Traditional molecular modeling is performed at atomic resolution, which relies on X-ray and NMR experiments to provide structural information. When dealing 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 as the EMAP module. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Other experiment such as transition metal ion FRET (tmFRET) is becoming a useful way to obtain protein structure information. A new focus of our research is to combine efficient simulation technique with structural information from experiment to assist high throughput protein structure determination. High resolution Structure determination from EM maps The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for -galactosidase bound to the inhibitor phenylethyl -D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at 1.5 resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design. Structure mechanism of Glutamate receptor activation Ionotropic glutamate receptors are cation channels that mediate signal transmission by depolarizing the postsynapitic membrane in response to L-glutamate release from the presynaptic neuron. Within the iGluR family of receptors are a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), kainite(KA), and N-methyl-D-aspartate (NMDA) subtypes, receptors that are all activated by glutamate and related in amino acid sequence, yet distinct in overall architecture, pharmacology, and biophysical characteristics. AMPA receptors are tetrameric complexes composed of subunits with a modular domain arrangement, beginning with the amino-terminal domain(ATD), the ligand- or agonist-binding domain (LBD), and the pore-forming transmembrane domain (TMD). Because AMPA receptors undergo rapid and nearly complete desensitization in the continued presence of agonist, it has proven difficult to elucidate high-resolution structures of agonist-bound, activated states and to define mechanism by which the chemical potential of agonist binding is transduced into the mechanical force of ion channel gating. The map-restrained self-guided Langevin dynamics (MapSGLD) simulation method we developed previously can utilize structural information embedded in a force field to flexibly fit macromolecular systems into low resolution maps to obtain energetically favored atomic structures that satisfy the maps. We perform flexible fitting with MapSGLD to obtain atomic structures of the glutamate receptor from EM maps. The open state atomic structure of the glutamate receptor shows the LBD in the clamshell closed conformation that agrees with the LBD x-ray structure. In addition to structural determination, MapSGLD provides dynamic information about the transition between different states. Comparing cryo-EM and X-ray water locations Correlation between water molecules identified in atomic models of -galactosidase determined by cryo-EM and X-ray crystallography. This work is a collaboration with a cryo-EM laboratory at the National Cancer institute, NIH. It is based on their recently published 2.2- resolution solution structure of -galactosidase, that contains resolved water densities. This study analyses how the water positions determined by cryo-EM compare to conserved water across all crystallography-determined structures. Interactions between water molecules and amino acids occur both at the surface of protein and within the structure, including areas such as subunit interfaces, catalytic sites, and other cavities. Identifying protein solvation profiles are critical to understand both protein structure and function, as well as for the design of high affinity lead compounds for drug discovery Kinesin walking mechanism from SGLD simulations. Kinesin belongs to a family of molecular motors characterized by unidirectional movement along microtubules from the center of a cell to its periphery. Numerous experimental and theoretical studies have been dedicated to kinesins since their discovery in 1985. Kinesin is a protein belonging to the class of Cytoskeletal motor proteins. Kinesin converts the energy of ATP hydrolysis into stepping movement along microtubules, which supports several vital cellular functions including mitosis, meiosis, and the transport of cellular cargo. Because kinesin is a fundamental protein, further research on the topic will provide important information as to how it functions. Combined with low resolution electron microscopic images, self-guided Langevin dynamics simulations are performed to study molecular motion and conformational change of kinesin motor domain in water and binding with microtubule. SGLD enable simulation to reach the time scale required for conformational change to understand the role of ATP binding and interaction with microtubules. Through flexible fitting of two newly release cryo-EM maps, we derived atomic structures of the kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states. To identify which head generating the cargo moving force, we designed atomic force simulations to examine the responses of the two heads to dragging forces. Our simulation results show the leading head can provide a necessary force to perform the power stroke while the trailing head cannot stand for even a 5pN dragging force. A structure comparison between two-head-bound and one-head bound states also supports the conclusion that the leading head is the source of the cargo moving force. Although the general features of the kinesin walking mechanism are becoming increasingly clear, some key questions remain unanswered, such as how they convert the chemical energy of ATP into mechanical energy and walk processively. In this study, through molecular simulations and free energy calculations, we found that in aqueous solution, kinesin favors an extended form with its microtubule-binding interface (MTBI) motif unfolded, as seen in a recent x-ray structure of kinesin-8. Through the flexible fitting of two newly released cryo-electron microscopy (cryo-EM) maps, we derived atomic structures of the kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states. Free energy calculations showed that kinesin bound to microtubules has a lower free energy than the extended form and that the free energy difference is in the range of the free energy released by ATP hydrolysis. The transition between the extended and compact forms, the structural differences of the leading and trailing heads, and atomic force simulations lead us to a completely new mechanism by which kinesin dimers walk on microtubules.
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