Cryo-electron Microscopy Single-particle Reconstruction Research Articles

ASOCEM: Automatic Segmentation Of Contaminations in cryo-EM

Particle picking is currently a critical step in the cryo-electron microscopy single particle reconstruction pipeline. Contaminations in the acquired micrographs severely degrade the performance of particle pickers, resulting in many “non-particles” in the collected stack of particles. In this paper, we present ASOCEM (Automatic Segmentation Of Contaminations in cryo-EM), an automatic method to detect and segment contaminations, which requires as an input only the approximate particle size. In particular, it does not require any parameter tuning nor manual intervention. Our method is based on the observation that the statistical distribution of contaminated regions is different from that of the rest of the micrograph. This nonrestrictive assumption allows to automatically detect various types of contaminations, from the carbon edges of the supporting grid to high contrast blobs of different sizes. We demonstrate the efficiency of our algorithm using various experimental data sets containing various types of contaminations. ASOCEM is integrated as part of the KLT picker (Eldar et al., 2020) and is available at https://github.com/ShkolniskyLab/kltpicker2.

Structures of rhodopsin in complex with G-protein-coupled receptor kinase 1.

G-protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate activated GPCRs, thereby priming them for desensitization1. Although it is unclear how GRKs recognize these receptors2-4, a conserved region at the GRK N terminus is essential for this process5-8. Here we report a series of cryo-electron microscopy single-particle reconstructions of light-activated rhodopsin (Rho*) bound to rhodopsin kinase (GRK1), wherein the N terminus of GRK1 forms a helix that docks into the open cytoplasmic cleft of Rho*. The helix also packs against the GRK1 kinase domain and stabilizes it in an active configuration. The complex is further stabilized by electrostatic interactions between basic residues that are conserved in most GPCRs and acidic residues that are conserved in GRKs. We did not observe any density for the regulator of G-protein signalling homology domain of GRK1 or the C terminus of rhodopsin. Crosslinking with mass spectrometry analysis confirmed these results and revealed dynamic behaviour in receptor-bound GRK1 that would allow the phosphorylation of multiple sites in the receptor tail. We have identified GRK1 residues whose mutation augments kinase activity and crosslinking with Rho*, as well as residues that are involved in activation by acidic phospholipids. From these data, we present a general model for how a small family of protein kinases can recognize and be activated by hundreds of different GPCRs.

Mechanism and inhibition of Streptococcus pneumoniae IgA1 protease

Opportunistic pathogens such as Streptococcus pneumoniae secrete a giant metalloprotease virulence factor responsible for cleaving host IgA1, yet the molecular mechanism has remained unknown since their discovery nearly 30 years ago despite the potential for developing vaccines that target these enzymes to block infection. Here we show through a series of cryo-electron microscopy single particle reconstructions how the Streptococcus pneumoniae IgA1 protease facilitates IgA1 substrate recognition and how this can be inhibited. Specifically, the Streptococcus pneumoniae IgA1 protease subscribes to an active-site-gated mechanism where a domain undergoes a 10.0 Å movement to facilitate cleavage. Monoclonal antibody binding inhibits this conformational change, providing a direct means to block infection at the host interface. These structural studies explain decades of biological and biochemical studies and provides a general strategy to block Streptococcus pneumoniae IgA1 protease activity to potentially prevent infection.

Freeze-Drying To Produce Efficacious CPMV Virus-like Particles.

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and functionof the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.

Pushing the resolution limit by correcting the Ewald sphere effect in single-particle Cryo-EM reconstructions

The Ewald sphere effect is generally neglected when using the Central Projection Theorem for cryo electron microscopy single-particle reconstructions. This can reduce the resolution of a reconstruction. Here we estimate the attainable resolution and report a “block-based” reconstruction method for extending the resolution limit. We find the Ewald sphere effect limits the resolution of large objects, especially large viruses. After processing two real datasets of large viruses, we show that our procedure can extend the resolution for both datasets and can accommodate the flexibility associated with large protein complexes.

Component tree analysis of cystovirus φ6 nucleocapsid Cryo-EM single particle reconstructions.

The 3-dimensional structure of the nucleocapsid (NC) of bacteriophage φ6 is described utilizing component tree analysis, a topological and geometric image descriptor. The component trees are derived from density maps of cryo-electron microscopy single particle reconstructions. Analysis determines position and occupancy of structure elements responsible for RNA packaging and transcription. Occupancy of the hexameric nucleotide triphosphorylase (P4) and RNA polymerase (P2) are found to be essentially complete in the NC. The P8 protein lattice likely fixes P4 and P2 in place during maturation. We propose that the viral procapsid (PC) is a dynamic structural intermediate where the P4 and P2 can attach and detach until held in place in mature NCs. During packaging, the PC expands to accommodate the RNA, and P2 translates from its original site near the inner 3-fold axis (20 sites) to the inner 5-fold axis (12 sites) with excess P2 positioned inside the central region of the NC.

