Marine mammals as indicators of Anthropocene Ocean Health.

The current state of marine mammal populations reflects increasing anthropogenic impacts on the global Ocean. Adopting a holistic approach towards marine mammal health, incorporating healthy individuals and healthy populations, these taxa present indicators of the health of the overall Ocean system. Their present deterioration at the animal, population and ecosystem level has implications for human health and the global system. In the Anthropocene, multiple planetary boundaries have already been exceeded, and quiet tipping points in the Ocean may present further uncertainties. Long and short-term monitoring of marine mammal health in the holistic sense is urgently required to assist in evaluating and reversing the impact on Ocean Health and aid in climate change mitigation.

The suitability of a monofunctional reagent of an undecagold cluster for phasing data collected from the large ribosomal subunits from Bacillus stearothermophilus.

An electron density map of the large ribosomal subunit from Bacillus stearothermophilus was obtained at 26 A resolution by single isomorphous replacement (SIR) from a derivative formed by specific quantitative labeling with a dense undecagold cluster. For derivatization, a monofunctional reagent of this cluster was bound to a sulfhydryl group of a purified ribosomal protein, which was in turn reconstituted with core particles of a mutant lacking this protein. The native, mutated, and derivatized 50S ribosomal subunits crystallize under the same conditions in the same space group. Under favorable conditions, crystals of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different packing. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the noncrystallographic symmetry found by Patterson searches as well as with the motif observed in electron micrographs of thin sections of the crystals. These studies established that phase information can be obtained from heavy metal clusters, even when the crystals under investigation are unstable and weakly diffracting. These results encouraged further effort at the construction of specifically derivatized crystals from other ribosomal particles that diffract to higher resolution.

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis.

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

The growth of ordered two-dimensional sheets of ribosomal particles from salt-alcohol mixtures.

A procedure for the in vitro growth of well-ordered two-dimensional sheets from ribosomal particles using salts and salt-alcohol mixtures has been developed. Employing this procedure, ordered two-dimensional sheets of the wild type as well as of mutated 50 S ribosomal subunits from Bacillus stearothermophilus can readily be obtained. These sheets, stained with uranyl acetate or gold-thioglucose, are suitable for three-dimensional image reconstruction. They consist of relatively small unit cells with dimensions of 160 +/- 15 and 365 +/- 20 A. Diffraction patterns of electron micrographs of these sheets contain features to 25 A resolution.

Reconstitution and crystallisation experiments with isolated split proteins from Bacillus stearothermophilus ribosomes.

Six proteins (B-L1, B-L6, B-L10, B-L11, B-L12 and B-L16) were removed from 50S ribosomal subunits of Bacillus stearothermophilus by treatment with ethanol and ammonium chloride. The proteins were isolated in a pure form, and one of them (B-L6) was crystallized. Five of the six proteins (in various combinations) were added back to the core particles, resulting in 50S subunits lacking one protein. The biological activities of these ribosomal particles as determined in the poly(U)-system varied over a wide range, depending on the protein which was omitted. The particles lacking one protein provide useful tools for heavy-atom derivation necessary for our crystallographic studies on the 50S subunits of Bacillus stearothermophilus.

Characterization of single crystals of the large ribosomal particles from Bacillus stearothermophilus.

Single, three-dimensional crystals of the 50 S ribosomal subunit from Bacillus stearothermophilus (strain NCA) have been characterized using a synchrotron X-ray source. The crystals are orthorhombic with unit cell dimensions: a = 350 A, b = 670 A, c = 905 A, and contain at least one 2-fold screw axis. With cooling to -2 degrees C, the large crystals (1.0 mm X 0.2 mm X 0.1 mm) diffract to 15 to 18 A resolution and are stable in the synchrotron beam for several hours. Despite the large cell dimensions, the reflections are readily resolved when the X-ray diffraction patterns are densitometered with a 25 microns faster.

A compact three-dimensional crystal form of the large ribosomal subunit from Bacillus stearothermophilus.

A new form of well-ordered three-dimensional crystals of intact 50 S ribosomal subunits from Bacillus stearothermophilus have been obtained. Electron micrographs of positively stained sections of these crystals revealed that the ribosomal particles are packed closely. The cell parameters have been determined. Representative electron micrographs and their computed contoured filtered images are shown.

Parameters for crystal growth of ribosomal subunits.

A systematic analysis of the parameters that control the crystal growth of the large subunit of ribosomes from B stearothermophilus has been carried out. Several parameters have been identified and classified according to their significance. It was found that only biologically active particles can crystallize and that the critical period for the crystallization process is the first few days, during which changes in the volume and content of the crystallization drop are of importance for both nucleation and crystal growth. Consequently, an experimental procedure for fine control of these variables has been developed. As a result of these studies, the reproducibility of crystal formation was increased, and larger and more stable crystals have been obtained.