Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

This is a crystallization protocol designed to optimize the growth of a protein crystal while simultaneously derivatizing the protein with a phasing molecule to determine the structure of a protein by x-ray crystallography. To determine the x-ray crystal structure of a completely novel protein, crystallization conditions are optimized to produce quality crystals. In the case of a completely novel protein, heavy atoms need to be introduced into the crystal to produce a derivatized crystal.

X-ray diffraction data is collected from the derivatized crystal and has to be computationally solved to produce an x-ray crystal structure. Crystallization and derivitization are both laborious processes. To address these bottlenecks, we have devised an optimized screening procedure that simultaneously achieves both of these objectives.

This protocol uses a previously developed technique known as random microseed matrix screening. Crystals or crystalline precipitate is crushed up into microseeds which can be used to start crystal growth in completely independent conditions. At the same time, we introduce a phasing molecule called I3C into the crystallization condition.

This molecule provides a large anomalous signal which allows the structure to be solved by a single wavelength anomalous discussion phasing. For users unfamiliar with experimental sizing of crystal structures we present a user-friendly software pipeline to partially automate the structure solution tailoring it to use the anomalous signal from I3C. The first step is to create an I3C stock.

Measure out 120 milligrams of I3C into a microcentrifuge tube. Add 200 microliters of two molar lithium hydroxide to the microcentrifuge tube. Heating the tube at 40 to 60 degrees and vortexing can be used to encouraged dissolution.

The one molar stock of lithium I3C solution should be brown. Two methods can be used to introduce I3C to the protein stock. Lithium I3C can be added directly to the protein stock.

3.75 microliters to 30 microliters of I3C can be added to 150 microliters of protein to give a concentration between five and 14 millimolar. Some proteins can precipitate upon contact with high concentrations of I3C. In these cases, lithium I3C can be added to a protein dilution buffer that is buffer matched the sample protein to a final concentration between 10 to 80 millimolar.

Add 150 microliters of the protein dilution buffer to 150 microliters of protein sample. A glass probe to crush crystals is made from a glass Pasteur pipette. With a Bunsen burner on the blue flame, heat the Pasteur pipette towards the middle.

Using a pair of tweezers, pull the ends of the Pasteur pipette out into a thin diameter of less than 0.3 millimeters. Hold that segment in the flame to separate the pipette at this point and round the end of the pipette to finish the glass probe. This is an example of how the glass pipette should look.

Crystal crushes might also be ordered from third party vendors as an alternative to making your own A microseed stock is generated by crushing up protein crystals or crystalline precipitate. Place five 1.5 mL microcentrifuge tubes on ice. Under a light microscope, select the best morphology crystals or crystalline precipitate that can be sacrificed.

For 96 well crystallization trays open the well by cutting the plastic sealing tape. For hanging drop trays, the cover slip can be removed using tweezers and inverted onto a flat surface. Transfer 70 microliters of reservoir solution to one of the microcentrifuge tubes and chill on ice.

To the remaining tubes, transfer 90 microliters of reservoir solution to it and return to ice to chill. If insufficient reservoir volume is present crystallization reservoir can be made by mixing the appropriate reagents and can be used instead. Agitate the crystals in the crystallization drop using the glass probe thoroughly crush it up.

Progress can be monitored under the microscope until no crystals or crystalline material is visible. Transfer all of the liquid from the drop to the microcentrifuge tube containing 70 microliters of reservoir solution. Mix by pipetting up and down.

Transfer two to three microliters of mixture back to the well and rinse the well by pipetting up and down. Transfer the mixture back to the microcentrifuge tube. This rinse step should be repeated once more.

The microcentrifuge tube should be kept cold from this point forward to avoid melting the microseeds. Vortex the tube at maximum speed at four degrees for about three minutes chilling the tube on ice regularly to prevent overheating. make one in 10 serial dilutions of the seed stock by sequentially transferring 10 microliters between the chilled reservoir solutions.

Between seed stock dilutions, the tube should be vortexed. Seed stocks that won't be immediately used, should be stored in a minus 80 degree ultra cold freezer. A 96 well random microseed matrix screen can be set up using a liquid dispensing robot.

Place the seed stock, protein stock solution supplemented with I3C and crystallization screen in the robot. Using the robot transfer 75 microliters from the crystallization screen to the 96 well tray. Add one microliter to the crystallization drop and 74 microliters to the reservoir.

Transfer one microliter of protein supplemented with I3C to the crystallization drop. Transfer 0.1 microliters of seed stock to the crystallization drop. Seal the plate using sealing tape.

