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09:52 min
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February 4th, 2021
DOI :
February 4th, 2021
•0:04
Introduction
0:44
Preparing a Seed Stock of Endothiapepsin Crystals
1:49
Morphogram Experiment
3:14
Morphogram Analysis
4:06
Scaling
5:56
Seeded‐Batch Protocol for 20 μm Endothiapepsin Crystals
7:11
Results: Representative Endothiapepsin Crystal Growth and Analysis
9:16
Conclusion
Transcribir
Serial crystallography experiments can be challenging. A key hurdle in their practice is the creation of microcrystalline samples. This protocol shows how such samples can be reliably created.
This method uses endothiapepsin to give a framework for optimizing large crystals grown in small-volume vapor diffusion plates into large-volume microcrystalline slurries. We cannot claim this protocol will be universally successful. However, we hope that the ideas and methods proposed here give others an experimental framework to employ on other proteins.
To begin, prepare a two-drop 96 well plate with the crystallization buffer using the Formulatrix robot. Using a liquid handling robot mix, mix 150 nanoliters of freshly defrosted endothiapepsin with 150 nanoliters of the crystallization buffer in each well. Seal the plate and allow the crystals to grow for 24 hours inside the Formulatrix Hotel.
After 24 hours, break up and remove the crystals from five wells of the 96-well crystallization plate. Place them in 250 microliters of the crystallization buffer in a 1.5 milliliter centrifuge tube placed in an ice bucket. Add 10 to 15 glass beads of one millimeter size into the centrifuge tube.
Vortex the crystals and beads at 1000 RPM for 30 seconds, and replace them on ice for 30 seconds. The seeds can then be used straight away or stored at minus 20 degrees Celsius. To perform a morphogram experiment, add five to 40%concentrations of PEG 6000 with one molar Tris-HCl at pH seven and 0.15 molar magnesium chloride along the plate columns of a two-drop 96-well grid screen Repair a sequential dilution of endothiapepsin in 0.1 molar sodium acetate from 100 to 12.5 milligrams per milliliter over eight steps.
Then pipette eight microliters of each dilution followed by two microliters of seed stock into the liquid handling robot plate. Using a liquid handling robot, dispense 150 nanoliters of each dilution of endothiapepsin into sub-wells one and two of the screen. Multi-aspirate 50 nanoliters of defrosted seed stock and 100 nanoliters of the well solution from sub-well two and dispense both into the protein solution.
Mix solutions three times upon addition of the crystallization buffer or crystallization buffer and seed mix. Now seal the plate and image at zero, three, six, 12, 18, and 24 hours of the first day every day for the first week and every week for the next four weeks at 20 degrees Celsius. After 24 hours, look at the plate under a microscope, estimate the number of crystals within each well and measure a sample of the crystals.
Record these estimates in the provided morphogram generator worksheet. Input the starting concentrations of endothiapepsin and PEG 6000 into the appropriate boxes. The worksheet will automatically plot the results in the traditional phase diagram format with the precipitant and protein concentrations on the X and Y axis respectively.
Well conditions that only give rise to crystals in their seeded drops indicate the diagram's MetaStable region, whereas conditions with crystals in both the seeded and non-seeded drops indicate the nucleation zone. From the morphogram analysis, select the most promising condition for scaling into 24 well plates. Then begin with 100 milligrams per milliliter of endothiapepsin and 35%PEG 6000.
Prepare five milliliters of crystallization buffer with 35%PEG 6000, 0.1 molar Tris-HCl with pH seven, and 0.15 molar magnesium chloride. Now place 0.5 milliliters of the crystallization buffer in three wells of a greased 24-well hanging drop plate. Mix 15 microliters of endothiapepsin with 15 microliters of crystallization solution on a glass cover slip.
Place the solution over the wells and leave the plate for 24 hours. After 24 hours, look at the plate under a microscope, estimate the number of crystals in the drop, and measure their size. If the crystals are incorrect, repeat this step, but change the protein concentration or add seeds if the crystal nucleation is not high enough.
Repeat the 24-well scaling experiment by adding seeds at a higher concentration of PEG 6000. Place 0.5 milliliters of the new crystallization buffer in three wells of a greased 24-well hanging drop plate. Place 15 microliters of endothiapepsin on a glass cover slip and mix five microliters of seed stock and 10 microliters of crystallization solution with the endothiapepsin solution.
Invert the cover slips, place them over the wells, and leave for 24 hours. Recheck the drops to see if the crystals are smaller and have a higher concentration. Estimate the number of crystals in the drop and measure their size again to see if they are now the correct size.
To perform a seeded-batch protocol, prepare the crystallization buffer of 40%PEG 6000, 0.1 molar Tris-HCl with a pH of seven, and 0.15 molar magnesium chloride. Pre-mix 50 microliters of seed stock and 100 microliters of crystallization solution, and mix with protein solution in the 1.5 milliliter centrifuge tube. Vortex the tube for 10 seconds and place it on a rocker or shaker at 20 degrees Celsius.
Take a 2.5 microliter aliquot of the batch solution every 20 minutes. Monitor the size and number of the crystals using a hemocytometer. Count the number of crystals and measure their size under the microscope.
When the crystals have reached about 15 micron dimension, quench the reaction with 150 microliters of 0.5 molar sodium acetate supplemented with 0.5 molar Tris-HCl, 0.075 molar magnesium chloride, and 20%PEG 6000. Recheck the crystal size and concentration using the hemocytometer. Store the crystals at 20 degrees Celsius.
The endothiapepsin morphogram analysis suggested that nucleation was influenced by both protein and precipitant concentrations. The MetaStable, nucleation, and precipitation regions of the diagram can be nicely distinguished. The addition of seeds significantly increased the crystal number compared to drops without seeds.
Analysis of the endothiapepsin micro crystallization in 200 to 300 microliter volumes revealed the changes in crystal nucleation and the longest dimension over time. Increasing the PEG concentration to 35%markedly improved crystallization, with a final crystal concentration of around 3.6 times 10 to the six per milliliter, and the longest crystal dimension of 42 micrometers. To reduce the size of the final crystals, a protein concentration reduction approach was applied, which unfortunately reduced the crystal concentration significantly, and ultimately produced even larger crystals.
However, the approach of increasing the PEG concentration to 40%yielded crystals around 3.1 times 10 to the six per milliliter and a size of approximately 39 micrometers. Further, adding seeds led to a significant increase in crystal concentration, around 1.1 times 10 to the eight per milliliter and smaller dimensions of approximately 4.2 micrometers. The quenching effect attempted in the crystallization reaction gave a final crystal concentration of around 2.6 times 10 to the six per milliliter and a size of approximately 15 micrometers.
The CC half plotted against the resolution from the x-ray data showed a slight decrease in crystal resolution from the 10 microliter to 200 to 300 microliter volumes. However, the crystal still diffracted to 1.5 angstrum, which was deemed sufficient. Endothiapepsin is a forgiving protein to work with.
We hope that trying this protocol of endothiapepsin will generate useful ideas and methods for other proteins. Particularly with crystallography, it's beneficial to see how things look and feel rather than trying to only follow a written protocol. This video will hopefully give you as some of that feel.
The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.
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