XTEND: Extending the depth of field in cryo soft X-ray tomography

We have developed a new data collection method and processing framework in full field cryo soft X-ray tomography to computationally extend the depth of field (DOF) of a Fresnel zone plate lens. Structural features of 3D-reconstructed eukaryotic cells that are affected by DOF artifacts in standard reconstruction are now recovered. This approach, based on focal series projections, is easily applicable with closed expressions to select specific data acquisition parameters.This work was partially supported by MINECO grants BFU2014-54181 to JLC and AIC-A-2011-0638, BIO2013-44647-R and BIO2016-76400-R to JMC, Madrid. Regional government grants S2013/MIT-2850 to JLC and S2010/BMD-2305 to JMC, National Science Foundation grant DMS-1114901 to GTH, the European Union through BioStruct-X Project 283570 and Horizon 2020 through grant iNEXT (INFRAIA-1-2014-2015, Proposal: 653706).S

Monitoring SARS-CoV-2 Surrogate TGEV Individual Virions Structure Survival under Harsh Physicochemical Environments

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides. Our data provide two-fold results on virus stability: First, while particles with larger size and lower packing fraction kept their morphology intact after successive mechanical aggressions, smaller viruses with higher packing fraction showed conspicuous evidence of structural damage and content release. Second, monitoring the structure of single TGEV particles in the presence of detergent and alcohol in real time revealed the stages of gradual degradation of the virus structure in situ. These data suggest that detergent is three orders of magnitude more efficient than alcohol in destabilizing TGEV virus particles, paving the way for optimizing hygienic protocols for viruses with similar structure, such as SARS-CoV-2.P. J. De Pablo acknowledges support by grants from the Spanish Ministry of Economy, Industry and Competitiveness projects (FIS2017- 89549-R and FIS2017–90701- REDT), the Human Frontiers Science Program (HFSPO RGP0012/2018) and Fondo SUPERA COVID19 Santander CRUE project TEXGRAF. J. Martín-Benito acknowledges support by grants from the Spanish Ministry of Science and Innovation projects (PID2020–117752RB-I00 and BFU2017–90018-R), CSIC project (PIE202020E079–6) and Fondo SUPERA COVID19 Santander CRUE project TEXGRAF. We acknowledge access to the cryoEM CNB-CSIC facility in the context of the CRIOMECORR project (ESFRI2019–01-CSIC16). The professional editing service NB Revisions was used for technical preparation of the text prior to submission

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.This study was funded by grants from Ministerio de Economía y Competitividad from Spain (AGL2010-21774, AGL2014-56193-R to E.M. and L.M.G., and BFU2013-43149-R to D.L.). Cryo-SXT experiments were funded by ALBA synchrotron from Barcelona, Spain (proposals 2016021614 and 2017022084) and performed at MISTRAL beamline at ALBA Synchrotron with the collaboration of ALBA staff. E.S. was awarded a research fellowship from Plan Estatal de Investigación Científica y Técnica y de Innovación.S

New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

<div><p>The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.</p></div

3DR of SA11 TLP from cryo-electron tomography.

<p>(A, C) Slice through the xy plane of the reconstructed cryo-electron tomograms of NTR- (A) and TR-TLP (C). The bar represents 100 nm. White arrowheads indicate examples of electrodense structures inside particles. Black arrowheads indicate examples of spikes on the outer particle surface of the virions. (B, D) Gallery of central slices through extracted NTR- (B) and TR-TLP (D). (E, G) Surface-rendered model of the averaged NTR- (E) and TR-TLP (G) calculated from the extracted subtomograms and viewed along an icosahedral 2-fold axis. The bar represents 100 Å. (F, H) Close up view of the NTR (C) and TR (F) spike represented as in E and G.</p

Model of the conformational states of the rotavirus spike in NTR- and TR-TLP.

<p>NTR spikes have a flexible VP4-C lectin domain at the base of the stalk (top, left). NTR Class 1 represents the average of more distal positions (black asterisks) and class 2 is generated by the average of more central positions (white asterisk) of the flexible lectin domain. The proteolytic processing of spike components releases the VP4-C lectin domain (if an additional cleavage at Lys²⁹ is produced) or increases its flexibility in TR class 1 spikes (bottom, left). The fraction of NTR (top, right) and TR (bottom, left) subvolumes in which no spike density is detected could correspond to a mixture of positions without spikes (or with damaged spikes) and with highly flexible spikes.</p

Tomogram averaging and classification of NTR and TR spikes.

<p>(A, B) Surface-rendered model of averaged tomograms, reference-free classified, for NTR (A) and TR (B) spikes. Top and middle rows show two side views of the averages related by a 90 degree rotation. The bottom row shows the top view of the averages. Arrowheads indicate an extra density at the base of the spike stalk in the class 2 NTR average, which is absent in NTR and TR class 1 averages. (C) Surface-rendered model of the class 1 TR spike fitted with the VP4 atomic model (PDB entry 3IYU). VP8* molecules A and B are in purple, VP5* molecules A and B are in red, and VP5* C is in orange. The last resolved residue of VP8* (Lys²⁹, purple) and the first resolved residue of VP5* (Glu²⁶⁴, orange) are indicated by spheres for the VP4-C molecule. (D) Surface-rendered model of the class 2 NTR spike fitted with the VP4 atomic model and represented as in C. (E) Close up view of the NTR spike fitted with a single VP8* lectin domain superimposed on the extra density detected at the base of the stalk. First (Leu⁶⁵, cyan) and last (Leu²²⁴, yellow) residues for the fitted domain are represented as spheres (arrowheads).</p

Infectivity assay of <i>in vitro</i> trypsin-treated TLP.

<p>(A, B) Coomassie blue-stained SDS-PAGE gels of purified SA11 (A) and OSU (B) TLP grown in the absence (NTR) or presence (TR) of trypsin. Samples were mock-incubated (-Trp) or incubated <i>in vitro</i> with 100 BAEE units/ml of trypsin (+Trp) (30 min, 37°C). The positions of the structural proteins (VP) are indicated. The unprocessed spike protein VP4 and its proteolytic products VP8* and VP5* are highlighted in grey. (C, D) Determination of specific infectivity of SA11 (C) and OSU (D) TLP by fluorescent focus assay in the absence of trypsin. (E, F) Determination of infectivity of SA11 (E) and OSU (F) TLP by plaque-forming assay in the presence of trypsin. Data are shown as mean ± SD. FFU, focus-forming units. PFU, plaque-forming units. * p<0.02, ** p<0.005.</p

Single particle three-dimensional structures of SA11 NTR- and TR-TLP.

<p>(A, D) Surface-shaded representation of the outer surfaces of NTR (A) and TR (D) particles, viewed along an icosahedral 2-fold axis. The surfaces are radially color-coded to represent VP4 or VP8*/VP5* spikes (red), VP7 (yellow), and VP6 (blue). The density is contoured at 1σ above the mean. The bar represents 100 Å. (B, E) Transverse sections, 2.8 Å thick, taken from the maps of NTR- (B) and TR-TLP (E), parallel but displaced 34 Å from the central section, viewed along a 2-fold axis (darker, denser). Arrows indicate spikes in the surface-shaded representations in A and D and their corresponding densities in B and E. For the NTR map, the relative density of the spike contained ∼50% of the shell density; for the TR map, relative density was ∼55%. (C, F) Close up view of the NTR (C) and TR (F) spike represented as in A and D.</p