Fermín Otálora

Counterdiffusion methods applied to protein crystallization.

Accumulated experience during the last years on counterdiffusion crystallization methods shows that they are a convenient and generally applicable way of optimizing solution crystal growth experiments. Irrespective of whether the objective of the experiment is to improve crystal quality or size, many experiments reporting a positive or neutral effect of counterdiffusion exists, but adverse effects are consistently absent. Thus counterdiffusion is viewed as a rational crystallization approach to minimize supersaturation and impurity levels at the crystal growth front and to ensure steadiness of both values. This control of the phase transition state is automatically achieved and sustained by a dynamic equilibrium between mass transport and aggregation kinetics. The course of this function can be implemented in any media permitting diffusive mass transport (gels, capillaries, microfluidic devices or microgravity). The counterdiffusion technique has been exploited in many recent applications revealing interesting effects on nucleation and polymorphic precipitation, hence opening further possibilities for innovative screening of crystallization conditions.

A supersaturation wave of protein crystallization

A microgravity protein crystallization experiment is described in which the existence of a supersaturation wave traveling across a diffusion-reaction system is experimentally demonstrated for the first time. The self-organized dynamics of the experimental setup were used to implement a crystallization technique able to search automatically through the crystallization parameter space for optimum nucleation and growth conditions. The crystals obtained by this automatic optimization produced the highest quality X-ray diffraction data ever collected from the model protein used in the experiment.

DENSITY-DEPENDENT AGE OF FIRST REPRODUCTION AS A BUFFER AFFECTING PERSISTENCE OF SMALL POPULATIONS

We documented a density-dependent age of first reproduction using a pop- ulation of Spanish Imperial Eagles (Aquila adalberti )i n Donana National Park (south- western Spain). Then, a stochastic individual-based simulation was designed to analyze the effect of variations in the age of first reproduction on the fluctuation of small populations. This is a discrete-time simulation model that uses the age structure, mortality rates, repro- ductive rate, and density-dependent relationships from empirical studies. Age of breeding in the real population was positively related to population density. The probability of persistence of simulated populations was affected by both breeding population ceiling and age of first breeding. Our simulations were compared to the historical evolution of the Donana population, assuming an age of first reproduction of three years. Density-dependent variation in age of first breeding increased the mean time of persistence, allowing the population to stay at carrying capacity longer. This buffer mechanism could be crucial for the persistence of small populations.

Formation of natural gypsum megacrystals in Naica, Mexico

Exploration in the Naica mine (Chihuahua, Mexico) recently unveiled several caves containing giant, faceted, and transparent single crystals of gypsum (CaSO4•2H2O) as long as 11 m. These large crystals form at very low supersaturation. The problem is to explain how proper geochemical conditions can be sustained for a long time without large fluctuations that would trigger substantial nucleation. Fluid inclusion analyses show that the crystals grew from low-salinity solutions at a temperature of ∼54 °C, slightly below the one at which the solubility of anhydrite equals that of gypsum. Sulfur and oxygen isotopic compositions of gypsum crystals are compatible with growth from solutions resulting from dissolution of anhydrite previously precipitated during late hydrothermal mineralization, suggesting that these megacrystals formed by a self-feeding mechanism driven by a solution-mediated, anhydrite-gypsum phase transition. Nucleation kinetics calculations based on laboratory data show that this mechanism can account for the formation of these giant crystals, yet only when operating within the very narrow range of temperature identified by our fluid inclusion study. These singular conditions create a mineral wonderland, a site of scientific interest, and an extraordinary phenomenon worthy of preservation.

Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques.

Granada Crystallisation Box (GCB) is a new crystallisation device designed to perform counter-diffusion experiments. Here we describe the device and its use for protein crystallisation purposes. GCB allows one to explore and exploit the coupling between crystallisation and diffusion. The role of viscous fluids, gels and/or microgravity can be enhanced by using capillary volumes, creating a perfect diffusive mass transport scenario. The use of capillaries also reduces the consumption of macromolecules and avoids the handling of crystals for X-ray diffraction data collection.

Environmental stochasticity in dispersal areas can explain the ‘mysterious’ disappearance of breeding populations

We present the results of an individual-based simulation model, showing that increasing the mortality of non-breeding dispersers within settlement areas can lead to the extinction of species and (meta)populations in a subtle way. This is because the areas where dispersers settle are generally unknown or difficult to detect. Consequently, fewer efforts are devoted to the conservation of these sites than to the conservation of breeding territories. Additionally, high mortality rates affecting the floater sector of a population become evident in the breeding sector only after several of years, when it is too difficult or too late to halt the decline. As a result, because most conservation projects on endangered species and populations mainly focus on breeding areas, many current efforts may be wasted in locations other than those in which conservation would be really necessary and effective.