Hanging drop screens can be set up in 24 well hanging drop crystallization trays. Grease the edges of the hanging drop wells. Transfer 500 microliters of crystallization solution to the reservoir.

Near the center of a glass cover slide place a one microliter drop of crystallization solution. To this drop, add one microliter of protein supplemented with lithium I3C and 0.1 microliters of seed stock. Invert the cover slide and seal the crystallization well by pushing the cover slide into the grease.

Hanging drop and sealing drop trays are incubated at a constant temperature to allow crystals to form. They should be regularly inspected under a microscope for crystal growth. After diffraction data has been obtained, integrated, and scaled as explained in the accompanied written protocol, the Auto-Rickshaw automated crystal structure determination pipeline can be used to solve the phase problem and model the protein.

At the Auto-Rickshaw landing page, click the proceed button. Enter your institutional email in the Auto-Rickshaw web form and click proceed with running Auto-Rickshaw. For proteins without a homology moto template run the SAD protocol of Auto-Rickshaw in advanced mode.

Enter the required parameters. Select protein as the molecule type. Enter the data collection wavelength in angstroms.

So like I as substructure element to indicate iodine atoms were used. Select I3C substructure type to indicate I3C was the phasing molecule. Select sub_direct as the substructure determination method.

Select three as the number of expected substructures per monomer. Enter one as the reservation cutoff of substructure search. This allows Auto-Rickshaw to automatically determine a suitable resolution cutoff.

Enter the number of residues in a single monomer, space group of the data set and number of molecules in the asymmetric unit based on the Matthews coefficient. Select the appropriate dissemination level of x-ray data that suits your needs. Input the anomalous data as an empty set file.

Input your protein sequence as an SEQ, PIR, or TXT file. Enter your institutional email address. Results are delivered to you via a web link sent to the email address provided.

If Auto-Rickshaw fails to solve the structure using its determined substructure validating the substructure could aid in troubleshooting structure solution. Download the list of heavy atoms sites from the Auto-Rickshaw results page. It is a hyperlink called heavy atom sites.

This will download a text file with the heavy atom sites. Change the file extension of the file from txt to pdb. Open the pdb file in Coot.

Turn on symmetry to see all the heavy atoms from neighboring asymmetric units. Measure the distances between the heavy atoms, including across asymmetric units. I3C will appear as an equilateral triangle with a side length of six angstroms.

The presence of a triangle with these dimensions indicates the placements of those heavy atoms are correct. If the Auto-Rickshaw run substructure is incorrect, other Auto-Rickshaw settings can be tested as discussed in the written protocol. The I3C RMMS method was tested on two proteins, hen egg white lysozyme using the in HT screen from Hampton Research and the domain of the Orf11 lysin protein from bacteriophage P68.

Full screens were set up for each protein corresponding to the control screen without I3C or microseed, screen with microseed added, screen with I3C, and finally the screen with I3C and microseed. Adding I3C to a crystal screen does not appear to increase the number of crystallization hits. With hen egg white lysozyme, the number of conditions fell from 31 to 26.

With the Orf11 domain a single hit condition was found in the control screen. Adding I3C to the screen also gave a single hit in the same conditions. Adding microseed to the well resulted in a significant increase in the number of hit conditions resulting in a 2.1 and six fold increase for hen egg white lysozyme and the Orf11 domain respectively.

This result is consistent with other studies testing RMMS. Most importantly, adding I3C and microseed to a screen also increased the number of hit conditions relative to the control screen demonstrating a 2.3 and seven fold increase for hen egg white lysozyme and the Orf11 domain respectively. This demonstrates that the I3C RMMS method can efficiently produce new conditions for derivatized crystals.

Crystals from these conditions could be harvested and used to solve the crystal structure. Both the structure of hen egg white lysozyme Orf11 domain could be solved with SAD phasing on the Auto-Rickshaw pipeline, using the anomalous signal from I3C demonstrating successful derivitization by the I3C RMMS screen. We have presented a simple and efficient protocol to produce high quality crystals derivatized with the phasing molecule I3C.

It minimizes the number of screens and crystalline handling steps, and thus is of particular interest to structural biologists studying novel proteins. We strongly recommend optimizing crystal size by testing different seed stock dilutions which were made during seed stock preparation. And this step is completed after initial screens have been done.

I3C is widely available and it's inexpensive to purchase and thus we believe this method is within the reach of most structural biology laboratories.