Perfect Crystals: microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography.

The NASA mission Perfect Crystals used the microgravity environment on the International Space Station (ISS) to grow crystals of human manganese superoxide dismutase (MnSOD)-an oxidoreductase critical for mitochondrial vitality and human health. The mission's overarching aim is to perform neutron protein crystallography (NPC) on MnSOD to directly visualize proton positions and derive a chemical understanding of the concerted proton electron transfers performed by the enzyme. Large crystals that are perfect enough to diffract neutrons to sufficient resolution are essential for NPC. This combination, large and perfect, is hard to achieve on Earth due to gravity-induced convective mixing. Capillary counterdiffusion methods were developed that provided a gradient of conditions for crystal growth along with a built-in time delay that prevented premature crystallization before stowage on the ISS. Here, we report a highly successful and versatile crystallization system to grow a plethora of crystals for high-resolution NPC.

The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis.

Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.

Are the St John's wort Hyp-1 superstructures different?

Two commensurately modulated structures (PDB entries 4n3e and 6sjj) were solved using translational noncrystallographic symmetry (tNCS). The data required the use of large supercells, sevenfold and ninefold, respectively, to properly index the reflections. Commensurately modulated structures can be challenging to solve. Molecular-replacement software such as Phaser can detect tNCS and either handle it automatically or, for more challenging situations, allow the user to enter a tNCS vector, which the software then uses to place the components. Although this approach has been successful in solving these types of challenging structures, it does not make it easy to understand the underlying modulation in the structure or how these two structures are related. An alternate view of this problem is that the atoms and associated parameters are following periodic atomic modulation functions (AMFs) in higher dimensional space, and what is being observed in these supercells are the points where these higher dimensional AMFs intersect physical 3D space. In this case, the two 3D structures, with a sevenfold and a ninefold superstructure, seem to be quite different. However, describing those structures within the higher dimensional superspace approach makes a strong case that they are closely related, as they show very similar AMFs and can be described with one unique (3+1)D structure, i.e. they are two different 3D intersections of the same (3+1)D structure.

Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.

Simulation of modulated protein crystal structure and diffraction data in a supercell and in superspace.

The toolbox for computational protein crystallography is full of easy-to-use applications for the routine solution and refinement of periodic diffraction data sets and protein structures. There is a gap in the available software when it comes to aperiodic crystallographic data. Current protein crystallography software cannot handle modulated data, and small-molecule software for aperiodic crystallography cannot work with protein structures. To adapt software for modulated protein data requires training data to test and debug the changed software. Thus, a comprehensive training data set consisting of atomic positions with associated modulation functions and the modulated structure factors packaged as both a three-dimensional supercell and as a modulated structure in (3+1)D superspace has been created. The (3+1)D data were imported into Jana2006; this is the first time that this has been performed for protein data.

Processing incommensurately modulated protein diffraction data with Eval15.

Recent challenges in biological X-ray crystallography include the processing of modulated diffraction data. A modulated crystal has lost its three-dimensional translational symmetry but retains long-range order that can be restored by refining a periodic modulation function. The presence of a crystal modulation is indicated by an X-ray diffraction pattern with periodic main reflections flanked by off-lattice satellite reflections. While the periodic main reflections can easily be indexed using three reciprocal-lattice vectors a*, b*, c*, the satellite reflections have a non-integral relationship to the main lattice and require a q vector for indexing. While methods for the processing of diffraction intensities from modulated small-molecule crystals are well developed, they have not been applied in protein crystallography. A recipe is presented here for processing incommensurately modulated data from a macromolecular crystal using the Eval program suite. The diffraction data are from an incommensurately modulated crystal of profilin-actin with single-order satellites parallel to b*. The steps taken in this report can be used as a guide for protein crystallographers when encountering crystal modulations. To our knowledge, this is the first report of the processing of data from an incommensurately modulated macromolecular crystal.

A very simple spiking neuron model that allows for modeling of large, complex systems.

This letter introduces a biologically inspired very simple spiking neuron model. The model retains only crucial aspects of biological neurons: a network of time-delayed weighted connections to other neurons, a threshold-based generation of action potentials, action potential frequency proportional to stimulus intensity, and interneuron communication that occurs with time-varying potentials that last longer than the associated action potentials. The key difference between this model and existing spiking neuron models is its great simplicity: it is basically a collection of linear and discontinuous functions with no differential equations to solve. The model's ability to operate in a complex network was tested by using it as a basis of a network implementing a hypothetical echolocation system. The system consists of an emitter and two receivers. The outputs of the receivers are connected to a network of spiking neurons (using the proposed model) to form a detection grid that acts as a map of object locations in space. The network uses differences in the arrival times of the signals to determine the azimuthal angle of the source and time of flight to calculate the distance. The activation patterns observed indicate that for a network of spiking neurons, which uses only time delays to determine source locations, the spatial discrimination varies with the number and relative spacing of objects. These results are similar to those observed in animals that use echolocation.