Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

Hello, and welcome to the laboratory architecture and reactivity of RNA at the Institute of Molecular and Cell Biology in Strasbourg. Growing well-defracting crystals is a critical step in any crystallographic study. In this video, we will demonstrate the use of a new microfluidic tool to grow crystals of a biomolecule and to determine its 3D structure by in situ X-ray diffraction.

The microfluidic device called ChipX presents several advantages. It is designed to miniaturize and facilitate crystallization assets by the method of counter-diffusion. Its set up is easy and performed with standard lab material.

It is also optimized for the direct characterization of crystals grown in the chip by serial X-ray crystallography. ChipX eliminates crystal handling and cryo-cooling steps and preserves the intrinsic quality of the crystals which are analyzed in situ and at room temperature. ChipX has the format of a microscope slide, 7.5 by 2.5 centimeters.

It contains eight microfluidic channels used as crystallization chambers with a section of 80 by 18 micrometres and a length of four centimeters. The inlet on the left-hand side allows the manual injection of the biomolecule solution which will fill the eight channels up to the reservoirs. Crystallization solutions are deposited in these reservoirs on the right-hand side.

The crystallization is triggered by a phenomenon called counter diffusion. When the crystallizing agent diffuses into the biomolecule solution and creates a concentration and a super saturation gradient. Labels and bost along the channels help locate the crystals more easily.

Setting up a chip requires five to six microliters of biomolecule solution at a typical concentration of five to 10 milligrams per milliliter. One microlitre paraffin oil, five microliters of crystallization solution per reservoir and adhesive tape. The loading of ChipX is performed manually with a micropipette and standard tips.

A blue sample solution is used in this example for a better visualization of the injection process. To avoid leakage, the tip is inserted in the sample inlet perpendicularly to the chip. One microliter of paraffin oil is loaded after the sample to separate the channels from each other.

The sample inlet is then sealed with tape. The eight reservoirs are loaded individually with five microliters of crystallizing agent solution. To do so, the pipette tip is placed close to the end of the channel at an angle of about 45 degrees.

This prevents the formation of an air bubble between the sample and the crystallizing agent. Reservoirs can be filled with cocktails containing various buffers, polymers or salts, et cetera, for screening or optimization. Reservoirs are sealed with tape.

As demonstrated here, setting up a chip is easy and takes only a few minutes. In ChipX, crystallization occurs by counter diffusion. Crystallizing agents deposited in the reservoirs diffuse into the channels containing the biomolecule.

This creates gradients of concentration and of super saturation which will trigger crystallization. These are examples of crystals of an enzyme obtained inside the channels. They may grow to fill the channels which have a cross section of 80 by 80 micrometers.

Crystals are easily detectable in ChipX by fluorescence microscopy using the natural fluorescence of tryptophan residues under UV light, or as shown here using a fluorescently labeled protein. This section shows a Chris telegraphic analysis performed at Swiss light source in filaggrins Switzerland. The chips can be carried to the synchrotron without any additional equipment or special care.

Let's enter the hall of Swiss light source. Electrons circulate in the central ring and generate a wide spectrum of electromagnetic waves, including x-rays exploited at different beamlines. This video was recorded at X06, which is a beamline dedicated to macro-molecular crystallography.

The protocol may change from one synchrotron facility to the other, but the overall principle remains the same. ChipX is mounted on a 3D printed holder. This holder is then attached to the magnet of a standard goniometer.

The serial analysis consistent collecting data from a series of crystals grown in the chip. Lists of crystal positions are established using the labels and bost along the chip channels. Researchers move the chip to center the crystals in the x-ray beam symbolized by the yellow window.

A fast centering procedure using the x-ray beam is launched. The beam stop goes up into place and the detector moves towards the sample. The grid screening is performed to ensure that the crystal is aligned with the beam.

The same procedure is repeated after rotating the chip by 30 degrees. The second step is important because the chip material creates a parallax effect which leads to a shift of the diffraction maximum with respect to the observed crystal position. As soon as the crystal is centered in the beam the data collection starts.

This sequence shows data collection in real time. The chip rotates by 30 degrees. Meanwhile, corresponding diffraction images are collected by the detector.

The characterization of this crystal is complete. The chip translated to the next crystal in the channel and the whole procedure is repeated. Note that this analysis is operated in situ without direct handling of the crystal and at room temperature.

Data collected on a series of crystals are then merged to obtain a complete diffraction dataset that is used to compute an electron density map, shown in blue, and to build the atomic model. The procedure described in this video was applied in the frame of the structural characterization of the tRNA modification enzyme. The CCA-adding enzyme from the cold adapted bacterium, Planococcus halocryophilus.

ChipX was used to crystallize the enzyme by counter diffusion in the presence of ammonium sulfide as a crystallizing agent. By-pyramidal crystals appeared along the channels, after a few days of incubation at 20 degrees Celsius. The fluorescent labeling of the protein facilitated the identification of protein crystals and their discrimination from salt crystals.

A series of crystals was analyzed in situ and at room temperature. Their diffraction data were merged and led to the crystal structure of the APOE enzyme at a resolution of 2.5 angstrom. In addition, the diffuse of environment in chip channels was exploited to deliver a substrate to the enzyme that builds up the crystals.

In the present case, a CTP analog was added to the reservoir solutions two days before the synchrotron analysis. This allowed the compound to diffuse and reach the catalytic site of the enzyme. As seen in the crystal structure of the complex determined at a resolution of 2.3 angstrom.

The protocol demonstrated in this video is generally applicable and has been tested on a variety of biomolecules. To conclude ChipX is a Lab chip tool that integrates all steps of a crystallographic study and allows you to go from the biomedical solution to the crystal structure in a unique microfluidic device. Thank you for watching this video.

and we hope we have convinced you of the benefits of using ChipX to crystallize and determine the crystal structure of your favorites by Biomolecule.