- Email: [email protected]
RNA Conformational Ensembles: Narrowing the GAP between Experiments and Simulations with Metadynamics
522a
Wednesday, March 2, 2016
living organisms noninvasively at high resolution in both space and time. However, the imaging of biological specimens involves inevitable tradeoffs of spatial resolution, speed, non-invasiveness, and imaging depth. I will describe three methods that balance these tradeoffs in different ways: structured illumination microscopy at 50-80 nm resolution, which we apply to study endocytic and cytoskeletal dynamics at the plasma membrane; lattice light sheet microscopy, which we use to image the rapid three-dimensional dynamics of single molecules, cells and embryos at hundreds of image planes per second; and adaptive optics, which we use to recover optimal resolution of fine neural processes deep in the brains of zebrafish and mice.
cells. We developed a live cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous b-actin genes in mouse embryonic fibroblasts, we observe that short (~8 s) Pol II clusters correlate with basal mRNA output. With serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with the proportionate increase in mRNAs synthesized minutes later. An additional burst of mRNA synthesis can be induced, at will, with a drug that stalls then releases Pol II clustering. Our findings reveal that transient clustering of Pol II constitutes a pre-transcriptional regulatory event which dynamically controls gene expression output.
2571-Symp Localizing Molecules in Cellular CT Scans Carolyn Larabell1,2, Gerry McDermott1,2, Mark Le Gros1,2. 1 Anatomy, University of California, San Francisco, San Francisco, CA, USA, 2 Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Much like medical CT scans reveal anatomical structures in the body, soft x-ray tomography (SXT) visualizes and quantifies the organization of sub-cellular structures within a cell. In SXT, the specimen is illuminated with x-ray photons from within a region of the spectrum known as the ‘water window’ (284 543eV). ‘Water window’ x-ray photons are absorbed an order of magnitude more strongly by carbon- and nitrogen-containing organic material than by water. Consequently, variation in biomolecule composition and concentration gives rise to quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells imaged by SXT are, therefore, highly representative of the cell in its native, functional state. Attenuation of soft x-rays, as they pass through the specimen, adheres to the BeerLambert Law. Attenuation is, therefore, a function of chemical composition and concentration of organic material, yielding unique quantitative Linear Absorption Coefficient (LAC) measurements for cellular components. LAC values are enormously powerful in terms of quantifying alterations in cell structures during events such as cell differentiation, progression or etiology of disease states, genetic manipulation, and application of exogenous agents. To localize molecules without perturbing cell structures, we use correlated high numerical aperture cryogenic fluorescence tomography (CFT). This multi-modal approach - imaging the same cell using both CFT and SXT - allows localization of labeled molecules directly in the context of a high-resolution 3-D tomographic reconstruction of the cell. I will show examples of data collected using these imaging technologies developed at the National Center for X-ray Tomography, an NIGMS-NIH supported Biomedical Technology Research Resource.
2574-Symp Single-Molecule Imaging of RNA in Live Cells Maria Carmo-Fonseca. Instituto de Medicina Molecular, University of Lisbon, Lisbon, Portugal. Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Previous studies have imaged RNAs in living cells by genetically inserting the binding sites for bacteriophage coat proteins in the RNA of interest. However, multiple nascent RNAs were simultaneously detected at the site of transcription, necessitating a modelling approach to infer kinetic information. To circumvent these significant limitations and potential problems in data interpretation, we developed a strategy that permits direct tracking of single nascent pre-mRNA molecules in live cells. We are using this approach to study how kinetic mechanisms impact on RNA biogenesis.
2572-Symp Matching Scales and Capabilities with Integrated Fluorescence and Electron Microscopy Jacob P. Hoogenboom. Imaging Physics, Delft University of Technology, Delft, Netherlands. Superresolution techniques have pushed the resolution of fluorescence microscopy (FM) towards that of electron microscopy (EM). Meanwhile, developments in scanning EM are revolutionizing EM, moving lateral image dimensions to typical FM fields-of-view and extending imaging capability into the third dimension and the live-cell regime. By correlating data from both techniques, molecules can be localized within the context of cells and tissue and with reference to their live dynamics, but throughput and quantification are hindered by elaborate, expert procedures involving separate microscopes. I will show integration of highnumerical aperture FM in a SEM, such that the electron beam can be positioned anywhere within the fluorescence field of view. Using electron-beam excited cathodoluminescence from the transparent sample substrate, we achieve automated FM-EM image registration with an accuracy that can be pushed to 5nm, i.e. equaling bio-molecular length scales. Besides integrated correlation microscopy, I will show our progress towards novel applications bridging fluorescence and electron microscopy, such as fluorescence-guided live-cell EM, and electronbeam identification and localization of labels, molecules, and cells.
