“Modeling of large macromolecular complexes.”
One of the major challenges in structural biology is to determine the structures of macromolecular complexes and to understand their function and mechanism of action. However, structural characterization of macromolecular assemblies is very difficult. A hybrid computational approach is required that will be able to incorporate spatial information from a variety of experimental methods into modeling procedure. Thus far, we developed PyRy3D, a method for building low-resolution models of large macromolecular complexes. The components (proteins, nucleic acids and any other type of physical objects including e.g. solid surfaces) can be represented as rigid bodies (e.g. based on atomic coordinates of structures determined experimentally or modeled computationally) or as flexible shapes (e.g. for parts, whose structure is dynamic or unknown). The model building procedure applies a Monte Carlo approach to sample the space of solutions. Spatial restraints are used to define components interacting with each other, and a a simple scoring function is applied to pack them tightly into contours of the entire complex (e.g. cryoEM density maps). This approach enables the construction of low-resolution models even for very large macromolecular complexes with components of unknown 3D structure, such as human mitochondrial RNA polymerase gamma.
PyRy3D can be integrated with user's code via Python scripts. The program is also supported by UCSF Chimera Extension which provides users with graphical interface for the program.
3D Modeling of Group I Intron structures by comparative modeling with ModeRNA and de
novo RNA folding with SimRNA
Deepak Kumar, Boniecki MJ, Janusz M. Bujnicki
Group I introns is a family of widespread non-coding RNA molecules well known for self-splicing from the host precursor RNA. Thus far only Azoarcus, Tetrahymena and Twort are known and well studied structures of this family. Commonly, group I introns are classified into 14 subfamilies  based on conserved core sequences and peripheral structures. However, introns from particular groups have high length diversity and weak sequence similarity, which makes structure prediction for these RNAs very difficult. To provide 3D structural models of representatives of group I introns from all families, we used a combination of comparative and de novo RNA structure modeling. We manually prepared alignment of 11 representatives (from subfamilies with unknown structures) with sequences and structures of representatives with known structures. ModeRNA  software was used to generate initial models of group I intron core structures by a comparative modeling approach. We defined the structural core based on the available secondary and tertiary structures, P4-P6 domain containing P4, P5 and P6 and P3-P9 domain containing P3, P7, P8 and P9. Azoarcus, Tetrahymena and Twort sharing significant similarity and common secondary structure with the representatives were chosen as templates for modeling. Fragments of models without counterparts in templates were then added and folded with a de novo modeling approach, as implemented in the SimRNA method (Boniecki, Bujnicki, and coworkers, manuscript in preparation). The generated models of group I intron structures accurately depict the global topology, secondary and tertiary interactions. Expectedly, the accuracy is highest in the core, with RMSD between 3-4 Å, whereas deviations are larger for peripheral regions that differ substantially between different introns. The results of this analysis provide a 3D perspective for studying group I introns and for interpretation of their sequence evolution in a structural context.
Modeling of human Splicing Factor 3b complex structure using PyRy3D software
Mateusz Dobrychłop, Joanna M. Kasprzak, Janusz M. Bujnicki
Determination of structures and mechanism of action of macromolecular complexes is one of major challenges in molecular biology. However, experimental structure determination for macromolecular assemblies is very difficult. For this reason a hybrid computational approach is used to incorporate spatial information from a variety of experimental methods into a modeling procedure. We developed PyRy3D (see http://www.genesilico.pl/pyry3d) - a computational tool that applies hybrid approach in order to build low-resolution models of large macromolecular complexes. The model building procedure applies a Monte Carlo approach to sample the space of solutions. Spatial restraints are used to define components interacting with each other, and a simple scoring function is applied to pack them tightly into contours of the entire complex (e.g. cryoEM density maps). Splicing Factor 3b (SF3b) is a protein complex responsible for recognition of the intron’s branch site in U2- and U12-dependent introns. Human SF3b complex consists of seven proteins (SF3b155, SF3b145, SF3b130, SF3b49, SF3b14a, SF3b14b and SF3b10). However, so far, only fragments of some SF3b components have been determined experimentally. For others computational models are available . Additionally electron density map for the whole complex was solved with 9.7 Å resolution, and the positions of RRM domains of SF3b14a and SF3b49 proteins were proposed . Despite intensive research on SF3b, its complete structure and mechanism of action remain unknown. We applied hybrid modeling approach implemented in PyRy3D software in order to build ensembles of structural models of the human SF3b complex, that fulfill currently available experimental and theoretical data. Our model recapitulated the predicted positions of RRM domains of SF3b14a and SF3b49 proposed by the Stark group and identified likely positions of other proteins that form the SF3b complex: SF3b155, SF3b145, SF3b130, SF3b14b and SF3b10.
