Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.11Ysciescopu

Citizen science’s transformative impact on science, citizen empowerment and socio-political processes

Citizen science (CS) can foster transformative impact for science, citizen empowerment and socio-political processes. To unleash this impact, a clearer understanding of its current status and challenges for its development is needed. Using quantitative indicators developed in a collaborative stakeholder process, our study provides a comprehensive overview of the current status of CS in Germany, Austria and Switzerland. Our online survey with 340 responses focused on CS impact through (1) scientific practices, (2) participant learning and empowerment, and (3) socio-political processes. With regard to scientific impact, we found that data quality control is an established component of CS practice, while publication of CS data and results has not yet been achieved by all project coordinators (55%). Key benefits for citizen scientists were the experience of collective impact (“making a difference together with others”) as well as gaining new knowledge. For the citizen scientists’ learning outcomes, different forms of social learning, such as systematic feedback or personal mentoring, were essential. While the majority of respondents attributed an important value to CS for decision-making, only few were confident that CS data were indeed utilized as evidence by decision-makers. Based on these results, we recommend (1) that project coordinators and researchers strengthen scientific impact by fostering data management and publications, (2) that project coordinators and citizen scientists enhance participant impact by promoting social learning opportunities and (3) that project initiators and CS networks foster socio-political impact through early engagement with decision-makers and alignment with ongoing policy processes. In this way, CS can evolve its transformative impact

Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency

Small Particles with Big Impact : Structural Studies of Viruses and Toxicological Studies of Nanodiamonds

Nanoparticles (NPs) can be found everywhere and their existence has both beneficial and harmful consequences for the environment and living beings. The investigations on which this thesis is based upon have contributed to an increased understanding of some of these particles and to the development of a method that could be used to study their structure. Three different NPs have been studied by different means. In the first study, I describe how single-particle cryo-electron microscopy was used to determine the atomic structure of an algal virus; Chaetoceros tenuissimus RNA virus type II. This virus is taxonomically classified in the order Picornavirales, which includes viruses that infect a wide range of organisms, including humans, plants and insects. By comparing the algal virus structure to structures of related viruses in the order, we could identify a number of traits that were likely acquired or lost among these viruses during the course of evolution. In the second study, rice dwarf virus was utilised as a test sample to develop a new structural biology method, single-particle coherent diffractive imaging (CDI). The method aims to study macromolecules in a single-particle fashion at room temperature with the help of an X-ray free-electron laser, thus enabling studies of fast dynamics without the need to crystallize or freeze the sample. The study was the first of several within a large international collaboration and the first single-particle CDI experiment reported using femtosecond hard X-ray pulses. Despite several advances by the team, many challenges remain for the method to reach its full potential. In the third study, I describe in vitro and in vivo toxicological studies of detonation nanodiamonds (DNDs). I could demonstrate that some DNDs are toxic and that the toxicity is dependent both on the core and surface of the particles. DNDs are suggested for numerous different biomedical applications that alternately utilise their toxic properties or require biocompatibility. The results presented show that these contrasting properties can be exhibited by similar DNDs and that thorough characterisation and close control of the manufacturing process is essential for biomedical applications. This thesis explores how studies of some of nature’s nanoparticles - viruses - can lead to biological insight, how virus NPs can play a role in developing new technologies that may enable an even deeper understanding and explores issues that need to be considered for NPs to reach their potential in biomedical applications

Small Particles with Big Impact : Structural Studies of Viruses and Toxicological Studies of Nanodiamonds

Nanoparticles (NPs) can be found everywhere and their existence has both beneficial and harmful consequences for the environment and living beings. The investigations on which this thesis is based upon have contributed to an increased understanding of some of these particles and to the development of a method that could be used to study their structure. Three different NPs have been studied by different means. In the first study, I describe how single-particle cryo-electron microscopy was used to determine the atomic structure of an algal virus; Chaetoceros tenuissimus RNA virus type II. This virus is taxonomically classified in the order Picornavirales, which includes viruses that infect a wide range of organisms, including humans, plants and insects. By comparing the algal virus structure to structures of related viruses in the order, we could identify a number of traits that were likely acquired or lost among these viruses during the course of evolution. In the second study, rice dwarf virus was utilised as a test sample to develop a new structural biology method, single-particle coherent diffractive imaging (CDI). The method aims to study macromolecules in a single-particle fashion at room temperature with the help of an X-ray free-electron laser, thus enabling studies of fast dynamics without the need to crystallize or freeze the sample. The study was the first of several within a large international collaboration and the first single-particle CDI experiment reported using femtosecond hard X-ray pulses. Despite several advances by the team, many challenges remain for the method to reach its full potential. In the third study, I describe in vitro and in vivo toxicological studies of detonation nanodiamonds (DNDs). I could demonstrate that some DNDs are toxic and that the toxicity is dependent both on the core and surface of the particles. DNDs are suggested for numerous different biomedical applications that alternately utilise their toxic properties or require biocompatibility. The results presented show that these contrasting properties can be exhibited by similar DNDs and that thorough characterisation and close control of the manufacturing process is essential for biomedical applications. This thesis explores how studies of some of nature’s nanoparticles - viruses - can lead to biological insight, how virus NPs can play a role in developing new technologies that may enable an even deeper understanding and explores issues that need to be considered for NPs to reach their potential in biomedical applications