2575-Symp Micromechanical Study of Mammalian Metaphase Chromosomes and Nuclei John F. Marko. Northwestern University, Evanston, IL, USA. Eukaryote chromosomes are organized into complex folded structures with remarkably different organization at different stages of the cell cycle. During interphase, chromosomes are organized into functional compartments and domains composed of dense heterochromatic regions and less compacted, transcriptionally active chromatin. Then, following DNA replication, chromosomes are entirely refolded into remarkably uniform, cylindrical mitotic chromosomes, which become individualized and ultimately split into sister chromatids. We have investigated the organization of both metaphase chromosomes and interphase nuclei from mammalian cells using micromechanical experiments. Metaphase chromosomes behave remarkably like crosslinked networks of chromatin, showing a wide range of elastic extensibility (Young modulus ~ 400 Pa), and with their elasticity dependent on connectivity of chromatin. Experiments where DNA is cleaved show an immediate loss of mechanical stability arguing against an underlying connected protein ‘‘scaffold’’ as an organizing principle. siRNA experiments interfering with production of condensin subunits lead to a loss of mechanical stability of metaphase chromosomes, indicative of the role of condensin as a major ‘‘cross-linker’’ of chromatin in metaphase chromosomes, and antibody staining of condensin indicates its organization into clusters that become visible when metaphase chromosomes are extended. We have also recently developed similar techniques for studying the mechanics of isolated human cell nuclei; DNA cleavage experiments indicate that chromatin itself contributes a large portion of the elastic modulus of the nucleus for small deformations, with the elasticity of the nuclear lamina becoming important at large extensions. Experiments increasing or decreasing the amount of heterochromatin or lamin A (a major intermediate component of the nuclear lamina) validate our conclusions concerning the differential importance of chromatin and the nuclear lamina to small and large nuclear deformations.
Symposium: Crowding and Order in the Genome
Platform: Molecular Dynamics II
2573-Symp Predictability and Control of Gene Bursting in Live Mammalian Cells Ibrahim Cisse´. Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. Transcriptional bursting is a hallmark for cellular variability across species. Whether predictability and output control exist within the stochastic bursting process is unknown. Here, we advance that collective behaviors from transient bio-molecular interactions help confer gene expression control in mammalian
2576-Plat RNA Conformational Ensembles: Narrowing the GAP between Experiments and Simulations with Metadynamics Alejandro Gil-Ley, Sandro Bottaro, Giovanni Bussi. Physics, SISSA, Trieste, Italy. The computational study of conformational transitions in nucleic acids still faces many challenges. For example, in the case of single stranded RNA tetranucleotides, agreement between simulations and experiments is not satisfactory due to inaccuracies in the force fields commonly used in molecular dynamics.
Wednesday, March 2, 2016 Improvement of force fields is however hindered by the difficulties of decoupling those errors from the statistical errors caused by insufficient sampling. We here tackle both problems simultaneously by introducing a novel enhancing sampling method and using experimental data to improve RNA force fields. In this novel method, concurrent well-tempered metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strength of the bias potential exploiting the tunability of well-tempered metadynamics so as to scale barriers on individual collective variables [1]. At the same time, the metadynamics algorithm is modified so as to allow enforcing a target distribution of backbone and sugar-base torsion angles taken from experimental structures, using a procedure related to two recently introduced techniques. Replica-exchange simulations of several RNA tetranucleotides with experimental corrections show significantly better agreement with NMR experimental data and suggest a systematic procedure for force field refinement. [1] Gil-Ley, A.; Bussi, G. J. Chem. Theory Comput. 2015, 11, 1077-1085. 2577-Plat Application of the String and 2D Hamiltonian Replica Exchange Umbrella Sampling Methods for the Study of Conformational Changes in the Bacterial Aspartate Transporter Glt(Ph) Hristina R. Zhekova1, Bogdan Lev2, Sergei Noskov1. 