Biblioteca Digital

A Doença de Chagas, causada pelo parasita Trypanosoma cruzi, atinge cerca de um quarto da população da América Latina. Os fármacos disponíveis para o tratamento desta doença são inapropriados, apresentam baixa eficácia e sérios efeitos colaterais que limitam o seu uso. Esse grave panorama torna urgente a descoberta de novos agentes quimioterápicos para o tratamento seguro e eficaz da doença. A via glicolítica é principal forma de obtenção de energia de tripanosomatídeos. Um alvo molecular atrativo desta via bioquímica que desempenha papel essencial no controle do fluxo glicolítico do Trypanosoma cruzi, a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH), foi selecionada neste trabalho de Tese para estudos em biologia estrutural e química medicinal visando à identificação e planejamento de novos inibidores enzimáticos. Neste contexto, triagens biológicas resultaram na identificação de compostos de origem natural e sintética com atividade inibitória in vitro frente à GAPDH de T. cruzi, ampliando a diversidade química de moduladores seletivos deste alvo. Estudos cinéticos e estruturais demonstraram o comportamento não cooperativo entre os sítios ativos da enzima GAPDH de T. cruzi em relação à interação com o cofator NAD+...

The ability to adopt complex three-dimensional (3D) structures that can rapidly interconvert between multiple functional states (folding and dynamics) is vital for the proper functioning of RNAs. Consequently, RNA structure and dynamics necessarily determine their biological function. In the post-genomic era, it is clear that RNAs comprise a larger proportion (>50%) of the transcribed genome compared to proteins (≤2%). Yet the determination of the 3D structures of RNAs lags considerably behind those of proteins and to date there are even fewer investigations of dynamics in RNAs compared to proteins. Site specific incorporation of various structural and dynamic probes into nucleic acids would likely transform RNA structural biology. Therefore, various methods for introducing probes for structural, functional, and biotechnological applications are critically assessed here. These probes include stable isotopes such as 2H, 13C, 15N, and 19F. Incorporation of these probes using improved RNA ligation strategies promises to change the landscape of structural biology of supramacromolecules probed by biophysical tools such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and Raman spectroscopy. Finally, some of the structural and dynamic problems that can be addressed using these technological advances are outlined.

P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a β-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.

The Protein Structure Initiative’s Structural Biology Knowledgebase (SBKB, URL: http://sbkb.org) is an open web resource designed to turn the products of the structural genomics and structural biology efforts into knowledge that can be used by the biological community to understand living systems and disease. Here we will present examples on how to use the SBKB to enable biological research. For example, a protein sequence or Protein Data Bank (PDB) structure ID search will provide a list of related protein structures in the PDB, associated biological descriptions (annotations), homology models, structural genomics protein target status, experimental protocols, and the ability to order available DNA clones from the PSI:Biology-Materials Repository. A text search will find publication and technology reports resulting from the PSI’s high-throughput research efforts. Web tools that aid in research, including a system that accepts protein structure requests from the community, will also be described. Created in collaboration with the Nature Publishing Group, the Structural Biology Knowledgebase monthly update also provides a research library, editorials about new research advances, news, and an events calendar to present a broader view of structural genomics and structural biology.

The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB (http://sbkb.org), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a Functional Sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.

Current developments in the computational structural biology framework OpenStructure are presented.

The integration of structural data on atomistic to cellular scales with genetic, taxonomic and functional information is discussed. The challenges to the PDB and EMDB archives and some pertinent developments at PDBe to address these are discussed.

An introductory overview to the special issue papers on diffraction structural biology in this issue of the journal.

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X-ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well-suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR...

Carbon nanotubes represent ideal probes for high-resolution structural and chemical imaging of biomolecules with atomic force microscopy. Recent advances in fabrication of carbon nanotube probes with sub-nanometer radii promise to yield unique insights into the structure, dynamics and function of biological macromolecules and complexes.; Chemistry and Chemical Biology

Understanding how proteins facilitate signaling and substrate transport across biological membranes is an important frontier of structural biology. Membrane proteins are the doors and windows of cells: many membrane proteins are gates of entry into or exit from cells or cellular compartments, and others allow cells to sense their environment. One important multifunctional family of membrane proteins is the transient receptor potential (TRP) family of ion channels. TRP channels have recently been the subject of multiple structural analyses, both low resolution electron microscopy studies (reviewed by Moiseenkova-Bell and Wensel in this issue [p. 239]) and the divide and conquer approach of determining high resolution crystal structures of channel fragments, reviewed here.; Molecular and Cellular Biology

Early stage experimental data in structural biology is generally unmaintained and inaccessible to the public. It is increasingly believed that this data, which forms the basis for each macromolecular structure discovered by this field, must be archived and, in due course, published. Furthermore, the widespread use of shared scientific facilities such as synchrotron beamlines complicates the issue of data storage, access and movement, as does the increase of remote users. This work describes a prototype system that adapts existing federated cyberinfrastructure technology and techniques to significantly improve the operational environment for users and administrators of synchrotron data collection facilities used in structural biology. This is achieved through software from the Virtual Data Toolkit and Globus, bringing together federated users and facilities from the Stanford Synchrotron Radiation Lightsource, the Advanced Photon Source, the Open Science Grid, the SBGrid Consortium and Harvard Medical School. The performance and experience with the prototype provide a model for data management at shared scientific facilities.

