Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Optimization of Crystal Growth for Neutron Macromolecular Crystallography.

The 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.

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

A 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.

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction.

This paper reports a versatile microfluidic chip developed for on-chip crystallization of proteins through the dialysis method and in situ X-ray diffraction experiments. A microfabrication process enabling the integration of regenerated cellulose dialysis membranes between two layers of the microchip is thoroughly described. We also describe a rational approach for optimizing on-chip protein crystallization via chemical composition and temperature control, allowing the crystal size, number and quality to be tailored. Combining optically transparent microfluidics and dialysis provides both precise control over the experiment and reversible exploration of the crystallization conditions. In addition, the materials composing the microfluidic chip were tested for their transparency to X-rays in order to assess their compatibility for in situ diffraction data collection. Background scattering was evaluated using a synchrotron X-ray source and the background noise generated by our microfluidic device was compared to that produced by commercial crystallization plates used for diffraction experiments at room temperature. Once crystals of 3 model proteins (lysozyme, IspE, and insulin) were grown on-chip, the microchip was mounted onto the beamline and partial diffraction data sets were collected in situ from several isomorphous crystals and were merged to a complete data set for structure determination. We therefore propose a robust and inexpensive way to fabricate microchips that cover the whole pipeline from crystal growth to the beam and does not require any handling of the protein crystals prior to the diffraction experiment, allowing the collection of crystallographic data at room temperature for solving the three-dimensional structure of the proteins under study. The results presented here allow serial crystallography experiments on synchrotrons and X-ray lasers under dynamically controllable sample conditions to be observed using the developed microchips.

A crystallization apparatus for temperature-controlled flow-cell dialysis with real-time visualization.

Many instrumentation developments in crystallization have concentrated on massive parallelization assays and reduction of sample volume per experiment to find initial crystallization conditions. Yet improving the size and diffraction quality of the crystals for diffraction studies often requires decoupling of crystal nucleation and growth. This in turn requires the control of variables such as precipitant and protein concentration, equilibration rate, and temperature, which are all difficult parameters to control in the existing setups. The success of the temperature-controlled batch method, originally developed to grow very large crystals for neutron crystallography, demonstrated that the rational optimization of crystal growth has potential in structural biology. A temperature-controlled dialysis button has been developed for our previous device, and 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. The presented approach differs from the current paradigm, since it involves serial instead of parallel experiments, exploring multiple crystallization conditions with the same protein sample. The sample is not consumed in the experiment and the conditions can be changed in a reversible fashion, using dialysis with a flowing precipitant reservoir as well as precise temperature control. The control software allows visualization of the crystals, as well as control of the temperature and composition of the crystallization solution. The rational crystallization optimization strategies presented here allow tailoring of crystal size, morphology and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.