1 Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Department of Physics, RMIT University, Melbourne, Australia. Glt(Ph) is a homotrimeric protein involved in the sodium coupled transport of aspartate through the cellular membrane of Pyrococcus horikoshii. It belongs to the solute carrier 1 (SLC1) family, which comprises of a variety of proteins responsible for the trans-membrane transport of acidic or neutral amino acids in prokaryotes and eukaryotes. An important member of this family is the human glutamate transporter, which regulates the concentration of the neurotransmitter glutamate in the central nervous system. Unfortunately, its crystal structure is currently unknown. As Glt(Ph) has well resolved crystal structures in different functionally significant conformations, it has been studied extensively as a structural and functional analogue of the human glutamate transporter. Previous experimental and molecular dynamics studies have shown that substrate binding and the presence or absence of a lipid bilayer induce considerable conformational changes in Glt(Ph) which have been linked to the aspartate movement through the membrane. In this work two different sampling methods - 2D Hamiltonian Replica Exchange Umbrella Sampling (2DHREUS) and the String method - are used for the evaluation of potentials of mean force of a gate movement in Glt(Ph) associated with substrate binding to the active site. The gate opening has a low energy barrier and can be observed in the apo state even in the absence of a substrate. The String method compares well to the results of our benchmark 2DHREUS calculations and captures the most important structural and thermodynamical aspects of the conformational changes. 2578-Plat Quantifying Macromolecular Transition Paths with Path Similarity Analysis Sean L. Seyler, Avishek Kumar, Taylor Colburn, Michael F. Thorpe, Oliver Beckstein. Physics, Arizona State University, Tempe, AZ, USA. Diverse classes of proteins function through large-scale conformational changes; sophisticated enhanced sampling methods have been proposed to generate these macromolecular transition paths. As such paths are curves in a high-dimensional space, they have been difficult to compare quantitatively, a prerequisite to, for instance, assess the quality of different sampling algorithms. The Path Similarity Analysis (PSA) approach [1] alleviates these difficulties without relying on low-dimensional projections by utilizing the full information in 3N-dimensional trajectories in configuration space. PSA employs the Hausdorff or Fre´chet path (distance) metrics—adopted from computational geometry—enabling us to quantify path (dis)similarity, while the new concept of a Hausdorff-pair map permits the extraction of atomic-scale determinants responsible for path differences. Combined with clustering techniques, PSA facilitates the comparison of many paths, including collections of transition ensembles. We use the closed-to-open transition of the enzyme adenylate kinase (AdK)—a commonly used testbed for the assessment enhanced sampling algorithms [2]—to examine multiple microsecond equilibrium molecular dynamics (MD) transitions of AdK in its substrate-free form alongside transition ensembles from the MD-based dynamic importance sampling (DIMS-MD) and targeted MD (TMD) methods, and a geometrical targeting algorithm (FRODA). A Hausdorff pairs analysis of these ensembles revealed, for instance, that differences in DIMS-MD and FRODA paths were mediated by a set of conserved salt bridges whose charge-charge interactions are fully
523a
modeled in DIMS-MD but not in FRODA. We illustrate how existing trajectory analysis methods relying on pre-defined collective variables (such as native contacts or geometric quantities) can be used synergistically with PSA, as well as how PSA can be applied to more complex systems such as membrane transporter proteins. [1] Seyler, Kumar, Thorpe, Beckstein. PLoS Comput Biol (2015), http://dx.doi. org/10.1371/journal.pcbi.1004568; [2] Seyler and Beckstein. Mol Simul 40:855-877 (2014), http://dx.doi.org/10.1080/08927022.2014.919497. 2579-Plat Investigating Kinetics of Conformational Change using Molecular Dynamics and Milestoning Hiroshi Fujisaki1, Ayori Mitsutake2. 1 Nippon Medical School, Musashino, Tokyo, Japan, 2Keio University, Yokohama, Japan. To understand the functional roles of biomolecules, it is necessary to study both stable and metastable states in conformational space and how they are connected. Because of the advance of hardware and software, molecular dynamics (MD) simulation methods have become a feasible tool for this purpose, resulting in rather quantitative conclusions, which can be compared with experiment. MD simulation methods, however, still have a bottleneck due to ‘‘rare events’’ connecting stable states, and such a rare event can occur with millisecond timescales in a large biomolecule, which hampers a direct application of MD simulations to the rare event of biomolecules. This is why we need some novel ideas and algorithms to overcome this problem of rare events, and many researchers have been developing promising methods, mainly focusing on conformational sampling methods. These include very powerful replica exchange and multicanonical methods, but time information is lost in these methods and dynamic characterization of a rare event is difficult. In this presentation, we apply the milestoning method devised by Elber, Vanden-Eijnden and others to conformational change of a small peptide, chignolin, at the folding temperature (~420K). The milestoning method is regarded as an alternative of Markov state modeling, and by defining ‘‘milestones (collections of small dividing surfaces)’’ in some order parameter space and counting the crossing of milestones by a trajectory, mean first passage times (MFPTs) between two milestones can be obtained and a kinetic characterization of conformational change can be achieved. Here we will examine how the choice of order parameter space affects the results of MFPTs, and also analyze the effects of non-Markovianity for this process using Zuckerman’s method. 