Research projects in structural biology increasingly rely on combinations of heterogeneous sources of information, e.g. evolutionary information from multiple sequence alignments, experimental evidence in the form of density maps and proximity constraints from proteomics experiments. The OpenStructure software framework, which allows the seamless integration of information of different origin, has previously been introduced. The software consists of C++ libraries which are fully accessible from the Python programming language. Additionally, the framework provides a sophisticated graphics module that interactively displays molecular structures and density maps in three dimensions. In this work, the latest developments in the OpenStructure framework are outlined. The extensive capabilities of the framework will be illustrated using short code examples that show how information from molecular-structure coordinates can be combined with sequence data and/or density maps. The framework has been released under the LGPL version 3 license and is available for download from http://www.openstructure.org.

This ethnography tracks visualization and pedagogy in the burgeoning field of structural biology. Structural biologists are a multidisciplinary group of researchers who produce models and animations of protein molecules using three-dimensional interactive computer graphics. As they ramp up the pace of structure determination, modeling a vast array of proteins, these researchers are shifting life science research agendas from decoding genetic sequence data to interpreting the multidimensional forms of molecular life. One major hurdle they face is training a new generation of scientists to work with multi-dimensional data forms. In this study I document the formation and propagation of tacit knowledge in structural biology laboratories, in classrooms, and at conferences. This research shows that structural biologists-in-training must cultivate a feel for proteins in order to visualize and interpret their activity in cells. I find that protein modeling relies heavily on a set of practices I call the body-work of modeling. These tacit skills include: a) forms of kinesthetic knowledge that structural biologists gain through building and manipulating molecular models, and by using their own bodies as mimetic models to help them figure out how proteins move and interact; and b) narrative strategies that assume a teleological relationship between form and function...

By mid-2007, the three-dimensional (3D) structures of some 45,000 proteins have been solved, over a period where the linear structures of millions of genes have been defined. Technical challenges associated with X-ray crystallography are being overcome and high-throughput methods both for crystallization of proteins and for solving their 3D structures are under development. The question arises as to how structural biology can be integrated with and adds value to functional genomics programs. Structural biology will assist in the definition of gene function through the identification of the likely function of the protein products of genes. The 3D information allows protein sequences predicted from DNA sequences to be classified into broad groups, according to the overall ‘fold’, or 3D shape, of the protein. Structural information can be used to predict the preferred substrate of a protein, and thereby greatly enhance the accurate annotation of the corresponding gene. Furthermore, it will enable the effects of amino acid substitutions in enzymes to be better understood with respect to enzyme function and could thereby provide insights into natural variation in genes. If the molecular basis of transcription factor– DNA interactions were defined through precise 3D knowledge of the protein–DNA binding site...

Visualization of protein structures using stereoscopic systems is frequently needed by structural biologists working to understand a protein's structure-function relationships. Often several scientists are working as a team and need simultaneous interaction with each other and the graphics representations. Most existing molecular visualization tools support single-user tasks, which are not suitable for a collaborative group. Expensive caves, domes or geowalls have been developed, but the availability and low cost of high-definition televisions (HDTVs) and game controllers in the commodity entertainment market provide an economically attractive option to achieve a collaborative environment. This paper describes a low-cost environment, using standard consumer game controllers and commercially available stereoscopic HDTV monitors with appropriate signal converters for structural biology collaborations employing existing binary distributions of commonly used software packages like Coot, PyMOL, Chimera, VMD, O, Olex2 and others.

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography...

A selection of World Wide Web sites relevant to reviews published in this issue of Current Opinion in Structural Biology.

A selection of World Wide Web sites relevant to reviews published in this issue of Current Opinion in Structural Biology.

Taufer, Michela; Today, petascale distributed memory systems perform large-scale simulations and generate massive amounts of data in a distributed fashion at unprecedented rates. This massive amount of data presents new challenges for the scientists analyzing the data. In order to classify and cluster this data, traditional analysis methods require the comparison of single records with each other in an iterative process and therefore involve moving data across nodes of the system. When both the data and the number of nodes increase, classification and clustering methods can put increasing pressure on the system's storage and bandwidth. Thus, the methods become inefficient and do not scale. New methodologies are needed to analyze data when it is distributed across nodes of large distributed memory systems. In general, when analyzing such scientific data, we focus on specific properties of the data records. For example, in structural biology datasets, properties include the molecular geometry or the location of a molecule in a docking pocket. Based on this observation, we propose a methodology that enables the scalable analysis for large datasets, composed of millions of individual data records, in a distributed manner on large distributed memory systems. The methodology comprises two general steps. The first step extracts concise properties or features of each data record in isolation and represents them as metadata in parallel. The second step performs the analysis (i.e....