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Order Reovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
At last, Zhang et al. (2016) recorded the electron cryomicroscopy images of intact BTV virions with a direct electron detector operated at super-resolution counting mode and obtained a 3.5 Å-resolution structure of the virion by single-particle analysis. Figure 13.4 demonstrates the obtained structure. Thus, the BTV virion contained an outer layer of 60 VP2 trimers and 120 VP5 trimers; a middle layer with 260 VP7 trimers; and an inner layer formed by 120 VP3 monomers. Each VP2 trimer bound atop four VP7 trimers. Situated at a six-coordinated position of the icosahedral lattice, each VP5 trimer bridged the channel formed by six surrounding VP7 trimers (Zhang et al. 2016). Remarkably, the structural features of the middle and inner layers, including aa side chains, matched the atomic structures of core proteins VP3 and VP7 solved previously by x-ray crystallography (Grimes et al. 1998) and presented in Figure 13.3.
The proteasome as a target for protozoan parasites
Published in Expert Opinion on Therapeutic Targets, 2019
Stanley C. Xie, Lawrence R. Dick, Alexandra Gould, Stephen Brand, Leann Tilley
Efforts to achieve selective inhibition of enzyme targets are greatly facilitated by structure-guided design. High-resolution structures of the P. falciparum 20S have been determined using cryo-electron microscopy (cryo-EM) and single particle analysis. The structure of Pf20S with bound WLW-vs (PDB: 5 fmg) was solved at 3.6 Å [35]. The structure of Pf20S without bound ligand (PDB: 6muw; 3.6 Å) is also available [34]. The latter study reported the structure of Pf20S in complex with the activator, PfPA28. CryoEM offers the exciting possibility of imaging different conformational states of a protein complex, and it was revealed that PfPA28 binds the core particle asymmetrically – strongly engaging subunits only on one side of the core. The PfPA28 cap undergoes a rocking motion on the 20S barrel, opening and closing a gap at the PfPA28/Pf20S interface, potentially facilitating release of peptide products [34].
Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration
Published in Journal of Extracellular Vesicles, 2020
Joshua A. Welsh, Edwin van der Pol, Britta A. Bettin, David R. F. Carter, An Hendrix, Metka Lenassi, Marc-André Langlois, Alicia Llorente, Arthur S. van de Nes, Rienk Nieuwland, Vera Tang, Lili Wang, Kenneth W. Witwer, Jennifer C. Jones
Bulk analysis techniques, such as Western blots, ELISAs, mass spectrometry, and sequencing, and bead capture assays, have been widely used and instrumental in the field to date, associating EV phenotype (molecular cargo) with function. However, bulk analysis methods cannot convey if a particular analyte is in or on all EVs or just a subset, reveal the distribution of markers within a positive subset, or identify the size distribution or concentration of the positive subset. Bulk techniques therefore lack the ability to characterize the heterogeneity of the EV population, which could be seen as critical for some of their intended uses in clinical chemistry. For this reason, there is a strong impetus to develop single-particle analysis techniques, and an increasing number of platforms have become available. As seen in Table 1, only electron microscopy and super-resolution microscopy are capable of phenotyping single EVs of the smallest diameter. Though these methods are specialized and low-throughput (time-intensive) and can analyse only a small portion of the population, possibly neglecting low abundance particles such as large EVs. High-throughput methods are therefore desirable for single-particle phenotyping. NTA can technically be used for high-throughput fluorescence-based phenotyping, but low detection sensitivity and fluorophore bleaching have limited its application. Flow cytometry is another high-throughput possibility, but a lack of minimum procedural and reporting guidelines for single EV flow cytometry, combined with variable equipment sensitivities, settings and staining methodologies, has led to a general lack of reproducible data. This has only recently been address in the form of the MIFlowCyt-EV framework [15].
High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies
Published in mAbs, 2019
T. Noelle Lombana, Marissa L. Matsumoto, Jack Bevers III, Amy M. Berkley, Evangeline Toy, Ryan Cook, Yutian Gan, Changchun Du, Peter Liu, Paul Schnier, Wendy Sandoval, Zhengmao Ye, Jill M. Schartner, Jeong Kim, Christoph Spiess
To identify such antibodies, we aimed to target epitopes that overlap with previously identified cleavage sites.6,7 We immunized mice with MIC protein(s) and retrieved a panel of > 50 antibodies that bound MICA/B. However, due to the limitations of established epitope mapping technologies, it was not readily possible to identify the antibodies that bound near the proteolysis sites. Using the high throughput method of antibody competition, we observed a correlation between antibodies that bound a similar region and shedding inhibition, but the low resolution of this method was unable to reveal any spatial epitope information. Epitope mapping technologies that provide high-resolution at the sequence level include alanine scanning mutagenesis, peptide mapping, oxidative footprinting by fast photochemical oxidation of proteins (FPOP), X-ray crystallography, and cryo-electron microscopy (cryo-EM). All of these methods, however, are low throughput and also suffer from intrinsic limitations. Alanine scanning mutagenesis, where each antigen residue is mutated to alanine and tested for antibody binding, provides epitope mapping at the single residue level, yet not all antigens can tolerate mutagenesis to alanine. Peptide mapping uses overlapping peptides of the antigen sequence to detect antibody binding, but conformational epitopes cannot be detected by this method. FPOP identifies antigen epitopes that are protected from oxidation when bound to an antibody, yet only has sequence resolution at the proteolytic peptide level. Moreover, not all amino acids are equally susceptible to oxidation,8 further limiting its application. With significant advancements in the field, EM is becoming more feasible for epitope mapping. Negative stain 2-dimentional EM analysis can be used in a high throughput manner to grossly map a panel of antibodies;9 however, it generally requires large antigen-binding fragment (Fab) complexes (≥ 100–200 kDa). While X-ray crystallography and single-particle analysis by cryo-EM capture the highest resolution epitope information with a snapshot of intact antigen bound to antibody, these are the most labor-intensive methods and typically reserved for select antibodies.