Capsid structure of a marine algal virus of the order Picornavirales

The order Picornavirales includes viruses that infect different kinds of eukaryotes and that share similar properties. The capsid proteins (CPs) of viruses in the order that infect unicellular organisms, such as algae, presumably possess certain characteristics that have changed little over the course of evolution, and thus these viruses may resemble the Picornavirales ancestor in some respects. Herein, we present the capsid structure of Chaetoceros tenuissimus RNA virus type II (CtenRNAV-II) determined using cryo-electron microscopy at a resolution of 3.1 Å, the first alga virus belonging to the family Marnaviridae of the order Picornavirales. A structural comparison to related invertebrate and vertebrate viruses revealed a unique surface loop of the major CP VP1 that had not been observed previously, and further, revealed that another VP1 loop obscures the so-called canyon, which is a host-receptor binding site for many of the mammalian Picornavirales viruses. VP2 has an N-terminal tail, which has previously been reported as a primordial feature of Picornavirales viruses. The above-mentioned and other critical structural features provide new insights on three long-standing theories about Picornavirales: (i) the canyon hypothesis, (ii) the primordial VP2 domain swap, and (iii) the hypothesis that alga Picornavirales viruses could share characteristics with the Picornavirales ancestor. IMPORTANCE Identifying the acquired structural traits in virus capsids is important for elucidating what functions are essential among viruses that infect different hosts. The Picornavirales viruses infect a broad spectrum of hosts, ranging from unicellular algae to insects and mammals and include many human pathogens. Those viruses that infect unicellular protists, such as algae, are likely to have undergone fewer structural changes during the course of evolution compared to those viruses that infect multicellular eukaryotes and thus still share some characteristics with the Picornavirales ancestor. This article describes the first atomic capsid structure of an alga Marnavirus, CtenRNAV-II. A comparison to capsid structures of the related invertebrate and vertebrate viruses identified a number of structural traits that have been functionally acquired or lost during the course of evolution. These observations provide new insights on past theories on the viability and evolution of Picornavirales viruses

Structural Insights into Common and Host-Specific Receptor-Binding Mechanisms in Algal Picorna-like Viruses

Marnaviridae viruses are abundant algal viruses that regulate the dynamics of algal blooms in aquatic environments. They employ a narrow host range because they merely lyse their algal host species. This host-specific lysis is thought to correspond to the unique receptor-binding mechanism of the Marnaviridae viruses. Here, we present the atomic structures of the full and empty capsids of Chaetoceros socialis forma radians RNA virus 1 built-in 3.0 Å and 3.1 Å cryo-electron microscopy maps. The empty capsid structure and the structural variability provide insights into its assembly and uncoating intermediates. In conjunction with the previously reported atomic model of the Chaetoceros tenuissimus RNA virus type II capsid, we have identified the common and diverse structural features of the VP1 surface between the Marnaviridae viruses. We have also tested the potential usage of AlphaFold2 for structural prediction of the VP1s and a subsequent structural phylogeny for classifying Marnaviridae viruses by their hosts. These findings will be crucial for inferring the host-specific receptor-binding mechanism in Marnaviridae viruses

Structural Insights into Common and Host-Specific Receptor-Binding Mechanisms in Algal Picorna-like Viruses

Marnaviridae viruses are abundant algal viruses that regulate the dynamics of algal blooms in aquatic environments. They employ a narrow host range because they merely lyse their algal host species. This host-specific lysis is thought to correspond to the unique receptor-binding mechanism of the Marnaviridae viruses. Here, we present the atomic structures of the full and empty capsids of Chaetoceros socialis forma radians RNA virus 1 built-in 3.0 Å and 3.1 Å cryo-electron microscopy maps. The empty capsid structure and the structural variability provide insights into its assembly and uncoating intermediates. In conjunction with the previously reported atomic model of the Chaetoceros tenuissimus RNA virus type II capsid, we have identified the common and diverse structural features of the VP1 surface between the Marnaviridae viruses. We have also tested the potential usage of AlphaFold2 for structural prediction of the VP1s and a subsequent structural phylogeny for classifying Marnaviridae viruses by their hosts. These findings will be crucial for inferring the host-specific receptor-binding mechanism in Marnaviridae viruses

High-resolution comparative atomic structures of two Giardiavirus prototypes infecting G. duodenalis parasite.

The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis