Elaine C. Meng

UCSF CHIMERA-A VISUALIZATION SYSTEM FOR EXPLORATORY RESEARCH AND ANALYSIS

The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large‐scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real‐world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.

DOCK 6: Combining techniques to model RNA–small molecule complexes

With an increasing interest in RNA therapeutics and for targeting RNA to treat disease, there is a need for the tools used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids. Here, we have compiled a test set of RNA-ligand complexes to validate the ability of the DOCK suite of programs to successfully recreate experimentally determined binding poses. With the optimized parameters and a minimal scoring function, 70% of the test set with less than seven rotatable ligand bonds and 26% of the test set with less than 13 rotatable bonds can be successfully recreated within 2 A heavy-atom RMSD. When DOCKed conformations are rescored with the implicit solvent models AMBER generalized Born with solvent-accessible surface area (GB/SA) and Poisson-Boltzmann with solvent-accessible surface area (PB/SA) in combination with explicit water molecules and sodium counterions, the success rate increases to 80% with PB/SA for less than seven rotatable bonds and 58% with AMBER GB/SA and 47% with PB/SA for less than 13 rotatable bonds. These results indicate that DOCK can indeed be useful for structure-based drug design aimed at RNA. Our studies also suggest that RNA-directed ligands often differ from typical protein-ligand complexes in their electrostatic properties, but these differences can be accommodated through the choice of potential function. In addition, in the course of the study, we explore a variety of newly added DOCK functions, demonstrating the ease with which new functions can be added to address new scientific questions.

Tools for integrated sequence-structure analysis with UCSF Chimera

BackgroundComparing related structures and viewing the structures in the context of sequence alignments are important tasks in protein structure-function research. While many programs exist for individual aspects of such work, there is a need for interactive visualization tools that: (a) provide a deep integration of sequence and structure, far beyond mapping where a sequence region falls in the structure and vice versa; (b) facilitate changing data of one type based on the other (for example, using only sequence-conserved residues to match structures, or adjusting a sequence alignment based on spatial fit); (c) can be used with a researchers own data, including arbitrary sequence alignments and annotations, closely or distantly related sets of proteins, etc.; and (d) interoperate with each other and with a full complement of molecular graphics features. We describe enhancements to UCSF Chimera to achieve these goals.ResultsThe molecular graphics program UCSF Chimera includes a suite of tools for interactive analyses of sequences and structures. Structures automatically associate with sequences in imported alignments, allowing many kinds of crosstalk. A novel method is provided to superimpose structures in the absence of a pre-existing sequence alignment. The method uses both sequence and secondary structure, and can match even structures with very low sequence identity. Another tool constructs structure-based sequence alignments from superpositions of two or more proteins. Chimera is designed to be extensible, and mechanisms for incorporating user-specific data without Chimera code development are also provided.ConclusionThe tools described here apply to many problems involving comparison and analysis of protein structures and their sequences. Chimera includes complete documentation and is intended for use by a wide range of scientists, not just those in the computational disciplines. UCSF Chimera is free for non-commercial use and is available for Microsoft Windows, Apple Mac OS X, Linux, and other platforms from http://www.cgl.ucsf.edu/chimera.

BayGenomics: a resource of insertional mutations in mouse embryonic stem cells

The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

UCSF Chimera, MODELLER, and IMP: an integrated modeling system.

Structural modeling of macromolecular complexes greatly benefits from interactive visualization capabilities. Here we present the integration of several modeling tools into UCSF Chimera. These include comparative modeling by MODELLER, simultaneous fitting of multiple components into electron microscopy density maps by IMP MultiFit, computing of small-angle X-ray scattering profiles and fitting of the corresponding experimental profile by IMP FoXS, and assessment of amino acid sidechain conformations based on rotamer probabilities and local interactions by Chimera.

Similar Structures and Shared Switch Mechanisms of the β2-Adrenoceptor and the Parathyroid Hormone Receptor Zn(II) BRIDGES BETWEEN HELICES III AND VI BLOCK ACTIVATION

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996)Nature 383, 347–350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the β2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.

Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.

Receptor activation: what does the rhodopsin structure tell us?

G-protein-coupled receptors (GPCRs) are a large family of seven-transmembrane-helix proteins that mediate responses to hormones, neurotransmitters and, in the case of rhodopsin, photons. The recent determination of the structure of rhodopsin at atomic resolution opens avenues to a deeper understanding of GPCR activation and transmembrane signaling. Data from previous crosslinking, spin labeling and scanning accessibility experiments on rhodopsin have been mapped onto the high-resolution structure. These data correlate well and are consistent with the structure, and suggest that activation by light opens a cleft at the cytoplasmic end of the seven-helix bundle of rhodopsin. Furthermore, lessons learned from rhodopsin might also apply to other members of this essential family of receptors. (For an animation of the crystal structure of rhodopsin see http://archive.bmn.com/supp/tips/tips2211a.html)

The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse

Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising ∼45 000 well-characterized ES cell lines which currently represent ∼40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website () allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function.

C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144–164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.