Maxim V. Petoukhov

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, a program package for small-angle scattering data analysis

The program package ATSAS 2.1 for small-angle X-ray and neutron scattering data analysis is presented. The programs included in the package cover the major processing and interpretation steps from primary data reduction to three-dimensional modelling. This system is primarily oriented towards the analysis of biological macromolecules, but could also be used for non-biological isotropic and partially ordered objects (nanoparticle systems, colloidal solutions, polymers in solution and bulk). Recent developments in the programs included in ATSAS 2.1 are highlighted. The main programs run on multiple hardware platforms, including Windows PC, Linux RedHat and Suse, DEC Alpha, SGI IRIX and Mac OSX.

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.

MASSHA - a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data

A program, MASSHA, for three-dimensional rendering and rigid-body refinement is presented. The program allows display and manipulation of high-resolution atomic structures and low-resolution models represented as smooth envelopes or ensembles of beads. MASSHA is coupled with a computational module to align structural models of different nature and resolution automatically. The main feature is the possibility for rigid-body refinement of the quaternary structure of macromolecular complexes. If high- or low-resolution models of individual subunits are available, the complex can be constructed on the computer display and refined to fit the experimental solution scattering data. MASSHA provides both interactive and automated refinement modes to position subunits in a heterodimeric complex (general case) and in a homodimeric complex with a twofold symmetry axis. Examples of application of the program (running on IBM PC compatible machines under Windows 9x/NT/2000) are given.

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.

Applications of small-angle X-ray scattering to biomacromolecular solutions.

Small-angle scattering of X-rays (SAXS) is an established method for low-resolution structural characterization of biological macromolecules in solution. Being complementary to the high resolution methods (X-ray crystallography and NMR), SAXS is often used in combination with them. The technique provides overall three-dimensional structures using ab initio reconstructions and hybrid modeling, and allows one to quantitatively characterize equilibrium mixtures as well as flexible systems. Recent progress in SAXS instrumentation, most notably, high brilliance synchrotron sources, has paved the way for high throughput automated SAXS studies allowing screening of external conditions (pH, temperature, ligand binding etc.). The modern approaches for SAXS data analysis are presented in this review including rapid characterization of macromolecular solutions in terms of low-resolution shapes, validation of high-resolution models in close-to-native conditions, quaternary structure analysis of complexes and quantitative description of the oligomeric composition in mixtures. Practical aspects of SAXS as a standalone tool and its combinations with other structural, biophysical or bioinformatics methods are reviewed. The capabilities of the technique are illustrated by a selection of recent applications for the studies of biological molecules. Future perspectives on SAXS and its potential impact to structural molecular biology are discussed.

The Active Conformation of Glutamate Synthase and its Binding to Ferredoxin

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.

X-Ray Scattering Study of Activated Arp2/3 Complex with Bound Actin-WCA

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.

Chemical Basis of Peptidoglycan Discrimination by PrkC, a Key Kinase Involved in Bacterial Resuscitation from Dormancy

Bacterial Ser/Thr kinases modulate a wide number of cellular processes. In Bacillus subtilis , the Ser/Thr kinase PrkC has been shown to induce germination of bacterial spores in response to DAP-type but not Lys-type cell wall muropeptides. Muropeptides are a clear molecular signal that growing conditions are promising, since they are produced during cell wall peptidoglycan remodeling associated with cell growth and division of neighboring bacteria. However, whether muropeptides are able to bind the protein physically and how the extracellular region is able to distinguish the two types of muropeptides remains unclear. Here we tackled the important question of how the extracellular region of PrkC (EC-PrkC) senses muropeptides. By coupling NMR techniques and protein mutagenesis, we exploited the structural requirements necessary for recognition and binding and proved that muropeptides physically bind to EC-PrkC through DAP-moiety-mediated interactions with an arginine residue, Arg500, belonging to the protein C-terminal PASTA domain. Notably, mutation of this arginine completely suppresses muropeptide binding. Our data provide the first molecular clues into the mechanism of sensing of muropeptides by PrkC.

Joint use of small-angle X-ray and neutron scattering to study biological macromolecules in solution

Novel techniques for simultaneous analysis of X-ray and neutron scattering patterns from macromolecular complexes in solution are presented. They include ab initio shape and internal structure determination of multicomponent particles and more detailed rigid body modeling of complexes using high resolution structures of subunits. The methods fit simultaneously X-ray and neutron scattering curves including contrast variation data sets from selectively deuterated complexes. Biochemically sound interconnected models without steric clashes between the components displaying a pre-defined symmetry are generated. For rigid body modeling, distance restraints between specified residues/nucleotides or their ranges are taken into account. The efficiency of the methods is demonstrated in model examples, and potential sources of ambiguity are discussed.