Análisis estructural de los chrysovirus mediante criomicroscopía electrónica tridimensional: estuctura a resolución cuasi-atómica del virus de Penicillium chrysogenum

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 16-04-201

Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia Necatrix Quadrivirus 1

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization

DeepEMhacer: a deep learning solution for cryo-EM volume post-processing

Cryo-electron microscopy (cryo-EM) maps are among the most valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed before modeling in order to improve their interpretability. To that end, approaches based on B-factor correction are the most popular choices, yet they suffer from some limitations such as the fact that the correction is applied globally, ignoring the presence of heterogeneity in the map local quality that cryo-EM reconstructions tend to exhibit. With the aim of overcoming these limitations, here we present DeepEMhacer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental cryo-EM maps and maps sharpened by LocScape using their respective atomic models, DeepEMhacer has automatically learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhacer has been evaluated on a testing set of 20 different experimental maps, showing its ability to obtain much cleaner and detailed versions of the experimental maps, thus, improving their interpretability. Additionally, we have illustrated the benefits of DeepEMhacer with a use case in which the structure of the SARS-CoV 2 RNA polymerase is improved.This work is supported by the the Comunidad de Madrid through grant CAM (S2017/BMD-3817), the Spanish Ministry of Economy and Competitiveness (BIO2016-76400-R). J.V. acknowledges economical support from the Ramón y Cajal 2018 program (RYC2018-024087- I). R.S. is recipient of an FPU fellowship.Peer reviewe

Local computational methods to improve the interpretability and analysis of cryo-EM maps

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, ‘over-sharpening’ due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.N

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T=3 capsid protein leads to larger T=4 capsids

et al.Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution. © 2012, American Society for Microbiology.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2009-09331 to R.M., BFU2008-02328/BMC to J.L.C., BIO2008-02556 to N.V., AGL2010-22200-C02-02 to J.B., and BIO2008-02361 and BFU2011-25902 to J.R.C.), NADIR-UE-228394 (to J.B.), and the NIH Intramural Research Program with support from the Center for Information Technology (to B.L.T.).Peer Reviewe

Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins

International audienceOf five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast-and atadenoviruses, induced by the presence of an extended polypeptide. This polypeptide, and -helical clusters beneath the facet, likely correspond to genus-specific proteins LH2 and p32k. Another genus-specific protein, LH3, with a fold typical of bacteriophage tailspikes, contacts the capsid surface via a triskelion structure identical to that used by mastadenovirus protein IX, revealing a conserved capsid-binding motif and an ancient gene duplication event. Our data also suggest that mastadenovirus E1B-55 K was exapted from the atadenoviruslike LH3 protein. This work provides new information on the evolution of adenoviruses, emphasizing the importance of minor coat proteins for determining specific physicochemical properties of virions and most likely their tropism

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functionalities from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.Peer reviewe

Inflation and unemployment in Latvia

Bakalaura darbs ” Inflācija un bezdarbs Latvijā” tika izstrādāts 2011.gadā. Bakalaura darba mērķis ir uz Latvijas ekonomiska stāvokļa analīzes pamatā izvirzīt priekšlikumus inflācijas un bezdarba rādītāju samazināšanai. Izejot no bakalaura darba tēmas, bakalaura darba uzdevumi ir: 1) apskatīt teoriju par inflācijas un bezdarba būtību, tās rašanās iemesliem un sekām; 2) veikt Latvijas ekonomiskas stuācijas analīzi: -izanalizēt bezdarba un inflācijas rādītāju izmaiņas, -apskatīt kā mainījās inflācijas līmenis ES dalībvalstīs, kādi apstakļi to ietekmēja un salīdzīnāt ar Latvijas rādītājiem, -izvērtēt vai Latvijā pastāv sakarība starp inflāciju un bezdarbu; 3) Izvērtēt valdības 2007.gadā izstrādāto inflācijas apkarošanas plānu, apskatīt, kā ES dalībvalstīs cīnījas ar inflāciju un bezdarbu; 4) uz analīzes pamata izstrādāt secinājumus un priekšlikumus inflācijas un bezdarba problēmas risināšanai. Bakalaura darbā visbiežāk tiek izmantoti tādi vārdi, kā: inflācijas līmenis, bezdarbs, ES dalībvalstis, prognozes, apkarošanas plāns.The bachelor project “Inflation and Unemployment in Latvia” was done in 2011. The goals of the bachelor work are to analyze the Latvian economical situation and develop proposition for lower inflation and unemployment indicators. The main tasks in the bachelor work were: 1) analyze theory of inflation and unemployment, the reasons why it appears and how it affects. 2) analyze the Latvian economical situation: -analyze changes of inflation and unemployment; -review how inflation and unemployment level were changed in European countries, what affect it, and compare with Latvian indicators; -estimate whether the relationship between unemployment and inflation in Latvia; 3) asses the governments plan for 2007 for inflation and unemployment decrease, review how European countries struggled with inflation and unemployment; 4) based on the analyses done conclusion and develop proposition for inflation and unemployment in Latvia. In Bachelor project usually words used: inflation level, unemployment, European union countries, forecast, governments plan