2580-Plat Improved Kinetics of Molecular Simulations using Biased Markov State Models Joseph F. Rudzinski, Kurt Kremer, Tristan Bereau. Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. Molecular simulations can provide microscopic insight into the physical and chemical driving forces of complex molecular processes. Despite tremendous advances in the accuracy of models as well as in the sophistication of simulation methodology, force-field errors can always potentially cause inconsistencies between simulated and experimentally-measured observables. The present work presents a robust and systematic framework to reweight the ensemble of dynamical paths sampled in a molecular simulation in order to be consistent with a set of kinetic observables. The method employs the well-developed Markov state modeling framework in order to efficiently treat simulated dynamical paths. By biasing the Markov state model to reproduce a small number of kinetic constraints, we not only improve the kinetic description of the system, but also the equilibrium distributions. The method is illustrated on two distinct coarse-grained peptide models, for which we observe clear improvements of the equilibrium distributions as well as the largest eigenvalues and corresponding eigenvectors. The latter quantities describe the time scales and associated flux between microstates for the slowest kinetic processes sampled in the underlying simulation trajectory. Biasing a Markov state model with coarse dynamical information provides a robust, generic, and simple way to systematically refine the microscopic description of a simulation model. 2581-Plat Upside: A New Dynamics Method Capable of Cooperative De Novo Protein Folding in CPU-Hours Tobin R. Sosnick1, John M. Jumper2, Karl F. Freed2. 1 Biochemistry/Inst. Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 2Chemistry, University of Chicago, Chicago, IL, USA.
Wednesday, March 2, 2016
living organisms noninvasively at high resolution in both space and time. However, the imaging of biological specimens involves inevitable tradeoffs of spatial resolution, speed, non-invasiveness, and imaging depth. I will describe three methods that balance these tradeoffs in different ways: structured illumination microscopy at 50-80 nm resolution, which we apply to study endocytic and cytoskeletal dynamics at the plasma membrane; lattice light sheet microscopy, which we use to image the rapid three-dimensional dynamics of single molecules, cells and embryos at hundreds of image planes per second; and adaptive optics, which we use to recover optimal resolution of fine neural processes deep in the brains of zebrafish and mice.
cells. We developed a live cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous b-actin genes in mouse embryonic fibroblasts, we observe that short (~8 s) Pol II clusters correlate with basal mRNA output. With serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with the proportionate increase in mRNAs synthesized minutes later. An additional burst of mRNA synthesis can be induced, at will, with a drug that stalls then releases Pol II clustering. Our findings reveal that transient clustering of Pol II constitutes a pre-transcriptional regulatory event which dynamically controls gene expression output.
2571-Symp Localizing Molecules in Cellular CT Scans Carolyn Larabell1,2, Gerry McDermott1,2, Mark Le Gros1,2. 1 Anatomy, University of California, San Francisco, San Francisco, CA, USA, 2 Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Much like medical CT scans reveal anatomical structures in the body, soft x-ray tomography (SXT) visualizes and quantifies the organization of sub-cellular structures within a cell. In SXT, the specimen is illuminated with x-ray photons from within a region of the spectrum known as the ‘water window’ (284 543eV). ‘Water window’ x-ray photons are absorbed an order of magnitude more strongly by carbon- and nitrogen-containing organic material than by water. Consequently, variation in biomolecule composition and concentration gives rise to quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells imaged by SXT are, therefore, highly representative of the cell in its native, functional state. Attenuation of soft x-rays, as they pass through the specimen, adheres to the BeerLambert Law. Attenuation is, therefore, a function of chemical composition and concentration of organic material, yielding unique quantitative Linear Absorption Coefficient (LAC) measurements for cellular components. LAC values are enormously powerful in terms of quantifying alterations in cell structures during events such as cell differentiation, progression or etiology of disease states, genetic manipulation, and application of exogenous agents. To localize molecules without perturbing cell structures, we use correlated high numerical aperture cryogenic fluorescence tomography (CFT). This multi-modal approach - imaging the same cell using both CFT and SXT - allows localization of labeled molecules directly in the context of a high-resolution 3-D tomographic reconstruction of the cell. I will show examples of data collected using these imaging technologies developed at the National Center for X-ray Tomography, an NIGMS-NIH supported Biomedical Technology Research Resource.
