ORCID Profile
0000-0003-0410-538X
Current Organisation
ALBA Synchrotron Light Facility
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Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH14455
Abstract: The use of X-ray crystallography for the structure determination of biological macromolecules has experienced a steady expansion over the last 20 years with the Protein Data Bank growing from deposited structures in 1992 to 000 in 2014. The large number of structures determined each year not only reflects the impact of X-ray crystallography on many disciplines in the biological and medical fields but also its accessibility to non-expert laboratories. Thus protein crystallography is now largely a mainstream research technique and is routinely integrated in high-throughput pipelines such as structural genomics projects and structure-based drug design. Yet, significant frontiers remain that continuously require methodological developments. In particular, membrane proteins, large assemblies, and proteins from scarce natural sources still represent challenging targets for which obtaining the large diffracting crystals required for classical crystallography is often difficult. These limitations have fostered the emergence of microcrystallography, novel approaches in structural biology that collectively aim at determining structures from the smallest crystals. Here, we review the state of the art of macromolecular microcrystallography and recent progress achieved in this field.
Publisher: Elsevier BV
Date: 07-2013
DOI: 10.1016/J.JMB.2013.03.034
Abstract: Increasing amounts of data show that conformational dynamics are essential for protein function. Unveiling the mechanisms by which this flexibility affects the activity of a given enzyme and how it is controlled by other effectors opens the door to the design of a new generation of highly specific drugs. Viral RNA-dependent RNA polymerases (RdRPs) are not an exception. These enzymes, essential for the multiplication of all RNA viruses, catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA-template-dependent fashion. Inhibition of RdRP activity will prevent genome replication and virus multiplication. Thus, RdRPs, like the reverse transcriptase of retroviruses, are validated targets for the development of antiviral therapeutics. X-ray crystallography of RdRPs trapped in multiple steps throughout the catalytic process, together with NMR data and molecular dynamics simulations, have shown that all polymerase regions contributing to conserved motifs required for substrate binding, catalysis and product release are highly flexible and some of them are predicted to display correlated motions. All these dynamic elements can be modulated by external effectors, which appear as useful tools for the development of effective allosteric inhibitors that block or disturb the flexibility of these enzymes, ultimately impeding their function. Among all movements observed, motif B, and the B-loop at its N-terminus in particular, appears as a new potential druggable site.
Publisher: Public Library of Science (PLoS)
Date: 05-01-2012
Publisher: Proceedings of the National Academy of Sciences
Date: 12-05-2014
Abstract: Viruses that are seemingly unrelated in genomic studies, and which infect hosts in different domains of life, show similarities in virion structure that indicate deep evolutionary relationships. We report the cryo-EM structure, at near-atomic resolution, of the fungal dsRNA Penicillium chrysogenum virus. Its capsid protein is a duplication of a single primordial α-helical domain. This domain has a fold that is conserved among dsRNA viruses it has increased its complexity through an early gene duplication event, followed by insertion of distinct segments in preferential “hotspots.” We show evidence that this preserved hallmark indicates an ancestral fold, and we suggest a relationship among current viral lineages.
Publisher: American Society for Microbiology
Date: 15-07-2010
DOI: 10.1128/JVI.00432-10
Abstract: Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 Å resolution. The capsid protein has a high content of rod-like densities characteristic of α-helices, forming a repeated α-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the α-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an un ided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.
Publisher: Springer Science and Business Media LLC
Date: 22-06-2017
DOI: 10.1038/S41598-017-04345-W
Abstract: Recent studies reveal that the mechanical properties of virus particles may have been shaped by evolution to facilitate virus survival. Manipulation of the mechanical behavior of virus capsids is leading to a better understanding of viral infection, and to the development of virus-based nanoparticles with improved mechanical properties for nanotechnological applications. In the minute virus of mice (MVM), deleterious mutations around capsid pores involved in infection-related translocation events invariably increased local mechanical stiffness and interfered with pore-associated dynamics. To provide atomic-resolution insights into biologically relevant changes in virus capsid mechanics, we have determined by X-ray crystallography the structural effects of deleterious, mechanically stiffening mutations around the capsid pores. Data show that the cavity-creating N170A mutation at the pore wall does not induce any dramatic structural change around the pores, but instead generates subtle rearrangements that propagate throughout the capsid, resulting in a more compact, less flexible structure. Analysis of the spacefilling L172W mutation revealed the same relationship between increased stiffness and compacted capsid structure. Implications for understanding connections between virus mechanics, structure, dynamics and infectivity, and for engineering modified virus-based nanoparticles, are discussed.
Publisher: MDPI AG
Date: 20-10-2021
DOI: 10.3390/V13112109
Abstract: Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo–soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological s les to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for s le manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo–soft X-ray tomography, its s le requirements, its advantages and its limitations. To highlight the potential of this technique, ex les of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.
Publisher: American Society for Microbiology
Date: 28-01-2021
DOI: 10.1128/JVI.02166-20
Abstract: The birnavirus moonlighting protein VP3 plays crucial roles in interacting with the dsRNA genome segments to form stable ribonucleoprotein complexes and controlling host cell immune responses, presumably by binding to and shielding the dsRNA from recognition by the host silencing machinery. The structural, biophysical, and functional data presented in this work identified the N-terminal domain of VP3 as being responsible for the dsRNA-binding and silencing suppression activities of the protein in drosophila X virus.
