Anomalous small angle x-ray scattering simulations: proof of concept for distance measurements for nanoparticle-labelled biomacromolecules in solution.

Anomalous small angle X-ray scattering can in principle be used to determine distances between metal label species on biological molecules. Previous experimental studies in the past were unable to distinguish the label-label scattering contribution from that of the molecule, because of the use of atomic labels; these labels contribute only a small proportion of the total scattering signal. However, with the development of nanocrystal labels (of 50-100 atoms) there is the possibility for a renewed attempt at applying anomalous small angle X-ray scattering for distance measurement. This is because the contribution to the scattered signal is necessarily considerably stronger than for atomic labels. Here we demonstrate through simulations, the feasibility of the technique to determine the end-to-end distances of labelled nucleic acid molecules as well as other internal distances mimicking a labelled DNA binding protein if the labels are dissimilar metal nanocrystals. Of crucial importance is the ratio of mass of the nanocrystals to that of the labelled macromolecule, as well as the level of statistical errors in the scattering intensity measurements. The mathematics behind the distance determination process is presented, along with a fitting routine than incorporates maximum entropy regularisation

Of Bits and Bugs — On the Use of Bioinformatics and a Bacterial Crystal Structure to Solve a Eukaryotic Repeat-Protein Structure

Pur-α is a nucleic acid-binding protein involved in cell cycle control, transcription, and neuronal function. Initially no prediction of the three-dimensional structure of Pur-α was possible. However, recently we solved the X-ray structure of Pur-α from the fruitfly Drosophila melanogaster and showed that it contains a so-called PUR domain. Here we explain how we exploited bioinformatics tools in combination with X-ray structure determination of a bacterial homolog to obtain diffracting crystals and the high-resolution structure of Drosophila Pur-α. First, we used sensitive methods for remote-homology detection to find three repetitive regions in Pur-α. We realized that our lack of understanding how these repeats interact to form a globular domain was a major problem for crystallization and structure determination. With our information on the repeat motifs we then identified a distant bacterial homolog that contains only one repeat. We determined the bacterial crystal structure and found that two of the repeats interact to form a globular domain. Based on this bacterial structure, we calculated a computational model of the eukaryotic protein. The model allowed us to design a crystallizable fragment and to determine the structure of Drosophila Pur-α. Key for success was the fact that single repeats of the bacterial protein self-assembled into a globular domain, instructing us on the number and boundaries of repeats to be included for crystallization trials with the eukaryotic protein. This study demonstrates that the simpler structural domain arrangement of a distant prokaryotic protein can guide the design of eukaryotic crystallization constructs. Since many eukaryotic proteins contain multiple repeats or repeating domains, this approach might be instructive for structural studies of a range of proteins

Preparation and Characterization of the Extracellular Domain of Human Sid-1

In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9–12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective

Coiled coils unspring protein origami

Self-assembling sequences of protein coiled coils create polyhedral nanostructures for advanced applications in biomedicine, chemistry and materials science

Structural transition of nanogel star polymers with pH by controlling PEGMA interactions with acid or base copolymers

We use small angle X-ray scattering (SAXS) to characterise a class of star diblock polymers with a nanogel core on which the outer block arms are comprised of random copolymers of temperature sensitive PEGMA with pH sensitive basic (PDMAEMA) and acidic (PMAA) monomers. The acquired SAXS data show that many of the nanogel star polymers undergo a sharp structural transition over a narrow range of pH, but with unexpectedly large shifts in the apparent pKa with respect to that of the acidic or basic monomer unit, the linear polymer form or even an alternate star polymer with a tightly cross-linked core chemistry. We have demonstrated a distinct and quantifiable structural response for the nanogel star copolymers by altering the core or by pairing the monomers PDMAEMA–PEGMA and PMAA–PEGMA to achieve structural transitions that have typically been observed in stars through changes in arm length and number

High-Throughput SAXS for the Characterization of Biomolecules in Solution: A Practical Approach

The recent innovation of collecting X-ray scattering from solutions containing purified macromolecules in high-throughput has yet to be truly exploited by the biological community. Yet, this capability is becoming critical given that the growth of sequence and genomics data is significantly outpacing structural biology results. Given the huge mismatch in information growth rates between sequence and structural methods, their combined high-throughput and high success rate make high-throughput small angle X-ray scattering (HT-SAXS) analyses increasingly valuable. HT-SAXS connects sequence as well as NMR and crystallographic results to biological outcomes by defining the flexible and dynamic complexes controlling cell biology. Commonly falling under the umbrella of bio-SAXS, HT-SAXS data collection pipelines have or are being developed at most synchrotrons. How investigators practically get their biomolecules of interest into these pipelines, balance sample requirements and manage HT-SAXS data output format varies from facility to facility. While these features are unlikely to be standardized across synchrotron beamlines, a detailed description of HT-SAXS issues for one pipeline provides investigators with a practical guide to the general procedures they will encounter. One of the longest running and generally accessible HT-SAXS endstations is the SIBYLS beamline at the Advanced Light Source in Berkeley CA. Here we describe the current state of the SIBYLS HT-SAXS pipeline, what is necessary for investigators to integrate into it, the output format and a summary of results from 2 years of operation. Assessment of accumulated data informs issues of concentration, background, buffers, sample handling, sample shipping, homogeneity requirements, error sources, aggregation, radiation sensitivity, interpretation, and flags for concern. By quantitatively examining success and failures as a function of sample and data characteristics, we define practical concerns, considerations, and concepts for optimally applying HT-SAXS techniques to biological samples