PDB Reference: nicotinic acid mononucleotide adenylyltransferase, 3dv2, r3dv2sf.Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD + and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R free of 0.228 and 0.263, respectively, at 2.3 Å resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNATrecently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.

To investigate the effect of macromolecular transport and the incorporation of protein aggregate impurities in growing crystals, experiments were performed on the International Space Station (ISS) and compared with control experiments performed in a 1G laboratory environment. Crystal growth experiments for hen egg-white lysozyme (HEWL) and Plasmodium falciparum glutathione S-transferase (PfGST) were monitored using the ISS Light Microscopy Module (LMM). Experiments were performed applying the liquid–liquid counter diffusion crystallization method using rectangular, optically transparent capillaries. To analyze the quantity of impurity incorporated into growing crystals, stable fluorescently labeled protein aggregates were prepared and subsequently added at different percent concentrations to nonlabeled monomeric protein suspensions. For HEWL, a covalent cross-linked HEWL dimer was fluorescently labeled, and for PfGST, a stable tetramer was prepared. Crystallization solutions containing different protein aggregate ratios were prepared. The frozen samples were launched on 19.02.2017 via SpaceX-10 mission and immediately transferred to a -80°C freezer on the ISS. Two series of crystallization experiments were performed on ISS, one during 26.02.2017 to 10.03.2017 and a second during 16.06.2017 to 23.06.2017. A comparison of crystal growth rate and size showed different calculated average growth rates as well as different dimensions for crystals growing in different positions along the capillary. The effect of macromolecular mass transport on crystal growth in microgravity was experimentally calculated. In parallel, the percentage of incorporated fluorescent aggregate into the crystals was monitored utilizing the fluorescent LMM and ground-based fluorescent microscopes.

Microgravity conditions have been used to improve protein crystallization from the early 1980s using advanced crystallization apparatuses and methods. Early microgravity crystallization experiments confirmed that minimal convection and a sedimentation-free environment is beneficial for growth of crystals with higher internal order and in some cases, larger volume. It was however realized that crystal growth in microgravity requires additional time due to slower growth rates. The progress in space research via the International Space Station (ISS) provides a laboratory-like environment to perform convection-free crystallization experiments for an extended time. To obtain detailed insights in macromolecular transport phenomena under microgravity and the assumed reduction of unfavorable impurity incorporation in growing crystals, microgravity and unit gravity control experiments for three different proteins were designed. To determine the quantity of impurity incorporated into crystals, fluorescence-tagged aggregates of the proteins (acting as impurities) were prepared. The recorded fluorescence intensities of the respective crystals reveal reduction in the incorporation of aggregates under microgravity for different aggregate quantities. The experiments and data obtained, provide insights about macromolecular transport in relation to molecular weight of the target proteins, as well as information about associated diffusion behavior and crystal lattice formation. Results suggest one explanation why microgravity-grown protein crystals often exhibit higher quality. Furthermore, results from these experiments can be used to predict which proteins may benefit more from microgravity crystallization.

Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6 3 22, with unit-cell parameters a = 124.25, c = 77.83 Å . There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.