Topography and high resolution diffraction studies in tetragonal lysozyme

Abstract Two complementary approaches are used to enhance the usefulness of X-ray topographies obtained from protein crystals. First, the use of thin plate-like crystals in conjunction with a high intensity, collimated and small source size synchrotron beam produces a large beneficial effect on the level of detail and contrast of topographies for the quantification of local misalignment in the crystal lattice. Second, the recording of topography series along the rocking curve of a diffraction peak is proposed as a technique to combine the benefits of both rocking curves and topographies and produce very detailed data (“rocking maps”) on the spatial distribution of lattice misalignments and mosaic spread (“local rocking curves”). The most important crystal features controlling the observed contrast are growth sectors and inter-sector boundaries, clearly observed in the topographies. Systems of parallel fringes are observed in many of the topographies. Two alternative explanations for these fringes are discussed: (a) as moire interference fringes or (b) as Pendellosung fringes in a wedge shaped crystal volume. In both cases, growth sectors play a central role in the physics of fringe generation. Many observations suggest the presence of a relatively large component of dynamical diffraction in these crystals; the consequences of this new scenario are discussed.

Reinforced protein crystals

Large tetragonal hen egg white lysozyme single crystals (up to 16 mm 3 ) can be obtained by the counter-diffusion method, using high concentration silica gels. The protein crystal lattice is able to incorporate large amounts of silica while still maintaining its short-range crystallographic order. The crystal morphology is controlled by the concentration of the silica gel, which can reduce surface energy anisotropy to such an extent that spherical single crystals can be obtained as growth forms. The mechanical properties and the stability of the crystals against dehydration are improved by the incorporated hydrophilic silica polymeric network. This makes it possible to record full diffraction data sets with a resolution better than 1.5 A from crystals glued to glass fibers. Such reinforcement of the crystals facilitates their handling at ambient conditions and opens new possibilities for the measurement of physical properties of large biological macromolecules as well as for their technological applications.

Crystallization and cryocrystallography inside X-ray capillaries

This paper presents a modification of the gel acupuncture method to grow isolated crystals inside X-ray capillaries. Protein crystals are grown from 2–12 µl of gelled agarose–protein solution, cryoprotected and immobilized by the gel matrix. The same X-ray capillary that acts as a crystallization reactor is used to transport the crystals to the X-ray source and to collect data at both room temperature and 100 K, without any post-crystallization manipulation. To enhance the flash-cooling stage, two additional elements are proposed for inclusion in the cryosystems currently in use: a laser pointer to illuminate the crystal to be flash-cooled and a trap to divert the N2 flow and switch from room temperature to 100 K without misalignment of the crystal. With the proposed implementation, data can be collected at different temperatures from the same crystal in exactly the same orientation. This permits the study, at lattice level, of changes in unit-cell parameters, mosaic spread and crystal quality induced by cryogenic temperatures and annealing techniques.

Crystal growth studies in microgravity with the APCF. I. Computer simulation of transport dynamics

Abstract A computer program has been designed to simulate the pre-nucleation history of protein crystal growth experiments in the Advanced Protein Crystallization Facility developed by ESA to conduct crystallization experiments under microgravity conditions. The program was fed with the initial conditions of a set of experiments performed by the ESA Diagnostic Group during the SpaceHab-01 mission. The time histories of the salt and protein concentrations and supersaturation at any point inside the reactor were obtained. The differences between Dialysis and Free Interface Diffusion techniques are discussed. Both DIA and FID expriments performed in the short protein chamber of the APCF are basically very slow mixing batch experiments although differences exist in the equilibration behavior. The kinetics of protein nucleation coupled with transport phenomena are analyzed, showing that the supersaturation rate modifies the supersaturation value at which nucleation occurs as well as waiting times for nucleation. Reactor geometry and initial conditions can be optimized. As a general rule, higher protein concentrations will produce crystals that nucleate at lower supersaturation and supersaturation rates, provided that the growth chamber is long enough to implement a true diffusive set-up. When these conditions are fulfilled, the growth system becomes self regulated and crystals nucleate under different supersaturation and supersaturation rate conditions, performing a kind of automatic, single-reactor screening experiment.