of attaining near atomic resolution structure with cryoET-STA, especially the effect of electron damage through the tilt series and inelastic scattering at various ice thickness, we encourage a customized tilt series collection strategy for efficient throughput. This study provides a biophysical basis for the application of cryoET-STA (for highly symmetric molecules like MmCpn) toward high resolution and the rationales in using cryoET-STA to achieve an efficient outcome at the desired resolution.

Photodissociation dynamics of H2S+ via A2A1 (0, 11, 0) state

Time-sliced velocity map ion imaging (VMI) experiments were performed to investigate the photodissociation of H2S+X2B1, via the excitation to the A2A1 (0, 11, 0) state. Experimental images of the S+ (4Su) products were recorded near 349.60 nm for the K = 1 band, and near 344.30 nm for the K = 2 band. The derived product total kinetic energy release (TKER) spectra exhibit partially rotationally resolved structures corresponding to the H2(X1Σg+) co-products. The observed product state and angular distributions both exhibit sensitive dependence on the photolysis wavelength at near 349.60 nm and 344.30 nm. These phenomena indicate the underlying rich dynamic details and the role of the rotational excitation of H2S+.

Revisiting huntingtin activity and localization signals in the context of protein structure

Protein localization signals and activity motifs have been defined within huntingtin since 2003. Advances in technology in protein structure determination by cryo-electron microscopy (EM) have led to 2.6 Å resolution structures of huntingtin and HAP40 for the majority of the protein, although structure of the amino terminus with the polyglutamine expansion remains elusive in the context of full-length huntingtin. Recent advances in protein modeling using neural network algorithms trained on a database of known protein structures has resulted in structure predictions that are useful for researchers but need experimental validation. Here, we use both structures solved by cryo-EM as well as modeling centered around experimental structural data to retrospectively revisit huntingtin protein localization signals identified prior to the cryo-EM and AI-enabled structural revolutions. We interrogate these models as well as put forward testable hypotheses of allosteric changes in huntingtin and how they could be affected by polyglutamine expansion. We also extended this methodology to another polyglutamine disease protein, ataxin-1, expanded in Spinocerebellar Ataxia Type 1 (SCA1).

Cryo-EM structure of a novel α-synuclein filament subtype from multiple system atrophy.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease characterized by accumulation of α-synuclein cross-β amyloid filaments in the brain. Previous structural studies of these filaments by cryo-electron microscopy (cryo-EM) revealed three discrete folds distinct from α-synuclein filaments associated with other neurodegenerative diseases. Here, we use cryo-EM to identify a novel, low-populated MSA filament subtype (designated Type I2) in addition to a predominant class comprising MSA Type II2 filaments. The 3.3-Å resolution structure of the Type I2 filament reveals a fold consisting of two asymmetric protofilaments, one of which adopts a novel structure that is chimeric between two previously reported protofilaments. These results further define MSA-specific folds of α-synuclein filaments and have implications for designing MSA diagnostics and therapeutics.

Research on rotational rollback suppression of diamond-shaped active locking flexure mechanism (ALFM) driven by piezoelectric wafers

Abstract Piezoelectric actuators have a wide range of applications in many areas due to their advantages of fast response speed, high resolution, compact and simple structure, diverse configurations, and resistance to electromagnetic interference. However, existing piezoelectric actuators generally have large fallback, and a few researchers have applied the ALFM to the fallback suppression of actuators. In this paper, an ALFM using a piezoelectric wafer as the driving source is innovatively designed, and the structure and dimensions are simulated and optimized using Finite Element Method, according to which the piezoelectric wafer-type ALFM is used to design a new inertial piezoelectric actuator that can suppress backward movement. The motion principle of the piezoelectric actuator is theoretically analyzed, followed by the establishment of the machine dynamics model of the piezoelectric actuator and simulation with MATLAB/simulink to verify the reasonableness of the dynamics model. Finally, a series of experiments are carried out on the processed drive model, and the results show that the maximum accuracy of the actuator is 8.5 μrad, and the maximum load capacity is 160 g. The comparison experiments at 30 Hz and 40 Hz with and without the ALFM prove that the locking mechanism does supress the actuator from backing off to a certain extent, which verifies the reasonableness of the scheme proposed in this paper.

Dimensionality and Molecular Packing Control of Covalent Organic Frameworks through Pendant Group Design.

Tuning the dimensions and molecular packing geometry of crystalline organic frameworks and polymers represents an important challenge for reticular chemistry. Here we show that for extended structures made of 1,3,6,8-tetrakis(4-aminophenyl)pyrene (PyTTA) linked with methoxy group functionalized terephthalaldehyde aldehydes, simple substituents on the aldehyde linker can have profound structure directing effects due to noncovalent interactions. Specifically, reacting 2,3-dimethoxyterephthalaldehyde with PyTTA gives a 2D covalent organic framework with unique AA-inclined-AA stacking and bilayer pyrene motifs, whereas reacting 2,5-dimethoxyterephthalaldehyde with PyTTA gives a 1D crystalline polymer with AB stacking and isolated pyrene motifs. Both materials show high crystallinity, allowing for atomic resolution structure determination using three-dimensional electron diffraction, and the similarity of their fluorescence properties shows the electronic structures of pyrene-based extended structures mostly depends on the in-plane structures, which is supported by density functional theory calculations. These two pyrene-based extended structures also show different fluorescence responses to organic vapors due to different pore environments. The current work shows the potential of noncovalent interactions in the reticular design of functional organic materials.

Infectious parvovirus B19 circulates in the blood coated with active host protease inhibitors

The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study, we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly, two host protease inhibitors (PIs), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3, were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious, and the capsid-associated PIs retain activity; however, upon virion interaction with target cells, the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity.

2-D Displacement Sensor With High Resolution and Simple Structure

2-D Displacement Sensor With High Resolution and Simple Structure

Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles

Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures of membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs of these vesicles can be challenging due to the strong signal contributed from the lipid bilayer. This challenge often requires iterative and laborious particle selection workflows to generate a dataset of high-quality particle images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on the Segment Anything model. Vesicle Picker enables automatic identification of vesicles in cryo-EM micrographs with high recall and precision. It then exhaustively selects all potential particle locations, either at the perimeter or uniformly over the surface of the projection of the vesicle. The program is designed to interface with cryoSPARC, which performs both upstream micrograph processing and downstream single particle image analysis. We demonstrate Vesicle Picker’s utility by determining a high-resolution map of the vacuolar-type ATPase from micrographs of native synaptic vesicles (SVs) and identifying an additional protein or protein complex in the SV membrane.