Procedure for making the microseed stock

 

·         Any crystalline protein material can be used for microseeding, including fine needles, “spherulites”, microcrystals, and irregular poorly-formed crystals. 

·         Note that the microcrystals in the seed stock are not stable because the seed stock contains very little protein.  Therefore the seed stock should be kept on ice and frozen as soon as possible, preferably at -80ºC.

·         If you have plenty of crystals, use the Seed Bead from Hampton Research, HR2-320, see http://hamptonresearch.com . 

·         If you have only a few small crystals, modify the procedure below by crushing the crystals with a probe and making a smaller volume of seed stock (e.g. 15 µl) without using the Seed Bead.

 

The method is based on the method of D’Arcy et al., adapted from Luft and DeTitta, references below.

 

 

1.       Put a Seed Bead (in its test-tube) into a bucket of ice to cool it.

2.       Open the well containing seed crystals, and crush the crystals with a probe to check that the crystals can be shattered (i.e. that they are not cross-linked).  Also check that they do not crush with a click that can be heard and felt, which would indicate that they are salt.  See below for instructions for making a suitable glass probe.

3.       Put 50 µl of the reservoir solution that gave the hit into the Seed Bead tube, keeping it on ice.

4.       Remove about 6 µl of reservoir solution from the tube, and transfer it to the well with crystals.  Dispense and suck back into the tip several times.  Change the volume setting on the pipette to e.g. 7 µl and transfer the mixture back to the Seed Bead tube.

5.       Wash the well with another 6 µl of solution from the Seed Bead tube, again transferring the washings back to the Seed Bead tube.

6.       Vortex the Seed Bead in its tube for two minutes.

7.       Use this (undiluted) seed stock for rMMS microseeding (seeding into a random screen).  For rMMS not dilute 1:100 as instructed in the Hampton Research Seed Bead instructions.  The more crystals there are in your seed stock the more hits you will obtain.  Recommended volumes to use are 0.3 µl of protein, 0.2 µl of reservoir, and 0.1µl of seed stock for robotic work.  Before using the seed stock, agitate it in case the suspended crystals have settled in the tube.

8.       However, we suggest that you make a dilution series straight away, up to 1 in 10,000.  Use these diluted seed stocks in later experiments if too many crystals are obtained.  Freeze all seed stocks immediately at -80ºC (or -20ºC if not available).

9.       rMMS can be performed by hand (increase the volume) or with a robot that uses contact dispensing.

10.   Immediately after use, freeze the undiluted seed stock at -80ºC / -20 ºC.

 

Notes

1.    Crystals can be crushed in their wells using a rounded glass probe.  Such a probe can be made from a glass rod, pipette or capillary using a blow-torch or Bunsen flame.  Thin-walled pipettes may be too flexible, so use a thick-walled pipette or capillary.  First heat the glass rod near the middle until it becomes flexible, then quickly remove the rod from the flame and draw it out by pulling apart the ends.  (If you keep the rod in the flame while pulling it will generally break.)  Aim to pull the glass down to a diameter below 0.25 mm.  Break the glass at the point where it is around 0.25 mm, and briefly plunge the broken end into the flame.  Repeat this until a blob of glass with a diameter of about 0.5 mm is formed on the end.  This probe is useful for crushing crystals because it is easier to hit crystals, and because it does not damage the plastic bottom of the well.

2.      Several groups have reported that, for some proteins, only fresh crystals work.  Crystals that have been in the lab for a few weeks may not work, even though the crystals still diffract.

3.      In cases where plenty of seed crystals are available, seed stock can be prepared simply by crushing the crystals in several large drops and collecting them in their mother liquor without any additions.  The seed bead can be used, although it has the disadvantage that some liquid tends to be removed and wasted when you take the seed bead out of the tube.  If the seed bead is not used, crush the crystals thoroughly in their wells with a glass probe.  If required, the volume of seed stock added to each drop can be reduced to as little as 10 nl using any of the Oryx systems.   This means that only about 1.5 µl of seed stock is required for 96 wells.

4.    Seed crystals of membrane proteins are particularly unstable.  This may be because of the need to keep the crystals in contact with relatively high concentrations of detergent.  Also, fewer crystals of membrane proteins may be available to make the seed stock.  We recommend that seed crystals of membrane crystals are crushed in their wells and harvested without any additions, as in point (2) above.  Again, the small quantity of seed stock used by Oryx is an important advantage.

5.      If you are attempting to crystallize a complex, you should avoid high salt conditions (see ref. by Radaev and Sun below).  Try suspending crystals in e.g. 40% PEG 3000 instead of the reservoir solution.  To find out if the seed crystals will be stable in PEG, incubate uncrushed crystals in PEG for 24 hours.  If the crystals do not crack, shatter or dissolve the solution can almost certainly be used to prepare a seed stock.

6.    Similarly, high salt conditions can be unhelpful for heavy atom derivatization and small molecule complexes, so you may also wish to suspend seeds in PEG in these cases.  Moreover, this approach will reduce the number of salt crystals obtained.

7.      "Preseeding the protein stock" (suspending crushed crystals in the normal protein stock) can be beneficial but it is less effective than using a separate seed stock.

8.      Seed stocks can be obtained by harvesting crystals from microfluidic devices and capillaries.

 

References:

 

Luft, J. R. & DeTitta, G. T. (1999). ‘A method to produce microseed stock for use in the crystallization of biological macromolecules’.  Acta Cryst. D55, 988–993.

 

Allan D’Arcy, Frederic Villarda, May Marsh.  'An automated microseed matrix-screening method for protein crystallization'.  Acta Crystallographica section D63 (2007), 550–554.  On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 

 

S. Radaev and P. D. Sun. 'Crystallization of protein-protein complexes.' Journal of Applied Crystallography, 35 (2002) 674-676. On-line at http://scripts.iucr.org/cgi-bin/paper?S0021889802013973

 

Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'.  Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442.  (To download free of charge click here.)

 

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