Paul Murphy

Automated prediction of CASP‐5 structures using the Robetta server

Robetta is a fully automated protein structure prediction server that uses the Rosetta fragment‐insertion method. It combines template‐based and de novo structure prediction methods in an attempt to produce high quality models that cover every residue of a submitted sequence. The first step in the procedure is the automatic detection of the locations of domains and selection of the appropriate modeling protocol for each domain. For domains matched to a homolog with an experimentally characterized structure by PSI‐BLAST or Pcons2, Robetta uses a new alignment method, called K*Sync, to align the query sequence onto the parent structure. It then models the variable regions by allowing them to explore conformational space with fragments in fashion similar to the de novo protocol, but in the context of the template. When no structural homolog is available, domains are modeled with the Rosetta de novo protocol, which allows the full length of the domain to explore conformational space via fragment‐insertion, producing a large decoy ensemble from which the final models are selected. The Robetta server produced quite reasonable predictions for targets in the recent CASP‐5 and CAFASP‐3 experiments, some of which were at the level of the best human predictions. Proteins 2003;53:524–533.

Rosetta Predictions in CASP5: Successes, Failures, and Prospects for Complete Automation

We describe predictions of the structures of CASP5 targets using Rosetta. The Rosetta fragment insertion protocol was used to generate models for entire target domains without detectable sequence similarity to a protein of known structure and to build long loop insertions (and N‐and C‐terminal extensions) in cases where a structural template was available. Encouraging results were obtained both for the de novo predictions and for the long loop insertions; we describe here the successes as well as the failures in the context of current efforts to improve the Rosetta method. In particular, de novo predictions failed for large proteins that were incorrectly parsed into domains and for topologically complex (high contact order) proteins with swapping of segments between domains. However, for the remaining targets, at least one of the five submitted models had a long fragment with significant similarity to the native structure. A fully automated version of the CASP5 protocol produced results that were comparable to the human‐assisted predictions for most of the targets, suggesting that automated genomic‐scale, de novo protein structure prediction may soon be worthwhile. For the three targets where the human‐assisted predictions were significantly closer to the native structure, we identify the steps that remain to be automated. Proteins 2003;53:457–468.

Rosettascripts: A scripting language interface to the Rosetta Macromolecular modeling suite

Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.

Alteration of enzyme specificity by computational loop remodeling and design

Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme–substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein–ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-Å Cα RMSD of the computational model.

Prediction of structures of multidomain proteins from structures of the individual domains

We describe the development of a method for assembling structures of multidomain proteins from structures of isolated domains. The method consists of an initial low‐resolution search in which the conformational space of the domain linker is explored using the Rosetta de novo structure prediction method, followed by a high‐resolution search in which all atoms are treated explicitly and backbone and side chain degrees of freedom are simultaneously optimized. The method recapitulates, often with very high accuracy, the structures of existing multidomain proteins.

Use of adaptive thresholding to improve accuracy of small-aperture acoustic localization in shallow water

Passive acoustic localizing arrays are utilized in shallow water for applications ranging from environmental monitoring to marine security. Although large-aperture hydrophone arrays can provide accurate estimates of source position, they can be costly to deploy and maintain. Small-aperture arrays can be used to estimate the incident azimuth and elevation angles of acoustic sources, but their effectiveness may be hindered by multipath interference when deployed in reverberant environments. Here, a small-aperture hydrophone array is used to estimate the direction of acoustic sources with center frequencies of 69 and 120 kHz and durations less than 5 ms which were deployed at ranges of up to 75 m in a bay with a water depth on the order of 10 m. Time-differences of arrival are determined using an adaptive thresholding algorithm, which is shown to have superior performance to a cross-correlation method due to the prevalence of multipath interference in the signals. Errors in source bearing estimates and in the underlying time-differences of arrival calculations are evaluated by comparing to ground-truth values generated from the GPS position of the source.Passive acoustic localizing arrays are utilized in shallow water for applications ranging from environmental monitoring to marine security. Although large-aperture hydrophone arrays can provide accurate estimates of source position, they can be costly to deploy and maintain. Small-aperture arrays can be used to estimate the incident azimuth and elevation angles of acoustic sources, but their effectiveness may be hindered by multipath interference when deployed in reverberant environments. Here, a small-aperture hydrophone array is used to estimate the direction of acoustic sources with center frequencies of 69 and 120 kHz and durations less than 5 ms which were deployed at ranges of up to 75 m in a bay with a water depth on the order of 10 m. Time-differences of arrival are determined using an adaptive thresholding algorithm, which is shown to have superior performance to a cross-correlation method due to the prevalence of multipath interference in the signals. Errors in source bearing estimates and in th...

Acoustic characterization of wave energy converters

Wave energy converters produce sound as a consequence of their operation, but the specifics are not well-understood. Here, we present observations of two point-absorbing wave energy converters deployed at the US Navy’s Wave Energy Test Site in Kaneohe, HI. Measurements are obtained by free-drifting instrumentation packages which acoustical isolate the hydrophone from the surface expression to minimize masking by flow-noise (i.e., pseudo-sound generated by relative motion between hydrophone and water) and self-noise (i.e., propagating sound generated by the instrument package). Observations suggest that wave energy converters of different designs, even within the general class of point absorbers, produce different stereotypical sounds. Further, during normal operation, sound signatures can change substantially with sea state as new sound generation mechanisms come into play. One example of this is wave breaking around a shallow-hulled point absorber when waves exceed a critical steepness. The sound from bu...

Use of integrated instrumentation to detect and classify targets in shallow water

The detection and classification of targets in shallow water environments (e.g., depth < 20 m) is of increasing interest, both in security applications and more general environmental studies. Here, we report the ability of a cabled integrated instrumentation system to detect and classify targets in a shallow (15 m deep), narrow (150 m wide) channel influenced by moderate tidal currents (peak currents < 2 m/s). The instrumentation system incorporates a passive acoustic array, multibeam sonar, acoustic camera, stereo-optical camera, and acoustic Doppler current profiler. The system effectiveness is benchmarked using autonomous “cooperative” targets (e.g., drifters with known position and acoustic characteristics), divers, and opportunistic observations of fish and marine mammals. By fusing data streams from multiple sensors, detection and classification rates are improved, particularly for smaller targets. Approaches to data management (data rates exceed 80 MB/s), real-time classification, and de-noising of...

Acoustic characterization of a wave energy converter

As progress toward the commercial deployment of wave energy converters accelerates, it is important to ensure that these renewable energy systems do not have unintended, adverse environmental consequences. While the sound from wave energy converters is unlikely to cause acoustic injury to marine animals, it may affect their behavior. Here, we present measurements from a point-absorber wave energy converter at the U.S. Navy Wave Energy Test Site in Kaneohe Bay, HI. Measurements of wave converter sound are obtained for a range of sea states using a combination of free-drifting near-surface measurements and stationary bottom packages. The relative effectiveness of these systems are contrasted and the unique challenges associated with acoustic measurements at energetic sites discussed. For example, fixed measurements are found to be substantially contaminated by flow-noise (non-propagating sound) during long-period ocean swell, while free-drifting measurements require significant post-processing to avoid conv...