2574-Symp Single-Molecule Imaging of RNA in Live Cells Maria Carmo-Fonseca. Instituto de Medicina Molecular, University of Lisbon, Lisbon, Portugal. Expression of genetic information in eukaryotes involves a series of interconnected processes that ultimately determine the quality and amount of proteins in the cell. Many individual steps in gene expression are kinetically coupled, but tools are lacking to determine how temporal relationships between chemical reactions contribute to the output of the final gene product. Previous studies have imaged RNAs in living cells by genetically inserting the binding sites for bacteriophage coat proteins in the RNA of interest. However, multiple nascent RNAs were simultaneously detected at the site of transcription, necessitating a modelling approach to infer kinetic information. To circumvent these significant limitations and potential problems in data interpretation, we developed a strategy that permits direct tracking of single nascent pre-mRNA molecules in live cells. We are using this approach to study how kinetic mechanisms impact on RNA biogenesis.
2572-Symp Matching Scales and Capabilities with Integrated Fluorescence and Electron Microscopy Jacob P. Hoogenboom. Imaging Physics, Delft University of Technology, Delft, Netherlands. Superresolution techniques have pushed the resolution of fluorescence microscopy (FM) towards that of electron microscopy (EM). Meanwhile, developments in scanning EM are revolutionizing EM, moving lateral image dimensions to typical FM fields-of-view and extending imaging capability into the third dimension and the live-cell regime. By correlating data from both techniques, molecules can be localized within the context of cells and tissue and with reference to their live dynamics, but throughput and quantification are hindered by elaborate, expert procedures involving separate microscopes. I will show integration of highnumerical aperture FM in a SEM, such that the electron beam can be positioned anywhere within the fluorescence field of view. Using electron-beam excited cathodoluminescence from the transparent sample substrate, we achieve automated FM-EM image registration with an accuracy that can be pushed to 5nm, i.e. equaling bio-molecular length scales. Besides integrated correlation microscopy, I will show our progress towards novel applications bridging fluorescence and electron microscopy, such as fluorescence-guided live-cell EM, and electronbeam identification and localization of labels, molecules, and cells.
2575-Symp Micromechanical Study of Mammalian Metaphase Chromosomes and Nuclei John F. Marko. Northwestern University, Evanston, IL, USA. Eukaryote chromosomes are organized into complex folded structures with remarkably different organization at different stages of the cell cycle. During interphase, chromosomes are organized into functional compartments and domains composed of dense heterochromatic regions and less compacted, transcriptionally active chromatin. Then, following DNA replication, chromosomes are entirely refolded into remarkably uniform, cylindrical mitotic chromosomes, which become individualized and ultimately split into sister chromatids. We have investigated the organization of both metaphase chromosomes and interphase nuclei from mammalian cells using micromechanical experiments. Metaphase chromosomes behave remarkably like crosslinked networks of chromatin, showing a wide range of elastic extensibility (Young modulus ~ 400 Pa), and with their elasticity dependent on connectivity of chromatin. Experiments where DNA is cleaved show an immediate loss of mechanical stability arguing against an underlying connected protein ‘‘scaffold’’ as an organizing principle. siRNA experiments interfering with production of condensin subunits lead to a loss of mechanical stability of metaphase chromosomes, indicative of the role of condensin as a major ‘‘cross-linker’’ of chromatin in metaphase chromosomes, and antibody staining of condensin indicates its organization into clusters that become visible when metaphase chromosomes are extended. We have also recently developed similar techniques for studying the mechanics of isolated human cell nuclei; DNA cleavage experiments indicate that chromatin itself contributes a large portion of the elastic modulus of the nucleus for small deformations, with the elasticity of the nuclear lamina becoming important at large extensions. Experiments increasing or decreasing the amount of heterochromatin or lamin A (a major intermediate component of the nuclear lamina) validate our conclusions concerning the differential importance of chromatin and the nuclear lamina to small and large nuclear deformations.