Molecular architecture of the 90S small subunit pre-ribosome.

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

Structural biology: Catalytic spliceosome captured.

Spliceosome complexes remove non-coding sequences from RNA transcripts in two steps. A structure of a spliceosome after the first step reveals active-site interactions and evolutionary constraints on these non-coding regions. See Article p.197 The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 A. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

The architecture of the spliceosomal U4/U6.U5 tri-snRNP.

U4/U6.U5 tri-snRNP is a 1.5 MDa pre-assembled spliceosomal complex comprising U5 snRNA, extensively base-paired U4/U6 snRNAs and >30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a pre-mRNA substrate bound to U1 and U2 snRNPs and transforms into a catalytically active spliceosome following extensive compositional and conformational changes triggered by unwinding of the U4/U6 snRNAs. CryoEM single-particle reconstruction of yeast tri-snRNP at 5.9Å resolution reveals the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3′-stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the N-terminal domain of Prp8 position U5 snRNA to insert its Loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome.

Localization of the regulatory particle subunit Sem1 in the 26S proteasome

The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

Quantum dot-induced viral capsid assembling in dissociation buffer

Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD = 2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.

Dynein: A Force (Generation Mechanism) to Be Reckoned with

In this issue of Structure, Roberts etal. discuss how cryo-electron microscopy single particle reconstructionsof the microtubule-based motor dynein reveal dramatic nucleotide-dependent conformational changes. They provide insight into dynein force generation and hint at shared mechanisms with other AAA+ unfoldases.

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 A by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via alphav integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.

The Structure of Phosphorylase Kinase Holoenzyme at 9.9 Å Resolution and Location of the Catalytic Subunit and the Substrate Glycogen Phosphorylase

SummaryPhosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 Å resolution structure of PhK heterotetramer (αβγδ)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic γ subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 Å resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 Å resolution was consistent with a proteolysed activated form of PhK that had lost the α subunits and possibly the γ subunits.

Cryo-EM Structure of the DNA-Dependent Protein Kinase Catalytic Subunit at Subnanometer Resolution Reveals α Helices and Insight into DNA Binding

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the nonhomologous end joining pathway for repair of double-stranded DNA (dsDNA) breaks. Here, we present a 7A resolution structure of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction. This structure is composed of density rods throughout the molecule that are indicative of alpha helices and reveals structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within the density. Surprisingly, models for the kinase domain can be docked into either the crown or base of the molecule at this resolution, although real space refinement suggests that the base location is the best fit. We propose a model for the interaction of DNA with DNA-PKcs in which one turn of dsDNA enters the central channel and interacts with a resolved alpha-helical protrusion.

Three-dimensional display and arbitrary region interactive segmentation of high-resolution virus capsids from cryo-electron microscopy single particle reconstruction

Recent advances in cryo-electron microscopy (cryo-EM) instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3-D) structure determination of virus. In order to visualize and effectively understand the 3-D structure, we present a display method based on surface rendering, which has the function of 3-D arbitrary region interactive segmentation and quantitative analysis, and integrate them into a software package called CEM-3DVDSS (cryo-EM 3-D virus display and arbitrary region segmentation system). CEM-3DVDSS consists of a complete set of modular programs for 3-D display and segmentation of icosahedral virus, which is organized under a graphical user interface and provides user-friendly options. First, we convert volume data in the MRC format obtained by cryo-EM single particle reconstruction to the format of our own software; in the preprocessing step, the original volume data are compressed and a better vector dimension is found for controlling the speed and detail of display. Then, the new volume data can be displayed and segmented using CEM-3DVDSS. We demonstrate the applicability of CEM-3DVDSS by displaying the 3-D structures of 2.5 nm (resolution) BmCPV (Bombyx mori cytoplasmic polyhedrosis virus), 2.5 nm CSBV (Chinese Sacbrood bee virus) and 1.4 nm C6/36DNV (Densonucleosis virus). As a result, both the 3-D display speed and signal-to-noise ratio of CEM-3DVDSS are improved compared with the original method, and the segmentation results become precise and more intact with additional function of quantitative analysis of 3-D structure.