MetalionProt: a tool for prediction odmetal ion binding sites in protein structures
Interactions of proteins with metal ions play vital roles in many biological processes. Cations stabilize protein secondary, tertiary, and quaternary structure. Moreover, some enzymes require metal ions as cofactors for catalysis. Carbonic anhydrase, EcoRV, and carboxypeptidase are all examples of enzymes that need cations in order to catalyse chemical reactions. MetalionProt is a novel bioinformatics tool for predicting Zn 2+, Mg 2+, Na+, K+, and Fe 2+, 3+, 4+, 6+ cations binding sites in 3D protein structures. It employs a grid-based algorithm and a knowledge- based potential derived from metal ion binding sites from experimentally solved PDB structures of protein-metal ion complexes. MetalionProt determines most likely metal ion binding sites around an user-specified protein. MetalionProt can assist X-ray crystallographic structure determination, e.g. by identifying tentative metal ion sites to be further validated during structure refinement. It can also be used in a fully predictive mode to propose metal positions for structural models that usually lack ion coordinates, such as protein structures determined by nuclear magnetic resonance (NMR), spectroscopy or theoretical models.
Pyry3D UCSF Chimera extension and its application in prediction of macromolecular complexes’ structure
Mateusz Dobrychłop, Janusz M. Bujnicki, Joanna M. Kasprzak
PyRy3D is a software tool that creates ranked 3D models of macromolecular complexes based on experimental restraints and the shape of the whole complex. The program performs Monte Carlo simulations in order to find the best arrangement of the components inside a density map. To let the user compose his/her own arrangements of components inside the defined complex shape in the easiest and most intuitive way, we have created a tool that associates PyRy3D with UCSF Chimera, a popular program for interactive visualization and analysis of molecular structures. PyRy3D Chimera extension is a plugin, that provides a user-friendly graphical interface, letting the user to generate a set of PyRy3D input files interactively, or to calculate a score for a set of different components' arrangements, based on default or user-defined parameters, directly from the extension’s interface. The poster shows the PyRy3D extension’s basic features, and their exemplary use in predicting human DNA polymerase gamma holoenzyme’s three dimensional structure.
Comparative screening and validation as a novel tool to identify STAT-specific inhibitors
Anna Czerwoniec in collaboration with Małgorzata Szeląg, Joanna Wesoly and Hans A.R. Bluyssen
Based on newly developed 3D structure models for all human (h)STATs, we propose a pipeline approach that combines comparative in silico docking of STAT-SH2 models with an in vitro STAT phosphorylation assay, as a novel tool to screen multi-million compound libraries and identify specific inhibitors for different STATs. Identification of specific and effective STAT inhibitory compounds could provide a tool to increase our understanding of their functional role in different diseases, and serve as therapeutic strategies in cancer, inflammation and auto-immunity.
Structural bioinformatics of pre-mRNA editing complex in Trypanosoma brucei
Anna Czerwoniec, Joanna Kasprzak and Janusz M. Bujnicki
Mitochondrial pre-mRNA in trypanosomas kinetoplastids undergoes editing process to become translatable molecule. Despite intensive research on editing complexes their complete structure and components interactions remain unknown. Here we present structural analysis of 20S and 35S pre-mRNA editing complex.
Obtained homology models for components of 20S and 35S complexes will be used to analyze potential macromolecular interactions between individual elements and within whole editing complexes. Software – PyRy3D – developed in our laboratory will be used for building and visualizing very low-resolution models of large macromolecular complexes, where structural components are docked into a density map of a whole complex. Components will be represented by experimental structures (e.g. X-ray or NMR models), structural models (e.g. homology models) or flexible shapes.
Obtained results provide us with information about macromolecular interactions in pre-mRNA editing complex.
“RNA is an active player in many cellular processes.”
The “RNAse-Base” gathers information about metabolic pathways involving RNA. It aims to bridge the gap between the Modomics database of RNA modifications, and REPAIRtoire, a database of DNA repair pathways.
Prediction of RNA-ion interactions
“Ions are essential for folding and function of RNA structures.”
We created a knowledge-based potential from analyzing known ion binding sites. The potential takes the distance and angle to a pair of RNA atoms into account. We are developing an application to predict ion positions in a RNA structure.
“We can combine a template and alignment to create RNA 3D models.”
ModeRNA is a program for comparative modeling of RNA 3D structures. It generates a 3D model of a target RNA from a pairwise sequence alignment and a structural template. ModeRNA can be used via a script-based interface allowing to analyze and manipulate RNA structures.