Biology subjects

James Fraser

Publications and source records attributed to James Fraser.

6 recordsLinked to original sources

bioRxiv2016

Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

Cryo-EM has revealed many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5[A]), providing biological phenomena with molecular descriptions. However, at these resolutions accurately positioning individual atoms remains challenging and may be error-prone. Manually refining thousands of amino acids - typical in a macromolecular assembly - is tedious and time-consuming. We present an automated method that can improve the atomic details in models manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining or improving the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

Biochemistry↗

bioRxiv2015

Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting

Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies in S. cerevisiae indicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover \"shared sensitized positions\" localized to areas around the hydrophobic patch and to the C-terminus. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.

Systems Biology↗

bioRxiv2015

Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the \"flap\" regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.\n\nAuthor SummaryDescribing the multiple conformations of proteins is important for understanding the relationship between molecular flexibility and function. However, most methods for interpreting data from X-ray crystallography focus on building a single structure of the protein, which limits the potential for biological insights. Here we introduce an improved algorithm for using crystallographic data to model these multiple conformations that addresses two previously overlooked types of protein backbone flexibility: peptide flips and glycine movements. The method successfully models known examples of these types of multiple conformations, and also identifies new cases that were previously unrecognized but are well supported by the experimental data. For example, we discover glycine-driven peptide flips in the inhibitor-gating \"flaps\" of the drug target HIV protease that were not modeled in the original structures. Automatically modeling \"hidden\" multiple conformations of proteins using our algorithm may help drive biomedically relevant insights in structural biology pertaining to, e.g., drug discovery for HIV-1 protease and other therapeutic targets.

Biophysics↗

bioRxiv2015

Mapping the Conformational Landscape of a Dynamic Enzyme by XFEL and Multitemperature Crystallography

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free electron laser (XFEL) crystallography. The \"diffraction-before-destruction\" nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states for a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

Biophysics↗

bioRxiv2015

From deep TLS validation to ensembles of atomic models built from elemental motions

The widely used Translation Libration Screw (TLS) approximation describes concerted motions of atomic groups in X-ray refinement. TLS refinement often provides a better interpretation of diffraction data and the resulting rigid body motions may subsequently be assigned biochemical significance. In TLS refinement, three matrices (T, L and S) describe harmonic vibration, libration and their correlation. Because these matrices describe specific motions, they impose a number of conditions on their elements. Ignoring these conditions while refining the matrix elements may result in matrices that cannot be interpreted in terms of physically realistic motions. We describe a mathematical framework and the computational tools to analyze refined TLS matrices through their decomposition into descriptors of underlying motions. This allows for straightforward validation and identification of implausible TLS parameters. An algorithm for the generation of structural ensembles that are consistent with given TLS parameters, implemented as a part of the Phenix project, is also described.\n\nSynopsisProcedures for the decomposition of TLS matrices into elementary vibrations and librations indicates possible errors in the definition of these matrices and corrects them when possible. The program outputs the corresponding vibration-libration parameters and generates structural ensembles.

Biophysics↗

bioRxiv2015

Predicting X-ray Diffuse Scattering from Translation Libration Screw Structural Ensembles

Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. While Bragg diffraction describes the average positional distribution of crystalline atoms, many different models can fit this distribution equally well. Diffuse X-ray scattering can reduce this degeneracy by directly reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling and validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guiniers equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to Translation-Libration-Screw (TLS) refinement, which models rigid body displacement for segments of the macromolecule. To enable calculation of diffuse scattering from TLS refined structures, phenix.tls_models builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how X-ray diffuse scattering can extend macromolecular structural refinement, validation, and analysis.\n\nSynopsisA method of simulating X-ray diffuse scattering from multi-model PDB files is presented. Despite similar agreement with Bragg data, different Translation-Libration-Screw refinement strategies produce unique diffuse intensity patterns.

Biophysics↗