Publisher: American Society for Microbiology
Date: 15-07-2006
DOI: 10.1128/JVI.00368-06
Abstract: Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when in idually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 Å resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.
Publisher: International Union of Crystallography (IUCr)
Date: 29-09-2011
Publisher: Proceedings of the National Academy of Sciences
Date: 25-11-2013
Abstract: Human rhinoviruses (HRVs) cause the common cold and exacerbate chronic pulmonary diseases. Their single-stranded RNA genome is protected by an icosahedral capsid and must be released into the host cell cytosol for translation and replication. Using X-ray and cryo-EM analyses, we identified structural alterations that take place in the virus architecture during infection. In acidic endosomes in vivo and in our experimental conditions, the native virion is converted into the expanded, porous uncoating intermediate A-particle. This is accompanied by altered RNA–protein contacts at the inner capsid wall, leading to major changes in RNA conformation that result in a well-organized RNA layer. These rearrangements suggest that the RNA–protein interactions prepare RNA and facilitate its subsequent egress via a well-ordered mechanism.
Publisher: MyJove Corporation
Date: 21-07-2017
DOI: 10.3791/55793
Publisher: Microbiology Society
Date: 12-2004
Abstract: The features of Hepatitis C virus (HCV) quasispecies within an envelope segment including the hypervariable region 1 were analysed at an early time point post-infection in seven patients that acquired HCV from a single common donor during a nosocomial outbreak. The grouping of patients according to viral load was reflected in the structure of the quasispecies. A higher viral load correlated with the presence of a predominant HCV genome and a corresponding lower quasispecies complexity. The quasispecies complexity itself was not correlated with HCV clearance or persistence. Thus, the relationship between an intrapatient HCV quasispecies and the clinical outcome of an HCV infection is more complex than previously anticipated.
Publisher: International Union of Crystallography (IUCr)
Date: 30-03-2016
DOI: 10.1107/S2059798316002369
Abstract: While structure determination from micrometre-sized crystals used to represent a challenge, serial X-ray crystallography on microfocus beamlines at synchrotron and free-electron laser facilities greatly facilitates this process today for microcrystals and nanocrystals. In addition to typical microcrystals of purified recombinant protein, these advances have enabled the analysis of microcrystals produced inside living cells. Here, a pipeline where crystals are grown in insect cells, sorted by flow cytometry and directly analysed by X-ray diffraction is presented and applied to in vivo -grown crystals of the recombinant CPV1 polyhedrin. When compared with the analysis of purified crystals, in cellulo diffraction produces data of better quality and a gain of ∼0.35 Å in resolution for comparable beamtime usage. Importantly, crystals within cells are readily derivatized with gold and iodine compounds through the cellular membrane. Using the multiple isomorphous replacement method, a near-complete model was autobuilt from 2.7 Å resolution data. Thus, in favourable cases, an in cellulo pipeline can replace the complete workflow of structure determination without compromising the quality of the resulting model. In addition to its efficiency, this approach maintains the protein in a cellular context throughout the analysis, which reduces the risk of disrupting transient or labile interactions in protein–protein or protein–ligand complexes.
Publisher: Springer Netherlands
Date: 2013
DOI: 10.1007/978-94-007-6552-8_4
Abstract: For about 30 years X-ray crystallography has been by far the most powerful approach for determining virus structures at close to atomic resolutions. Information provided by these studies has deeply and extensively enriched and shaped our vision of the virus world. In turn, the ever increasing complexity and size of the virus structures being investigated have constituted a major driving force for methodological and conceptual developments in X-ray macromolecular crystallography. Landmarks of new virus structures determinations, such as the ones from the first animal viruses or from the first membrane-containing viruses, have often been associated to methodological breakthroughs in X-ray crystallography. In this chapter we present the common ground of proteins and virus crystallography with an emphasis in the peculiarities of virus studies. For ex le, the solution of the phase problem, a central issue in X-ray diffraction, has benefited enormously from the presence of non-crystallographic symmetry in virus crystals.
Publisher: Springer Science and Business Media LLC
Date: 20-10-2004
Publisher: Author(s)
Date: 2019
DOI: 10.1063/1.5084663
Publisher: Proceedings of the National Academy of Sciences
Date: 08-2018
Abstract: Most antibiotics do not interfere with viral infections. Rif icin is a notable exception, as it inhibits several poxviruses, including the causative agent of smallpox. However, the inhibition of viral assembly is unrelated to the antibacterial activity of rif icin against microbial RNA polymerases. Here, we reveal how the antibiotic prevents the recruitment of an essential scaffolding protein to nascent viral membranes. Based on these results, we provide a structural model of membrane assembly that is distinct from budding through cellular membranes and is most likely conserved in many large DNA viruses. Together, the mechanism of membrane assembly and structural models provide avenues to develop broad spectrum inhibitors against human and animal poxviruses.
Publisher: American Society for Microbiology
Date: 04-2015
DOI: 10.1128/JVI.02881-14
Abstract: The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel β-sheet that displays striking structural similarity to the β-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family.
Publisher: American Society for Microbiology
Date: 07-2007
DOI: 10.1128/JVI.00077-07
Abstract: Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an hipathic α-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal hipathic α-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the hipathic α-helix in the precursor capsid, as a five-α-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.
Location: No location found
No related grants have been discovered for Damià Garriga.