Controlling one protein crystal growth by droplet-based microfluidic system (original) (raw)
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Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies
Journal of Visualized Experiments, 2021
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 cutoff 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.
IUCrJ, 2014
An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.
Protein crystallization: from purified protein to diffraction-quality crystal
Nature Methods, 2008
Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth.
Growth and Characterization of High-quality Protein Crystals for X-ray Crystallography
Annals of the New York Academy of Sciences, 2009
Tetragonal hen egg white lysozyme is grown by the batch method in solution and gel media to study the influence of high magnetic fields on the quality of macromolecular crystals. The crystallographic quality of crystals grown in the absence and in the presence of 7-and 10-T fields are analyzed in terms of mosaicity and high-resolution X-ray imaging methods. Crystals grown by the batch method from solution showed a remarkable enhancement of the crystallographic quality, although the overall crystal quality was higher for gel-grown crystals than solution-grown crystals. The observed improvement in crystal quality can be attributed to the suppression of convective transport during the crystal growth process and the control of the nucleation kinetics by the use of a magnetic force.
In situ data collection and structure refinement from microcapillary protein crystallization
Journal of Applied Crystallography, 2005
In situX-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained fromin situdiffraction of crystals grown in microcapillaries.
Post-crystallization treatments for improving diffraction quality of protein crystals
Acta Crystallographica Section D Biological Crystallography, 2005
X-ray crystallography is the most powerful method for determining the three-dimensional structure of biological macromolecules. One of the major obstacles in the process is the production of high-quality crystals for structure determination. All too often, crystals are produced that are of poor quality and are unsuitable for diffraction studies. This review provides a compilation of post-crystallization methods that can convert poorly diffracting crystals into data-quality crystals. Protocols for annealing, dehydration, soaking and cross-linking are outlined and examples of some spectacular changes in crystal quality are provided. The protocols are easily incorporated into the structure-determination pipeline and a practical guide is provided that shows how and when to use the different post-crystallization treatments for improving crystal quality.
An Overview of Biological Macromolecule Crystallization
International Journal of Molecular Sciences, 2013
The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.
Crystallization of soluble proteins in vapor diffusion for x-ray crystallography
Nature Protocols, 2007
The preparation of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biological macromolecule. The complete automation of the crystallization procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. On the other hand, many research projects from departmental laboratories are devoted to the study of few specific proteins. Here, we try to provide a series of protocols for the crystallization of soluble proteins, especially the difficult ones, tailored for small-scale research groups. An estimate of the time needed to complete each of the steps described can be found at the end of each section.
High Resolution Protein Crystals Using an Efficient Convection-Free Geometry
Crystal Growth & Design, 2013
Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the "ceiling" of a growth-cell completely filled with the crystallization solution. The "ceiling crystals" experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optimization. In addition, we demonstrate that the ceiling crystallization method leads to crystals with much lower impurity incorporation.