Alexey Kikhney

New developments in the ATSAS program package for small-angle scattering data analysis.

The paper presents new developments and amendments to the ATSAS package (version 2.4) for processing and analysis of isotropic small-angle scattering data.

ATSAS 2.1 – towards automated and web-supported small-angle scattering data analysis

Small-angle scattering (SAS) is frequently employed for screening large numbers of samples and for studying these samples under different conditions, including space- and time-resolved analysis. These measurements produce immense amounts of data, especially on modern high-flux and high-brilliance sources (e.g. third-generation synchrotrons). In biological SAS, like high-throughput macromolecular crystallography, large-scale analysis of proteins and macromolecular complexes is also emerging. Automation of data analysis becomes an indispensable prerequisite for adequate evaluation of high-throughput SAS experiments. Here a prototype of an automated data-analysis system for isotropic solution scattering based on the further development of the programs belonging to the package ATSAS 2.1 is reported. This system allows the major analysis tasks starting from the raw data processing and, for monodisperse systems, finishing with a three-dimensional model, to be performed automatically. Convenient web interfaces for the online use of individual ATSAS programs are also provided.

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.

A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins.

Small‐angle X‐ray scattering (SAXS) is a biophysical method to study the overall shape and structural transitions of biological macromolecules in solution. SAXS provides low resolution information on the shape, conformation and assembly state of proteins, nucleic acids and various macromolecular complexes. The technique also offers powerful means for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs). Here, the basic principles of SAXS are presented, and profits and pitfalls of the characterization of multidomain flexible proteins and IDPs using SAXS are discussed from the practical point of view. Examples of the synergistic use of SAXS with high resolution methods like X‐ray crystallography and nuclear magnetic resonance (NMR), as well as other experimental andin silico techniques to characterize completely, or partially unstructured proteins, are presented.

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.

Instrumental setup for high-throughput small- and wide-angle solution scattering at the X33 beamline of EMBL Hamburg

A setup is presented for automated high-throughput measurements of small-angle X-ray scattering (SAXS) from macromolecular solutions on the bending-magnet beamline X33 of EMBL at the storage ring DORIS-III (DESY, Hamburg). A new multi-cell compartment allows for rapid switching between in-vacuum and in-air operation, for digital camera assisted control of cell filling and for colour sample illumination. The beamline is equipped with a Pilatus 1 M-W pixel detector for SAXS and a Pilatus 300 k-W for wide-angle scattering (WAXS), and results from the use of the Pilatus detectors for scattering studies are reported. The setup provides a broad resolution range from 100 to 0.36 nm without the necessity of changing the sample-to-detector distance. A new optimized robotic sample changer is installed, permitting rapid and reliable automated sample loading and cell cleaning with a required sample volume of 40 µl. All the devices are fully integrated into the beamline control software system, ensuring fully automated and user-friendly operation (attended, unattended and remote) with a throughput of up to 15 measurements per hour.

Peroxisomal Proteostasis Involves a Lon Family Protein That Functions as Protease and Chaperone

Background: A putative Lon protease has been identified in peroxisomes of various species (Pln). Results: Pln is an ATP-dependent protease that digests unfolded substrates e.g. oxidatively damaged catalase-peroxidase, and displays chaperone-like activity, circumventing accumulation of protein aggregates in peroxisomes that compromise organelle function. Conclusion: Pln is a bifunctional protein with chaperone and protease activities. Significance: Pln is crucial for peroxisome proteostasis. Proteins are subject to continuous quality control for optimal proteostasis. The knowledge of peroxisome quality control systems is still in its infancy. Here we show that peroxisomes contain a member of the Lon family of proteases (Pln). We show that Pln is a heptameric protein and acts as an ATP-fueled protease and chaperone. Hence, Pln is the first chaperone identified in fungal peroxisomes. In cells of a PLN deletion strain peroxisomes contain protein aggregates, a major component of which is catalase-peroxidase. We show that this enzyme is sensitive to oxidative damage. The oxidatively damaged, but not the native protein, is a substrate of the Pln protease. Cells of the pln strain contain enhanced levels of catalase-peroxidase protein but reduced catalase-peroxidase enzyme activities. Together with the observation that Pln has chaperone activity in vitro, our data suggest that catalase-peroxidase aggregates accumulate in peroxisomes of pln cells due to the combined absence of Pln protease and chaperone activities.

Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis. Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

The Switch that Does Not Flip: The Blue-Light Receptor YtvA from Bacillus subtilis Adopts an Elongated Dimer Conformation Independent of the Activation State as Revealed by a Combined AUC and SAXS Study

Photoreceptors play an important role in plants and bacteria by converting extracellular stimuli into intracellular signals. One distinct class are the blue-light-sensitive phototropins harboring a light-oxygen-voltage (LOV) domain coupled to various effector domains. Photon absorption by the chromophore within the LOV domain results in an activation of the output domain via mechanisms that are hitherto not well understood. The photoreceptor YtvA from Bacillus subtilis is a bacterial analog of phototropins, consists of an LOV and a sulfate transporter/anti-sigma factor antagonist domain, and is involved in the response of the bacterium to environmental stress. We present here analytical ultracentrifugation studies and small-angle X-ray scattering experiments, showing that YtvA is a dimer. On the basis of these results, we present a low-resolution model of the dimer in the dark and the lit state of the protein. In addition, we show that YtvA does not change its oligomerization state or its overall shape upon light activation.