Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

Average nutrient concentration, and growth characteristics of bacteria during USCGC Healy cruises in 2002 and 2004

Standing stocks and production rates for phytoplankton and heterotrophic bacteria were examined during four expeditions in the western Arctic Ocean (Chukchi Sea and Canada Basin) in the spring and summer of 2002 and 2004. Rates of primary production (PP) and bacterial production (BP) were higher in the summer than in spring and in shelf waters than in the basin. Most surprisingly, PP was 3-fold higher in 2004 than in 2002; ice-corrected rates were 1581 and 458 mg C/m**2/d respectively, for the entire region. The difference between years was mainly due to low ice coverage in the summer of 2004. The spatial and temporal variation in PP led to comparable variation in BP. Although temperature explained as much variability in BP as did PP or phytoplankton biomass, there was no relationship between temperature and bacterial growth rates above about 0°C. The average ratio of BP to PP was 0.06 and 0.79 when ice-corrected PP rates were greater than and less than 100 mg C/m**2/d, respectively; the overall average was 0.34. Bacteria accounted for a highly variable fraction of total respiration, from 3% to over 60% with a mean of 25%. Likewise, the fraction of PP consumed by bacterial respiration, when calculated from growth efficiency (average of 6.9%) and BP estimates, varied greatly over time and space (7% to >500%). The apparent uncoupling between respiration and PP has several implications for carbon export and storage in the western Arctic Ocean.

Espacio modernista. Una mirada abarcadora

El presente trabajo se propone una nueva mirada sobre el extenso corpus modernista hispanoamericano, narrativo y lírico, para descubrir en él las modulaciones del espacio poético y describir, evitando una formulación dogmática, el trazo de un periplo que comienza en el espacio americano, se sumerge en espacios interiores y artificiales, se evade en lo exótico y lo antiguo para regresar nuevamente a América con una técnica transformada. Se subraya la semejanza de tal periplo con la traslación física y espiritual de los poetas modernistas más significativos.

Del espacio vivido al espacio del texto. Significación histórica y literaria del estar en el mundo

En la línea de los estudios sobre el espacio iniciados por Arturo Ardao, el ensayo ..."Del espacio vivido al espacio del texto" prolonga esa reflexión gnoseológica desde una perspectiva latinoamericana. Los conceptos de punto de vista, horizonte, perspectiva, puntos cardinales configuran el "espacio que se es" construido tanto con el espacio exterior como interior, subjetividad inserta en una temporalidad histórica del "espacio vivido" en la que Ardao fundaba la tesis central de su obra Espacio e inteligencia. En la construcción del espacio latinoamericano propuesta en el presente ensayo, la literatura ha desempeñado un papel significativo al propiciar un pasaje del "espacio vivido" al "espacio del texto". Un texto eminentemente literario que ha incorporado el espacio y el paisaje como tema y motivo y donde los escenarios representativos de la historia latinoamericana "rezuman temporalidad",

Espacialización del contenido político en La furia de los vencidos de Carlos Alberto Arroyo

Ya desde su primera novela publicada (Barbarie, 1927), el novelista mendocino Carlos Alberto Arroyo se inscribe en una línea de especial relevancia dentro de nuestras letras provinciales: la que responde a una intencionalidad política, nutrida por las complejas alternativas de la historia lugareña, a tal punto que un vasto sector de su obra podría ser denominada "la saga de la Mendoza lencinista". Lo sucesos narrados en La furia de los vencidos giran alrededor de la Revolución del '30 y su repercusión en el ambiente político mendocino, en particular dentro del círculo de una tradicional familia mendocina, los Ansay, cuya casa es el escenario de gran parte de la acción. Sucesivas polaridades espaciales inscritas en los pasajes descriptivos tienden a resaltar el valor simbólico de la casa como ámbito de acción en La furia de los vencidos: la verticalidad que se establece a través del concepto de "clase" como estrato dirigente de la sociedad, respaldada en el ejercicio del poder político; la dialéctica entre el adentro y el afuera, vale decir, la intimidad familiar opuesta al espacio de actuación pública y la idea de lo cerrado, manifiesta en la construcción de un espacio doméstico, asociado a lo femenino -hortus conclusus descripto con una tópica consagrada por la tradición- son otras tantas equivalencias del actuar de los personajes, sometidos a una coyuntura de transformación social que influye de manera decisiva en su destino.

Desarrollo de herramientas y estrategias para el ordenamiento territorial

El presente no es un proyecto de investigación, sino de gestión y desarrollo de herramientas para la toma de decisiones. Su objetivo es colaborar dentro del marco de la "Red Nacional de Ordenamiento y Desarrollo Territorial" (Poder Ejecutivo Nacional, 2004) propuesta por el Gobierno Nacional, con los gobiernos provinciales y otros organismos en la planificación del uso de los recursos del territorio, procurando un manejo sostenible de los mismos, a fin de reducir en forma progresiva los desequilibrios espaciales, contribuyendo a elevar la calidad de vida de todos los habitantes del país. En las últimas décadas, ante la existencia de una creciente presión antrópica sobre el medio natural y, paralelamente, un mayor grado de conocimiento de las causas y efectos de los diferentes riesgos, éstos han comenzado a tener mayor influencia en la determinación de políticas y prioridades para inversiones o emprendimientos económicos en general y en la fijación de pautas de ocupación del territorio. La planificación aparece como una herramienta adecuada para orientar y organizar el desarrollo equitativo y sustentable de un territorio y la población que lo ocupa. El Ordenamiento Territorial (OT) significa disponer, con orden, la ocupación y usos del territorio según la mayor o menor aptitud de sus diferentes elementos constitutivos. Este proyecto se considera estratégico para el Programa Nacional Ecoregiones del INTA (PNECO), ya que encara los problemas territoriales desde un punto de vista global (aspectos económicos, sociales, productivos, culturales y ambientales), tradicionalmente tratados de forma sectorial, plantea directivas a mediano y largo plazo (escenarios) y guía la planificación regional y local. El enfoque central del OT es la visión participativa, acordando intereses contrapuestos (trade-offs1) y sinérgicos, transformando amenazas en oportunidades. Es este punto central el que permitirá vincular el PNECO con todos sus Proyecto Específicos (PE), considerando el marco de políticas públicas vinculadas al medio ambiente y la producción en cada ecoregión del país, asociado al Programa Nacional de Territorios del INTA