Cryo-electron Microscopy Image Research Articles

Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex.

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C-terminus of NuMA directly binds to and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited, but mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. The binding of the cortical protein LGN to the NuMA C-terminus results in preferential binding of NuMA to the microtubule plus-end. These results provide critical insights into the activation of NuMA and dynein for their functions in the spindle body and the cell cortex.

Structural dynamics of human ribosomes in situ reconstructed by exhaustive high-resolution template matching.

Protein synthesis is central to life and requires the ribosome, which catalyzes the stepwise addition of amino acids to a polypeptide chain by undergoing a sequence of structural transformations. Here, we employed high-resolution template matching (HRTM) on cryoelectron microscopy (cryo-EM) images of directly cryofixed living cells to obtain a set of ribosomal configurations covering the entire elongation cycle, with each configuration occurring at its native abundance. HRTM's position and orientation precision and ability to detect small targets (∼300kDa) made it possible to order these configurations along the reaction coordinate and to reconstruct molecular features of any configuration along the elongation cycle. Visualizing the cycle's structural dynamics by combining a sequence of >40 reconstructions into a 3D movie readily revealed component and ligand movements, some of them surprising, such as spring-like intramolecular motion, providing clues about the molecular mechanisms involved in some still mysterious steps during chain elongation.

Structural and Electrochemical Discussion on Direct Electron Transfer-Type Bioelectrocatalysis By Membrane-Bound Fructose Dehydrogenase from Gluconobacter Japonicus

Oxidoreductases have been utilized as bioelectrocatalysts to realize a variety of biotechnologies, such as biosensors, biofuel cells, solar fuel production, carbon dioxide capture and utilization, and cofactor-regeneration systems. These systems are based on bioelectrocatalysis which couples electrode and enzymatic reactions. Several metalloenzymes can communicate electronically with suitable electrodes without redox mediators. This phenomenon has been termed “direct electron transfer (DET)-type bioelectrocatalysis”. Owing to the mediator-less configuration, the reaction can offer the following benefits in future bioelectrochemical technologies: (i) minimized overvoltage, (ii) low cost, (iii) simple design, (iv) high degree of design freedom, and (v) nontoxic and environmentally friendly properties. However, there have been few reports of enzymes that can realize DET-type bioelectrocatalysis, thus the unique mechanism of the reaction has yet to be elucidated.We focused on a d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, which is known as a model enzyme for DET-type reactions. FDH is a unique enzyme with intense DET-type bioelectrocatalytic activity and has been extensively investigated from electrochemistry, protein engineering, and spectroscopy perspectives. FDH is a heterotrimeric membrane-bound protein with a molecular mass of ca. 138 kDa and is comprised of subunits I (67 kDa), II (51 kDa), and III (20 kDa). Subunit I contains a covalently bound flavin adenine dinucleotide (FAD), and subunit II carries three heme c moieties from its N-terminus called hemes 1c, 2c, and 3c. The redox potentials of hemes c in FDH and several variants were investigated using bioelectrochemical and spectroscopic methods. The DET pathway of FDH was examined with site-directed mutagenesis to replace the axial ligand of heme c or to delete the heme c moiety. These studies have shown that the electron is transferred from the reduced FAD through heme 3c to heme 2c and then to the electrode; heme 1c does not seem to be involved in the reaction. However, the entire three-dimensional (3D) structure of FDH and other DET-type membrane-bound quinohemoproteins, flavohemoproteins, and metallohemoproteins, remained unknown. Therefore, a quantitative discussion of their DET-type reaction was difficult.In the present study, we clarified the 3D structure of FDH using cryo-electron microscopy and single-particle image analysis with a resolution of 2.5 Å (PDB ID: 8JEJ). This is the first study to report the entire structures of membrane-bound flavohemoproteins, quinohemoproteins, and metallohemoproteins capable of DET-type reactions. The structure has revealed the 3Fe-4S iron-sulfur cluster (3Fe4S) in subunit I. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose through FAD, 3Fe4S, and hemes 3c, 2c, and 1c were examined based on Marcus’ theory. In addition, structural analysis has shown the localization of the electrostatic surface charges around heme 2c in subunit II, and experiments using functionalized electrodes with a controlled surface charge support the notion that heme 2c is the electrode-active site. Furthermore, two aromatic amino acid residues (Trp427 and Phe489) were located in a possible long-range ET pathway between heme 2c and the electrode. We constructed variants in which each of the corresponding residues was replaced with alanine (W427A and F489A) by site-directed mutagenesis, and their effects on DET-type activity were investigated by electrochemical measurements. Kinetic analysis of steady-state catalytic waves has revealed that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET between enzymes and an electrode.These groundbreaking findings provide vital information for searching for critical elements in DET-type reactions and a reasonable explanation for the outstanding DET-type activity of FDH. The appropriate mutation of aromatic residues to accelerate ET between an enzyme and an electrode will be a novel way to create new DET-type enzymes and innovative biomimetics. Figure 1

