Cy M. Jeffries

ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions

Developments and improvements of the ATSAS software suite (versions 2.5–2.8) for analysis of small-angle scattering data of biological macromolecules or nanoparticles are described.

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

An integrated environment for biological small-angle X-ray scattering (BioSAXS) at the high-brilliance P12 synchrotron beamline of the EMBL (DESY, Hamburg) allows for a broad range of solution scattering experiments. Automated hardware and software systems have been designed to ensure that data collection and processing are efficient, streamlined and user friendly.

SASBDB, a repository for biological small-angle scattering data

Small-angle X-ray and neutron scattering (SAXS and SANS) are fundamental tools used to study the global shapes of proteins, nucleic acids, macromolecular complexes and assemblies in solution. Due to recent advances in instrumentation and computational methods, the quantity of experimental scattering data and subsequent publications is increasing dramatically. The need for a global repository allowing investigators to locate and access experimental scattering data and associated models was recently emphasized by the wwPDB small-angle scattering task force (SAStf). The small-angle scattering biological data bank (SASBDB) www.sasbdb.org has been designed in accordance with the plans of the SAStf as part of a future federated system of databases for biological SAXS and SANS. SASBDB is a comprehensive repository of freely accessible and fully searchable SAS experimental data and models that are deposited together with the relevant experimental conditions, sample details and instrument characteristics. At present the quality of deposited experimental data and the accuracy of models are manually curated, with future plans to integrate automated systems as the database expands.

Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin.

Correlation Map, a goodness-of-fit test for one-dimensional X-ray scattering spectra.

Assessing similarity between data sets with the reduced χ2 test requires the estimation of experimental errors, which, if incorrect, may render statistical comparisons invalid. We report a goodness-of-fit test, Correlation Map (CorMap), for assessing differences between one-dimensional spectra independently of explicit error estimates, using only data point correlations. Using small-angle X-ray scattering data, we demonstrate that CorMap maintains the power of the reduced χ2 test; moreover, CorMap is also applicable to other physical experiments.

Effects of Macromolecular Crowding on an Intrinsically Disordered Protein Characterized by Small-Angle Neutron Scattering with Contrast Matching

Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage λ, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D(2)O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions.

Limiting radiation damage for high‐brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 10(12) photons s(-1), 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

Automated Pipeline for Purification, Biophysical and X-Ray Analysis of Biomacromolecular Solutions

Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg).

LIM domain binding proteins 1 and 2 have different oligomeric states.

LIM domain binding (Ldb) proteins are important regulators of LIM homeodomain and LIM-only proteins that specify cell fate in many different tissues. An essential feature of these proteins is the ability to self-associate, but there have been no studies that characterise the nature of this self-association. We have used deletion mutagenesis with yeast two-hybrid analysis to define the minimal self-association domains of Ldb1 and Ldb2 as residues 14-200 and 21-197, respectively. We then used a range of different biophysical methods, including sedimentation equilibrium and small-angle X-ray scattering to show that Ldb1(14-200) forms a trimer and Ldb2(21-197) undergoes a monomer-tetramer-octamer equilibrium, where the association in each case is of moderate affinity ( approximately 10(5) M(-1)). These modes of association represent a clear physical difference between these two proteins that otherwise appear to have very similar properties. The levels of association are more complex than previously assumed and emphasise roles of avidity and DNA looping in transcriptional regulation by Ldb1/LIM protein complexes. The abilities of Ldb1 and Ldb2 to form trimers and higher oligomers, respectively, should be considered in models of transcriptional regulation by Ldb1-containing complexes in a wide range of biological processes.

Stabilization of a binary protein complex by intein-mediated cyclization

The study of protein–protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein‐mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM‐only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X‐ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.