A high-level 3D visualization API for Java and ImageJ

BACKGROUND: Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set. RESULTS: Here we present a platform-independent framework based on Java and Java 3D for accelerated rendering of biological images. Our framework is seamlessly integrated into ImageJ, a free image processing package with a vast collection of community-developed biological image analysis tools. Our framework enriches the ImageJ software libraries with methods that greatly reduce the complexity of developing image analysis tools in an interactive 3D visualization environment. In particular, we provide high-level access to volume rendering, volume editing, surface extraction, and image annotation. The ability to rely on a library that removes the low-level details enables concentrating software development efforts on the algorithm implementation parts. CONCLUSIONS: Our framework enables biomedical image software development to be built with 3D visualization capabilities with very little effort. We offer the source code and convenient binary packages along with extensive documentation at http://3dviewer.neurofly.de

EasyModeller: A graphical interface to MODELLER

<p>Abstract</p> <p>Background</p> <p>MODELLER is a program for automated protein Homology Modeling. It is one of the most widely used tool for homology or comparative modeling of protein three-dimensional structures, but most users find it a bit difficult to start with MODELLER as it is command line based and requires knowledge of basic Python scripting to use it efficiently.</p> <p>Findings</p> <p>The study was designed with an aim to develop of "EasyModeller" tool as a frontend graphical interface to MODELLER using Perl/Tk, which can be used as a standalone tool in windows platform with MODELLER and Python preinstalled. It helps inexperienced users to perform modeling, assessment, visualization, and optimization of protein models in a simple and straightforward way.</p> <p>Conclusion</p> <p>EasyModeller provides a graphical straight forward interface and functions as a stand-alone tool which can be used in a standard personal computer with Microsoft Windows as the operating system.</p

Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

Using Situs for the integration of multi-resolution structures

Situs is a modular and widely used software package for the integration of biophysical data across the spatial resolution scales. It has been developed over the last decade with a focus on bridging the resolution gap between atomic structures, coarse-grained models, and volumetric data from low-resolution biophysical origins, such as electron microscopy, tomography, or small-angle scattering. Structural models can be created and refined with various flexible and rigid body docking strategies. The software consists of multiple, stand-alone programs for the format conversion, analysis, visualization, manipulation, and assembly of 3D data sets. The programs have been ported to numerous platforms in both serial and shared memory parallel architectures and can be combined in various ways for specific modeling applications. The modular design facilitates the updating of individual programs and the development of novel application workflows. This review provides an overview of the Situs package as it exists today with an emphasis on functionality and workflows supported by version 2.5

Analysis of the functional repertoire of a mutant form of survivin, K129E, which has been linked to lung cancer

Background Survivin is a protein that is normally present only in G2 and M-phases in somatic cells, however, in cancer cells, it is expressed throughout the cell cycle. A prosurvival factor, survivin is both an inhibitor of apoptosis and an essential mitotic protein, thus it has attracted much attention as a target for new oncotherapies. Despite its prevalence in cancer, reports of survivin mutations have mostly been restricted to loci within its promoter, which increase the abundance of the protein. To date the only published mutation within the coding sequence is an adenine > guanine substitution in exon 4. This polymorphism, which was found in a cohort of Korean lung cancer patients, causes a lysine > glutamic acid mutation (K129E) in the protein. However, whether it plays a causative role in cancer has not been addressed. Methods Using site directed mutagenesis we recapitulate K129E expression in cultured human cells and assess its anti-apoptotic and mitotic activities. Results K129E retains its anti-apoptotic activity, but causes errors in mitosis and cytokinesis, which may be linked to its reduced affinity for borealin. Conclusion K129E expression can induce genomic instability by introducing mitotic aberrations, thus it may play a causative role in cancer

Protein-protein modelling using cryo-EM restraints

The recent improvements in cryo-electron microscopy (cryo-EM) in the past few years are now allowing to observe molecular complexes at atomic resolution. As a consequence, numerous structures derived from cryo-EM are now available in the Protein Data Bank. However, if for some complexes atomic resolution is reached, this is not true for all. This is also the case in cryo-electron tomography where the achievable resolution is still limited. Furthermore the resolution in a cryo-EM map is not a constant, with often outer regions being of lower resolution, possibly linked to conformational variability. Although those low to medium resolution EM maps (or regions thereof) cannot directly provide atomic structure of large molecular complexes, they provide valuable information to model the individual components and their assembly into them. Most approaches for this kind of modelling are performing rigid fitting of the individual components into the EM density map. While this would appear an obvious option, they ignore key aspects of molecular recognition, the energetics and flexibility of the interfaces. Moreover, these often restricts the modelling to a unique source of data, the EM density map. In this chapter, we describe a protocol where an EM map is used as restraint in HADDOCK to guide the modelling process.Comment: 28 pages including 7 figure