We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

The database of molecular motions, MolMovDB (http://molmovdb.org), has been in existence for the past decade. It classifies macromolecular motions and provides tools to interpolate between two conformations (the Morph Server) and predict possible motions in a single structure. In 2005, we expanded the services offered on MolMovDB. In particular, we further developed the Morph Server to produce improved interpolations between two submitted structures. We added support for multiple chains to the original adiabatic mapping interpolation, allowing the analysis of subunit motions. We also added the option of using FRODA interpolation, which allows for more complex pathways, potentially overcoming steric barriers. We added an interface to a hinge prediction service, which acts on single structures and predicts likely residue points for flexibility. We developed tools to relate such points of flexibility in a structure to particular key residue positions, i.e. active sites or highly conserved positions. Lastly, we began relating our motion classification scheme to function using descriptions from the Gene Ontology Consortium.

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design. Parkinson's disease (PD) is a devastating neurological condition caused by the selective and progressive degeneration of dopaminergic neurons in the brain. Loss-of-function mutations in the PINK1 or PARKIN genes are the most common causes of recessively inherited PD. Together the encoded proteins coordinate a protective cellular quality control pathway that allows elimination of impaired mitochondria in order to prevent further cellular damage and ultimately death. Although it is known that the kinase PINK1 operates upstream and activates the E3 Ubiquitin ligase Parkin, the molecular mechanisms remain elusive. Here, we combined state-of-the art computational and functional biological methods to demonstrate that Parkin is sequentially activated through PINK1-dependent phosphorylation and subsequent structural rearrangement. The induced motions result in release of Parkin's closed, auto-inhibited conformation to liberate its enzymatic functions. We provide for the first time a complete protein structure of Parkin at an all atom resolution and a comprehensive molecular dynamics simulation of its activation and opening conformations. The generated models will allow uncovering the exact mechanisms of regulation and enzymatic activity of Parkin and potentially the development of novel therapeutics through a structure-function-based drug design.

Our understanding of RNA functions in the cell is evolving rapidly. As for proteins, the detailed three-dimensional (3D) structure of RNA is often key to understanding its function. Although crystallography and nuclear magnetic resonance (NMR) can determine the atomic coordinates of some RNA structures, many 3D structures present technical challenges that make these methods difficult to apply. The great flexibility of RNA, its charged backbone, dearth of specific surface features, and propensity for kinetic traps all conspire with its long folding time, to challenge in silico methods for physics-based folding. On the other hand, base-pairing interactions (either in runs to form helices or isolated tertiary contacts) and motifs are often available from relatively low-cost experiments or informatics analyses. We present RNABuilder, a novel code that uses internal coordinate mechanics to satisfy user-specified base pairing and steric forces under chemical constraints. The code recapitulates the topology and characteristic L-shape of tRNA and obtains an accurate noncrystallographic structure of theTetrahymenaribozyme P4/P6 domain. The algorithm scales nearly linearly with molecule size, opening the door to the modeling of significantly larger structures.