New developments in the program packageATSAS(version 2.4) for the processing and analysis of isotropic small-angle X-ray and neutron scattering data are described. They include (i) multiplatform data manipulation and display tools, (ii) programs for automated data processing and calculation of overall parameters, (iii) improved usage of high- and low-resolution models from other structural methods, (iv) new algorithms to build three-dimensional models from weakly interacting oligomeric systems and complexes, and (v) enhanced tools to analyse data from mixtures and flexible systems. The newATSASrelease includes installers for current major platforms (Windows, Linux and Mac OSX) and provides improved indexed user documentation. The web-related developments, including a user discussion forum and a widened online access to runATSASprograms, are also presented.

Dynamic ensembles of macromolecules mediate essential processes in biology. Understanding the mechanisms driving the function and molecular interactions of `unstructured' and flexible molecules requires alternative approaches to those traditionally employed in structural biology. Small-angle X-ray scattering (SAXS) is an established method for structural characterization of biological macromolecules in solution, and is directly applicable to the study of flexible systems such as intrinsically disordered proteins and multi-domain proteins with unstructured regions. TheEnsemble Optimization Method(EOM) [Bernadóet al.(2007).J. Am. Chem. Soc.129, 5656–5664] was the first approach introducing the concept of ensemble fitting of the SAXS data from flexible systems. In this approach, a large pool of macromolecules covering the available conformational space is generated and a sub-ensemble of conformers coexisting in solution is selected guided by the fit to the experimental SAXS data. This paper presents a series of new developments and advancements to the method, including significantly enhanced functionality and also quantitative metrics for the characterization of the results. Building on the original concept of ensemble optimization, the algorithms for pool generation have been redesigned to allow for the construction of partially or completely symmetric oligomeric models, and the selection procedure was improved to refine the size of the ensemble. Quantitative measures of the flexibility of the system studied, based on the characteristic integral parameters of the selected ensemble, are introduced. These improvements are implemented in the newEOMversion 2.0, and the capabilities as well as inherent limitations of the ensemble approach in SAXS, and ofEOM2.0 in particular, are discussed.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin‐based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin‐2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans‐synaptic neuroligin‐dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin‐based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses. Synopsis The postsynaptic cell adhesion protein neuroligin‐2 stabilizes the open/active conformation of collybistin to regulate gephyrin and GABA_A receptor clustering at postsynaptic plasma membranes. Collybistin regulates the recruitment of the postsynaptic scaffold protein gephyrin to inhibitory synapses in the brain, which, in turn, leads to the postsynaptic clustering of GABA_A (and glycine) receptors. Collybistin exhibits a dynamic equilibrium between closed/inactive and open/active conformations and requires a conformational activation for its phosphoinositide‐binding and gephyrin‐clustering activity. At synapses, the active conformation of collybistin is selected by interactions with the postsynaptic cell adhesion proteins neuroligin‐2 and neuroligin‐4 and is further stabilized by the subsequent formation of a neuroligin/gephyrin/collybistin holocomplex.

In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a KD value of 22±2nM for ZinT, while those with ZnuA point to one high affinity (KD<20nM) and one low affinity Zn(II) binding site (KD in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.