Semantic segmentation-based detection algorithm for challenging cryo-electron microscopy RNP samples.

In this study, we present a novel and robust methodology for the automatic detection of influenza A virus ribonucleoproteins (RNPs) in single-particle cryo-electron microscopy (cryo-EM) images. Utilizing a U-net architecture-a type of convolutional neural network renowned for its efficiency in biomedical image segmentation-our approach is based on a pretraining phase with a dataset annotated through visual inspection. This dataset facilitates the precise identification of filamentous RNPs, including the localization of the filaments and their terminal coordinates. A key feature of our method is the application of semantic segmentation techniques, enabling the automated categorization of micrograph pixels into distinct classifications of particle and background. This deep learning strategy allows to robustly detect these intricate particles, a crucial step in achieving high-resolution reconstructions in cryo-EM studies. To encourage collaborative advancements in the field, we have made our routines, the pretrained U-net model, and the training dataset publicly accessible. The reproducibility and accessibility of these resources aim to facilitate further research and validation in the realm of cryo-EM image analysis.

Size-Controllable and Monodispersed Lipid Nanoparticle Production with High mRNA Delivery Efficiency Using 3D-Printed Ring Micromixers.

Lipid nanoparticles (LNPs) are gaining recognition as potentially effective carriers for delivery of therapeutic agents, including nucleic acids (DNA and RNA), for the prevention and treatment of various diseases. Much effort has been devoted to the implementation of microfluidic techniques for the production of monodisperse and stable LNPs and the improvement of encapsulation efficiency. Here, we developed three-dimensional (3D)-printed ring micromixers for the production of size-controllable and monodispersed LNPs with a high mRNA delivery efficiency. The effects of flow rate and ring shape asymmetry on the mixing performance were initially examined. Furthermore, the physicochemical properties (such as hydrodynamic diameter, polydispersity, and encapsulation efficiency) of the generated LNPs were quantified as a function of these physical parameters via biochemical analysis and cryo-electron microscopy imaging. With a high production rate of 68 mL/min, our 3D-printed ring micromixers can be used to manufacture LNPs with diameters less than 90 nm, low polydispersity (<0.2), and high mRNA encapsulation efficiency (>91%). Despite the simplicity of the ring-shaped mixer structure, we can produce mRNA-loaded LNPs with exceptional quality and high throughput, outperforming costly commercial micromixers.

Mucin-Inspired Polymeric Fibers for Herpes Simplex Virus Type 1 Inhibition.

Mucus lines the epithelial cells at the biological interface and is the first line of defense against multiple viral infections. Mucins, the gel-forming components of mucus, are high molecular weight glycoproteins and crucial for preventing infections by binding pathogens. Consequently, mimicking mucins is a promising strategy for new synthetic virus inhibitors. In this work, synthetic mucin-inspired polymers (MIPs) as potential inhibitors of herpes simplex virus 1 (HSV-1) are investigated. By using a telechelic reversible addition-fragmentation chain-transfer (RAFT) polymerization technique, a new dendronized polysulfatep(G1AAm-OSO3)PDSwith an amide-backbone similar to the native mucin glycoproteins is synthesized.p(G1AAm-OSO3)PDSshows mucin-like elongated fiber structure, as revealed in cryo-electron microscopy (cryo-EM) imaging, and its HSV-1 inhibition activity together with its previously reported methacrylate analoguep(G1MA-OSO3)PDS is tested. Both of the sulfated MIPs show strong HSV-1 inhibition in plaque reduction assays with IC50 values in lower nanomolar range (<3× 10-9 m) and demonstrate a high cell compatibility (CC50>1.0mgmL-1) with lower anticoagulant activity than heparin. In addition, the prophylactic and therapeutic activity of both MIPs is assessed in pre- and post-infection inhibition assays and clearly visualize their high potential for application using fluorescent microscopy imaging of infected cells.