Symposium: Crowding and Order in the Genome
Platform: Molecular Dynamics II
2573-Symp Predictability and Control of Gene Bursting in Live Mammalian Cells Ibrahim Cisse´. Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA. Transcriptional bursting is a hallmark for cellular variability across species. Whether predictability and output control exist within the stochastic bursting process is unknown. Here, we advance that collective behaviors from transient bio-molecular interactions help confer gene expression control in mammalian
2576-Plat RNA Conformational Ensembles: Narrowing the GAP between Experiments and Simulations with Metadynamics Alejandro Gil-Ley, Sandro Bottaro, Giovanni Bussi. Physics, SISSA, Trieste, Italy. The computational study of conformational transitions in nucleic acids still faces many challenges. For example, in the case of single stranded RNA tetranucleotides, agreement between simulations and experiments is not satisfactory due to inaccuracies in the force fields commonly used in molecular dynamics.
Wednesday, March 2, 2016 Improvement of force fields is however hindered by the difficulties of decoupling those errors from the statistical errors caused by insufficient sampling. We here tackle both problems simultaneously by introducing a novel enhancing sampling method and using experimental data to improve RNA force fields. In this novel method, concurrent well-tempered metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strength of the bias potential exploiting the tunability of well-tempered metadynamics so as to scale barriers on individual collective variables [1]. At the same time, the metadynamics algorithm is modified so as to allow enforcing a target distribution of backbone and sugar-base torsion angles taken from experimental structures, using a procedure related to two recently introduced techniques. Replica-exchange simulations of several RNA tetranucleotides with experimental corrections show significantly better agreement with NMR experimental data and suggest a systematic procedure for force field refinement. [1] Gil-Ley, A.; Bussi, G. J. Chem. Theory Comput. 2015, 11, 1077-1085. 2577-Plat Application of the String and 2D Hamiltonian Replica Exchange Umbrella Sampling Methods for the Study of Conformational Changes in the Bacterial Aspartate Transporter Glt(Ph) Hristina R. Zhekova1, Bogdan Lev2, Sergei Noskov1. 1 Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Department of Physics, RMIT University, Melbourne, Australia. Glt(Ph) is a homotrimeric protein involved in the sodium coupled transport of aspartate through the cellular membrane of Pyrococcus horikoshii. It belongs to the solute carrier 1 (SLC1) family, which comprises of a variety of proteins responsible for the trans-membrane transport of acidic or neutral amino acids in prokaryotes and eukaryotes. An important member of this family is the human glutamate transporter, which regulates the concentration of the neurotransmitter glutamate in the central nervous system. Unfortunately, its crystal structure is currently unknown. As Glt(Ph) has well resolved crystal structures in different functionally significant conformations, it has been studied extensively as a structural and functional analogue of the human glutamate transporter. Previous experimental and molecular dynamics studies have shown that substrate binding and the presence or absence of a lipid bilayer induce considerable conformational changes in Glt(Ph) which have been linked to the aspartate movement through the membrane. In this work two different sampling methods - 2D Hamiltonian Replica Exchange Umbrella Sampling (2DHREUS) and the String method - are used for the evaluation of potentials of mean force of a gate movement in Glt(Ph) associated with substrate binding to the active site. The gate opening has a low energy barrier and can be observed in the apo state even in the absence of a substrate. The String method compares well to the results of our benchmark 2DHREUS calculations and captures the most important structural and thermodynamical aspects of the conformational changes. 2578-Plat Quantifying Macromolecular Transition Paths with Path Similarity Analysis Sean L. Seyler, Avishek Kumar, Taylor Colburn, Michael F. Thorpe, Oliver Beckstein. Physics, Arizona State University, Tempe, AZ, USA. Diverse classes of proteins function through large-scale conformational changes; sophisticated enhanced sampling methods have been proposed to generate these macromolecular transition paths. As such paths are curves in a high-dimensional space, they have been difficult to compare quantitatively, a prerequisite to, for instance, assess the quality of different sampling algorithms. The Path Similarity Analysis (PSA) approach [1] alleviates these difficulties without relying on low-dimensional projections by utilizing the full information in 3N-dimensional trajectories in configuration space. PSA employs the Hausdorff or Fre´chet path (distance) metrics—adopted from computational geometry—enabling us to quantify path (dis)similarity, while the new concept of a Hausdorff-pair map permits the extraction of atomic-scale determinants responsible for path differences. Combined with clustering techniques, PSA facilitates the comparison of many paths, including collections of transition ensembles. We use the closed-to-open transition of the enzyme adenylate kinase (AdK)—a commonly used testbed for the assessment enhanced sampling algorithms [2]—to examine multiple microsecond equilibrium molecular dynamics (MD) transitions of AdK in its substrate-free form alongside transition ensembles from the MD-based dynamic importance sampling (DIMS-MD) and targeted MD (TMD) methods, and a geometrical targeting algorithm (FRODA). A Hausdorff pairs analysis of these ensembles revealed, for instance, that differences in DIMS-MD and FRODA paths were mediated by a set of conserved salt bridges whose charge-charge interactions are fully
