New theoretical techniques are being developed and characterized.[unreadable] These efforts are usually coupled with software development, and involve the systematic testing and evaluation of new ideas. This development is driven by current needs and interests.[unreadable] Specific ongoing projects include:[unreadable] - Perturbational effects of point mutations on functional dynamics[unreadable] - Predicting protein conformational changes by fitting experimentally derived constraints[unreadable] - Electron Microscopy and Tomography Image Processing[unreadable] - Development of electric density map docking utility (EMAP)[unreadable] - The core-weighted fitting method to construct molecular assemblies from[unreadable] EM maps [unreadable] - Macromolecular tomography using low resolution maps[unreadable] - Hybrid Isotropic Periodic Sum method for the calculation of long range interactions [unreadable] - Development of methods for examining reaction mechanism in complex systems [unreadable] - Unbiased forced sampling of complex conformational transitions and estimation of the potential of mean force along the reaction pathway [unreadable] - Development of the REPLICA/PATH method for determining reaction paths in complex systems using simulated annealing with Q-Chem[unreadable] - Enhancements of QM/MM potentials (using Gaussian delocalize MM charges, double link atom method) [unreadable] - Q-Chem and CHARMM integration for QM/MM applications[unreadable] [unreadable] Perturbational effects of point mutations on functional dynamics: Dr. Zheng models point mutations in elastic network models by perturbations in the force constant of those springs connecting the mutated residue with its neighbors. The goal is to identify the dynamically important residues whose mutations may significantly affect the functional dynamics as described by the relevant normal modes. In a earlier study, this idea was tested in several polymerases. A network of dynamically important residues which are critical to the open/closed transition of polymerases, were found to be evolutionarily conserved, which validates their functional importance. In a follow-up study, It has been established that a key link between the robustness of the normal modes that describe functional motions of protein complexes at the mesoscopic level and the evolutionary sequence variations at the residue level. By analyzing three biological nanomachines (DNA polymerase, myosin motor, and the chaperonin GroEL) it is shown that the functionally relevant low-frequency modes are most robust to sequence variations.[unreadable] [unreadable] Predicting protein conformational changes by fitting experimentally derived constraints: Dr. Zheng has developed a method to predict the large-scale protein conformational changes by using a linear combination of the low-frequency normal modes derived from ENM to fit the pair-wise atomic distances from NMR or fast spectroscopy. This technique is particularly useful to probe the conformational changes toward transient states, which are elusive to standard X-ray crystallography. The earlier version of this method aimed to predict the directionality of a protein conformational change given the initial state crystal structure and several pair-wise distance constraints for the end state. The structural displacement is computed in response to a perturbation to the system energy that incorporates the given distance constraints as restraints. In a follow-up study. The method has been significantly improved to accurately model both the direction and amplitude of protein conformational changes. The new protocol iteratively minimizes the error of fitting up to 10 distance constraints using 10 lowest modes. It has achieved a near-optimal performance in almost all 16 test cases, and in many cases the final structural models have attained an accuracy of 1~2? of RMSD when applied to cases with known results. [unreadable] [unreadable] Structure determination through single-particle electron tomography [unreadable] Single-particle electron tomography can be used to obtain 3D volume data of molecular systems without averaging over many particles. Therefore, this technology has the potential to study multiple conformational states of single particles. High noise due to limited sampling and distorsion caused by missing widges are two difficulties for this technology. Normal fitting methods often fail when dealing with such a high noisy data. The core-weighted fitting method we developed and implemented into CHARMM can tolerate such high noise and produce reasonable fitting results. We are applying the core-weighted fitting method to derive complex structures from single-particle electron tomography data. PDH complexes based on icosahedral symmetry are among the largest cellular machines and are a good candidate for single-particle electron tomography study. With the tomography image, we successfully docked the E2CD core and E1 domains and obtained complexes that agree with the results from cro-EM studies. These results validate the experiment techniques as well as our fitting methods for tomography data.[unreadable] [unreadable] Protein-protein docking with Map Objects: Traditional molecular modeling is performed at atomic resolution, which relies on X-ray and NMR experiments to provide structural information. When deal with biomolecular assemblies of millions of atoms, atomic description of molecular objects becomes very computational inefficient. We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Particularly, we implemented the core-weighted correlation functions to effectively recognize correct fit of component maps in complex maps, and the grid-threading Monte Carlo search algorithm to efficiently construct complex structures from electron density maps. Dr. Gruschus is applying this method in his structure study of the perioxiredoxin complex. Peroxiredoxins (Prx) are on e of several classes of proteins that reduce perioxides, reactive oxygen species produced as a by-product of cellular metabolism. For modeling the complex, the ATP-bound human Sulfiredoxin (hSrx) was docked to hyperoxidized Prx II using EMAP. In the docked structures with the Prx cysteine-sulfinic residue closest to the hSrx reactive cysteine, Asn186 of Prx II is in contact with the hSrx-bound ATP beta and gamma phosphate groups.[unreadable] [unreadable] Mr. O'Brien's research has developed two theoretical/computational methods for ascertaining the effect of osmolytes and denaturants on protein tertiary stability and on the persistance length of structurally disordered peptides. In particular he has a developed a perturbation method which allows one to transform the protein's partition function at zero denaturant/osmolyte concentration to approximate the partition function at any concentration of denaturant/osmolyte - thereby getting the equilibrium properties of the protein at any denaturant type and concentration of interest. This method is efficient and saves a massive amount of computational time. Separate from this he has modified Flory's transfer matrix method to allow one to compute the effect of different denaturants/osmolytes on the persistence length of structurally disordered peptides.[unreadable] [unreadable] A hybrid method of the newly developed Isotropic Periodic Sum (IPS) method by Dr. Xiongwu Wu for Lennard-Jones interactions and Particle-mesh Ewald (PME) for electrostatic interactions is tested on interfacial systems, i.e., alkane-water, water-air, and lipid bilayers. This work has demonstrated the benefit of IPS/PME in accurately and efficiently obtaining long-range energies required in these simulations.