Accurate global and local 3D alignment of cryo-EM density maps using local spatial structural features.

Advances in cryo-electron microscopy (cryo-EM) imaging technologies have led to a rapidly increasing number of cryo-EM density maps. Alignment and comparison of density maps play a crucial role in interpreting structural information, such as conformational heterogeneity analysis using global alignment and atomic model assembly through local alignment. Here, we present a fast and accurate global and local cryo-EM density map alignment method called CryoAlign, that leverages local density feature descriptors to capture spatial structure similarities. CryoAlign is a feature-based cryo-EM map alignment tool, in which the employment of feature-based architecture enables the rapid establishment of point pair correspondences and robust estimation of alignment parameters. Extensive experimental evaluations demonstrate the superiority of CryoAlign over the existing methods in terms of both alignment accuracy and speed.

Square beams for optimal tiling in transmission electron microscopy

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem for dose-sensitive samples. Here, we introduce a square electron beam that can easily be retrofitted in existing microscopes, and demonstrate its application, showing that it can tile nearly perfectly and deliver cryo-electron microscopy imaging with a resolution comparable to conventional set-ups.

A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles

Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria.

The phytopathogen Paracidovorax citrulli possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if P. citrulli formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of npdA orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by P. citrulli AAC00-1 was confirmed by gene deletion mutagenesis (ΔnpdA), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of npdA were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in P. citrulli and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the P. citrulli S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.

Computational models of amorphous ice for accurate simulation of cryo-EM images of biological samples

Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.

Diffusion models in bioinformatics and computational biology.

Denoising diffusion models embody a type of generative artificial intelligence that can be applied in computer vision, natural language processing and bioinformatics. In this Review, we introduce the key concepts and theoretical foundations of three diffusion modelling frameworks (denoising diffusion probabilistic models, noise-conditioned scoring networks and score stochastic differential equations). We then explore their applications in bioinformatics and computational biology, including protein design and generation, drug and small-molecule design, protein-ligand interaction modelling, cryo-electron microscopy image data analysis and single-cell data analysis. Finally, we highlight open-source diffusion model tools and consider the future applications of diffusion models in bioinformatics.

Scipion-EM-ProDy: A Graphical Interface for the ProDy Python Package within the Scipion Workflow Engine Enabling Integration of Databases, Simulations and Cryo-Electron Microscopy Image Processing.

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.

The reduction of FIB damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis

Focused ion beam (FIB) is widely used for thinning frozen cells to produce lamellae for cryo-electron microscopy imaging and for protein structures study invivo. However, FIB damages the lamellae and a quantitative experimental analysis of the damage is lacking. We used a 30-keV gallium FIB to prepare lamellae of a highly concentrated icosahedral virus sample. The viruses were grouped according to their distance from the surface of lamellae and reconstructed. Damage to the approximately 20-nm-thick outermost lamella surface was similar to that from exposure to 16 e-/Å2 in a 300-kV cryo-electron microscope at high-resolution range. The damage was negligible at a depth beyond 50nm, which was reduced to 30nm if 8-keV Ga+ was used during polishing. We designed extra steps in the reconstruction refinement to maximize undamaged signals and increase the resolution. The results demonstrated that low-energy beam polishing was essential for high-quality thinner lamellae.

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin.

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

Many Roles of Carbohydrates: A Computational Spotlight on the Coronavirus S Protein Binding.

Glycosylation is one of the post-translational modifications with more than 50% of human proteins being glycosylated. The exact nature and chemical composition of glycans are inaccessible to X-ray or cryo-electron microscopy imaging techniques. Therefore, computational modeling studies and molecular dynamics must be used as a "computational microscope". The spike (S) protein of SARS-CoV-2 is heavily glycosylated, and a few glycans play a more functional role "beyond shielding". In this mini-review, we discuss computational investigations of the roles of specific S-protein and ACE2 glycans in the overall ACE2-S protein binding. We highlight different functions of specific glycans demonstrated in myriad computational models and simulations in the context of the SARS-CoV-2 virus binding to the receptor. We also discuss interactions between glycocalyx and the S protein, which may be utilized to design prophylactic polysaccharide-based therapeutics targeting the S protein. In addition, we underline the recent emergence of coronavirus variants and their impact on the S protein and its glycans.