523a
modeled in DIMS-MD but not in FRODA. We illustrate how existing trajectory analysis methods relying on pre-defined collective variables (such as native contacts or geometric quantities) can be used synergistically with PSA, as well as how PSA can be applied to more complex systems such as membrane transporter proteins. [1] Seyler, Kumar, Thorpe, Beckstein. PLoS Comput Biol (2015), http://dx.doi. org/10.1371/journal.pcbi.1004568; [2] Seyler and Beckstein. Mol Simul 40:855-877 (2014), http://dx.doi.org/10.1080/08927022.2014.919497. 2579-Plat Investigating Kinetics of Conformational Change using Molecular Dynamics and Milestoning Hiroshi Fujisaki1, Ayori Mitsutake2. 1 Nippon Medical School, Musashino, Tokyo, Japan, 2Keio University, Yokohama, Japan. To understand the functional roles of biomolecules, it is necessary to study both stable and metastable states in conformational space and how they are connected. Because of the advance of hardware and software, molecular dynamics (MD) simulation methods have become a feasible tool for this purpose, resulting in rather quantitative conclusions, which can be compared with experiment. MD simulation methods, however, still have a bottleneck due to ‘‘rare events’’ connecting stable states, and such a rare event can occur with millisecond timescales in a large biomolecule, which hampers a direct application of MD simulations to the rare event of biomolecules. This is why we need some novel ideas and algorithms to overcome this problem of rare events, and many researchers have been developing promising methods, mainly focusing on conformational sampling methods. These include very powerful replica exchange and multicanonical methods, but time information is lost in these methods and dynamic characterization of a rare event is difficult. In this presentation, we apply the milestoning method devised by Elber, Vanden-Eijnden and others to conformational change of a small peptide, chignolin, at the folding temperature (~420K). The milestoning method is regarded as an alternative of Markov state modeling, and by defining ‘‘milestones (collections of small dividing surfaces)’’ in some order parameter space and counting the crossing of milestones by a trajectory, mean first passage times (MFPTs) between two milestones can be obtained and a kinetic characterization of conformational change can be achieved. Here we will examine how the choice of order parameter space affects the results of MFPTs, and also analyze the effects of non-Markovianity for this process using Zuckerman’s method. 2580-Plat Improved Kinetics of Molecular Simulations using Biased Markov State Models Joseph F. Rudzinski, Kurt Kremer, Tristan Bereau. Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. Molecular simulations can provide microscopic insight into the physical and chemical driving forces of complex molecular processes. Despite tremendous advances in the accuracy of models as well as in the sophistication of simulation methodology, force-field errors can always potentially cause inconsistencies between simulated and experimentally-measured observables. The present work presents a robust and systematic framework to reweight the ensemble of dynamical paths sampled in a molecular simulation in order to be consistent with a set of kinetic observables. The method employs the well-developed Markov state modeling framework in order to efficiently treat simulated dynamical paths. By biasing the Markov state model to reproduce a small number of kinetic constraints, we not only improve the kinetic description of the system, but also the equilibrium distributions. The method is illustrated on two distinct coarse-grained peptide models, for which we observe clear improvements of the equilibrium distributions as well as the largest eigenvalues and corresponding eigenvectors. The latter quantities describe the time scales and associated flux between microstates for the slowest kinetic processes sampled in the underlying simulation trajectory. Biasing a Markov state model with coarse dynamical information provides a robust, generic, and simple way to systematically refine the microscopic description of a simulation model. 2581-Plat Upside: A New Dynamics Method Capable of Cooperative De Novo Protein Folding in CPU-Hours Tobin R. Sosnick1, John M. Jumper2, Karl F. Freed2. 1 Biochemistry/Inst. Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 2Chemistry, University of Chicago, Chicago, IL, USA.