Neutron Macromolecular Crystallography for Biological Samples—Current State and Future Perspectives

International audienceKnowledge of hydrogen locations and protonation states is critical for a fundamental understanding of biological macromolecular function/interactions, and neutron macromolecular crystallography (NMX) is uniquely suited among the experimental structural-determination methods to provide this information. However, despite its potential, NMX remains a relatively niche technique, due to substantial limitations. This review explores NMX’s role amongst the evolving landscape of structural biology, comparing and contrasting it to the historical gold standard of X-ray macromolecular crystallography (X-ray MX) and the increasingly prevalent electron-based methods—i.e., electron microscopy (EM) and electron diffraction (ED). Forthcoming developments (e.g., the European Spallation Source in Lund, Sweden, coming online) are expected to substantially address current limitations and ensure NMX will remain relevant in the coming decades

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

International audienceThe use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand. In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams

Microdialysis on-chip crystallization of soluble and membrane proteins with the MicroCrys platform and in situ X-ray diffraction case studies

The MicroCrys platform was developed to automate on-chip dialysis crystallization of proteins via chemical composition and temperature control, facilitating the optimization of crystallization conditions and the exploration of protein phase diagrams

Mixing Salts and Poly(ethylene glycol) into Protein Solutions: The Effects of Diffusion across Semipermeable Membranes and of Convection

International audienceGrowing a protein crystal starts by mixing a solution of the protein, with a solution of a precipitant—such as a salt or poly(ethylene glycol) (PEG). Mixing two solutions is a surprisingly complex process, but this complexity has not received much attention by those crystallizing proteins, despite crystallization being notoriously sensitive to solution conditions. We combine experimental data with modeling to improve our ability to predict mixing time scales for conditions typical of protein crystallization. We look at the effects of convection and of diffusion through semipermeable membranes. Our experiments are with dialysis chambers, where the crystallization chamber is separated from a precipitant reservoir by a semipermeable membrane. This membrane slows mixing down by factors that vary from ten, for smaller PEG and salts, to a hundred, for dilute larger PEG. This agrees with our model prediction that for larger polymers diffusion through the membrane is sensitive to both molecular weight and concentration. Both salt and PEG solutions are denser than dilute protein solutions, and this drives convection, which accelerates mixing. Convection is flow due to gravity acting on mass density differences. We show how to determine when convection occurs and how to estimate its effect on mixing times

Investigation of aprotinin (BPTI) solutions during nucleation

International audienceA combination of small angle X-ray scattering (SAXS) and gel techniques was used to follow the kinetics of aprotinin (BPTI) nucleation and crystal growth induced in NaCl solutions by increasing temperature. These experiments were carried out in order to determine if aggregation occurs in the solution during crystal growth. From these first series of experiments, we found a radius of gyration of 24.7 Å for intermediates species in solution, consistent with a mixture of decamers and monomers. Once formed, BPTI crystals obtained at acidic pH in NaCl kept this association and it is the decamer, which was found in the crystal packing in contrast to what happened at basic pH where only monomers are observed in the crystal

Comparison of solubility and interactions of aprotinin (BPTI) solutions in H2_2O and D2_2O

International audienceSmall-angle neutron scattering experiments are often performed with proteins solubilized in heavy water because of the large difference in neutron scattering properties of protons and deuterons. In order to characterize the effect of D$_2$O on physico-chemical properties of protein solutions, we investigated the effect of D$_2$O on the phase diagram and the interactions of bovine pancreatic trypsin inhibitor (BPTI) in solution. We measured the solubility in D$_2$O of BPTI solutions in the presence of NaCl (reverse solubility) and KSCN (direct solubility) and compared with the values measured by Lafont et al. in H$_2$O under the same conditions [Lafont et al., J. Crystal Growth 173 (1997) 132]. In the two salts, we found that BPTI solubility in D$_2$O is significantly lower than in H$_2$O. The curves representing the solubility of BPTI in KSCN are shifted by 7.2°C between light and heavy water, a shift obtained previously with lysozyme and representing the difference in the temperature of maximum density of both types of water [Gripon et al., J. Crystal Growth 177 (1997) 238; 178 (1997) 575]. In the case of BPTI in NaCl, we did not find this relationship between the solubility in H$_2$O and D$_2$O. We found, by dynamic light scattering, that BPTI attractive intermolecular interactions in the presence of NaCl in D$_2$O are significantly stronger than in H$_2$O. We investigated the association of BPTI molecules in crystallization conditions in the presence of NaCl in H$_2$O and D$_2$O by small-angle X-ray and neutrons scattering, respectively. In the presence of heavy water, the transition monomer–multimer is observed at about 2 mg/ml of BPTI in 1 M NaCl whereas in light water and in 1.4 M NaCl solution this transition is observed at about 15 mg/ml. These results clearly showed that BPTI in crystallization conditions is a multimer and confirm the importance of the isotopic nature of water in the crystallization of proteins. The replacement of H$_2$O by D$_2$O decreases the solubility and increases the attractive intermolecular interactions

Optimization of crystallization of biological macromolecules using dialysis combined with temperature control

International audienceA rational way to find the appropriate conditions to grow crystal samples for bio-crystallography is to determine the crystallization phase diagram, which allows precise control of the parameters affecting the crystal growth process. First, the nucleation is induced at supersaturated conditions close to the solubility boundary between the nucleation and metastable regions. Then, crystal growth is further achieved in the metastable zone – which is the optimal location for slow and ordered crystal expansion – by modulation of specific physical parameters. Recently, a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature–precipitant–concentration phase diagrams has been constructed. Here, it is demonstrated that a thorough knowledge of the phase diagram is vital in any crystallization experiment. The relevance of the selection of the starting position and the kinetic pathway undertaken in controlling most of the final properties of the synthesized crystals is shown. The rational crystallization optimization strategies developed and presented here allow tailoring of crystal size and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination

Crystallization of a recombinant form of the complete sequence of human γ\gamma-interferon: characterization by small-angle X-ray scattering, mass spectrometry and preliminary X-ray diffraction studies

International audienceThe crystallization conditions of a recombinant form of the complete sequence of human $\gamma$-interferon, designated r-hu IFN-$\gamma$ (RU 42369), have been determined after studying the behaviour of this protein in solution by small-angle X-ray scattering (SAXS) as a function of pH and salt type. IFN-$\gamma$ is difficult to crystallize without truncating at least the last five amino acids of the C-terminus; the SAXS results suggest viable crystallization conditions that led to crystals of r-hu IFN-$\gamma$ suitable for X-ray diffraction analysis. The crystals were grown in the presence of ammonium sulfate using vapour-diffusion techniques. The crystals, which diffract to 5 Å resolution at best, belong to the primitive tetragonal space group P42$_1$2 and have unit-cell parameters a = b = 123.4, c = 93.4 Å. The protein contained in these crystals was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which verified the presence of the complete amino-acid sequence of r-­hu IFN-$\gamma$