Determining protein structures in cellular lamella at pseudo-atomic resolution by GisSPA

Cryo-electron tomography is a major tool used to study the structure of protein complexes in situ. However, the throughput of tilt-series image data collection is still quite low. Here, we show that GisSPA, a GPU accelerated program, can translationally and rotationally localize the target protein complex in cellular lamellae, as prepared with a focused ion beam, using single cryo-electron microscopy images without tilt-series, and reconstruct the protein complex at near-atomic resolution. GisSPA allows high-throughput data collection without the acquisition of tilt-series images and reconstruction of the tomogram, which is essential for high-resolution reconstruction of asymmetric or low-symmetry protein complexes. We demonstrate the power of GisSPA with 3.4-Å and 3.9-Å resolutions of resolving phycobilisome and tetrameric photosystem II complex structures in cellular lamellae, respectively. In this work, we present GisSPA as a practical tool that facilitates high-resolution in situ protein structure determination.

Silk Assembly against Hydrophobic Surfaces─Modeling and Imaging of Formation of Nanofibrils.

A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, β-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of ∼7 nm and a width of ∼11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.

Structural basis for ligand modulation and partial agonism of the 5-HT3R.

Type-3 serotonin receptors (5-HT3Rs) are pentameric ligand gated ion channels that mediate fast synaptic signaling in response to binding of the neurotransmitter serotonin. 5-HT3Rs play a major role in regulating gut motility, secretion, and visceral perception. Hyperactivity of 5-HT3Rs underlies pathologies such as chemotherapy-induced nausea and vomiting, irritable bowel syndrome, depression, anxiety, bipolar disorder, and excessive visceral pain, making it an important drug target. Previous research has resulted in the structures of the 5-HT3AR in complex with the agonist serotonin and setrons, 5-HT3R antagonists. Recently, 5-HT3AR partial agonists have been proposed to treat 5-HT3R pathologies with less severe side effects than full antagonists. However, the molecular mechanisms of partial agonism by orthosteric ligands of the 5-HT3R are still poorly understood. Here we present structures of the 5-HT3AR in complex with two novel orthosteric partial agonists generated from cryo-electron microscopy imaging. Structural stability of the ligand binding poses was assessed by molecular dynamic simulations, and ligand function was assessed by two-electrode voltage clamp in wild type and mutant receptors. Using our ligand-bound models of the 5-HT3AR, we propose structure-based mechanisms for the functional differences between orthosteric partial agonists, full agonist, and antagonists.

Capturing Lipid Nanodisc Shape and Properties Using a Continuum Elastic Theory.

Lipid nanodiscs are nanometric bilayer patches enveloped by confining structures, commonly composed of membrane scaffolding proteins (MSPs). To resolve the interplay between MSP geometry, lipid confinement, and membrane material properties on the nanodisc shape, we apply a continuum elastic theory accounting for lipid bending, tilting, and area deformations. The equilibrium nanodisc shape is then determined by minimizing the elastic free energy functional. Analytic expressions derived under simplifying assumptions demonstrate that the nanodisc shape is sensitive to its size, lipid density, and the lipid tilt and thickness imposed at the contact with the MSP. Under matching physical parameters, these expressions quantitatively reproduce the shape of nanodiscs seen in molecular dynamics simulations, but only if lipid tilt is explicitly considered. We further demonstrate how the bending rigidity can be extracted from the membrane shape profile by fitting the numerically minimized full elastic functional to the membrane shape found in simulations. This fitting procedure faithfully informs on the bending rigidity of nanodiscs larger than ca. 5 nm in radius. The fitted profiles accurately reproduce the increase in bending modulus found using real-space fluctuation analysis of simulated nanodiscs and, for large nanodiscs, also accurately resolve its spatial variations. Our study shows how deformations in lipid patches confined in nanodiscs can be well described by a continuum elastic theory and how this fit can be used to determine local material properties from shape analysis of nanodiscs in simulations. This methodology could potentially allow direct determination of lipid properties from experiments, for example cryo-electron microscopy images of lipid nanodiscs, thereby allowing to guide the development of future nanodisc formulations with desired properties.