Hampton Research Seed Bead HR2-320 User manual
Seed Bead
User Guide HR2-320 (pg 1)
TM
Overview
This publication describes the use of the Seed Bead to create a seed stock for
performing subsequent seeding experiments. Seed Bead is based upon the
research of Joseph R. Luft and George T. DeTitta at the Hauptman Wood-
ward Medical Research Institute.3The Seed Bead Kit contains 24 Seed Beads
manufactured from PTFE, individually contained in a special 1.5 milliliter
microcentrifuge tube and instructions.
Background
A crystallization experiment typically begins with the sample in a stabilizing
solution of water and possibly other additives such as buffer, salt, reducing
agent, and other chemicals. Prior to mixing the sample with crystallization
reagent, this sample solution is undersaturated with respect to the macro-
molecule in question (sample). In an undersaturated sample solution, no
crystals can nucleate, nor can crystals grow from seeds. Upon addition of a
crystallization reagent the relative supersaturation of the sample is increased.
Assuming the crystallization reagent decreases the solubility of the sample
to increase the relative supersaturation, three events can take place. In the
first stage of supersaturation, the Metastable Zone, spontaneous homogenous
nucleation cannot occur, but crystal growth from seeds can occur. Moving
further into supersaturation, the Labile Zone, spontaneous homogeneous
nucleation and crystal growth can occur. Further into supersaturation, the
Precipitation Zone, precipitation of the sample from solution occurs. See Fig-
ure 1 below.
Seeding
Seeding allows one to grow crystals in the Metastable Zone, where sponta-
neous homogenous nucleation cannot occur, but crystal growth from seeds
can occur. Why would one want to do this? For control, reproducibility, and
to improve the likelihood of a successful crystallization experiment. In the
Metastable Zone crystals can grow from seeds but cannot spontaneously nu-
cleate. By placing a seed or solution of seeds in a drop which is saturated to
the Metastable Zone one can use the seeds to grow larger single crystals. By
controlling the number of seeds introduced into the Metastable Zone drop
one can control the number of crystals grown. It is not practically possible
to measure and know the number of seeds introduced to a drop, but by per-
forming serial dilutions from a concentrated seed stock one can control the
number of crystals grown in the Metastable Drop.
Preparing the Seed Stock – Contemporary Method
1. Place the Seed Bead (tube containing a bead) into a bucket of ice.
2. Open the drop well containing the crystals identified for creating seed
stock. Crush the crystals with a probe. If necessary add reservoir solution to
the drop to minimize and compensate for evaporation from the drop, depend-
ing upon the time spent crushing the crystals and the drop size. If the drop
well contains only a few small crystals, consider combining several drop wells
to increase the seed crystal concentration. Read Observations, Notes and Sug-
gestions #12 about combining drops.
3. Pipet 5 to 10 µl of crystallization reagent from the reservoir, and add it to
the drop well containing the crushed crystals. Aspirate and dispense the drop
several times. Use the pipet tip to dislodge crystals stuck to the plate. Pipet the
mixture from the drop well to the Seed Bead tube on ice.
4. Repeat step 3 for a total of five to ten times until all of the crushed crys-
tals have been transferred, and there are about 50 µl of solution containing
crushed crystals in the Seed Bead tube. Be sure to remove all crystals that
might be sticking to the plate.
5. Vortex the Seed Bead tube for three minutes, stopping every 30 seconds to
cool the tube on ice. This is your seed stock.
6. Use this undiluted seed stock for Microseed Matrix Screening (MMS) The
contemporary method uses a higher seed concentration than the classical
method, is amenable to automation due to the smaller volume of seed stock
and can produce more hits.
7. Manual MMS Set Up: 1.5 µl of protein, 1 µl of reservoir, and 0.5 µl of seed
stock. Before pipetting the seed stock, agitate the tube in case the suspended
crystals have settled in the tube.
8. Automated Contact Dispensing MMS Set Up: 0.3 µl of protein, 0.2 µl of
reservoir, and 0.1 µl of seed stock. Before pipetting the seed stock, agitate the
tube in case the suspended crystals have settled in the tube.
9. Before storing the seed stock, proceed with Serial Dilutions for seeding
now, up to 1 in 100,000. Fresh seeds are better than old seeds when creating
stocks. Use these diluted seed stocks in later experiments if too many crystals
are obtained. Freeze all seed stocks immediately at -80°C (or -20°C if not
available).
Preparing the Seed Stock - Classical Method
1. Pipet 50 microliters of crystal stabilizing solution into the microcentrifuge
tube with the Seed Bead. The stabilizing solution is a mixture of sample and
crystallization reagent in which the crystal will not dissolve nor continue to
grow, but is a solution which will support the stability of the crystal. A solu-
tion closely approaching that of the drop from which the crystal is removed
Solutions for Crystal Growth
Figure 1
The diagram is divided into four zones:
1. Stable: Undersaturated where crystal
nucleation and growth is not possible; clear
drops
2. Metastable: Supersaturated where
nuclei cannot form but crystals can grow.
3. Labile: Supersaturated where nucleic
can form and crystals can grow
4. Precipitation: Precipitation of sample
from solution, where crystal nucleation and
growth is not possible
User Guide HR2-320 (pg 2)
Seed BeadTM
is a good starting point for the stabilizing solution. The simplest option is to
use the crystallization reagent (reservoir solution) that produced the crystals.
A more complex option is to perform some empirical experimentation to de-
termine the reagent composition of the stabilizing solution. The stabilizing
solution will be a reagent composition somewhere between that of the reser-
voir used to obtain the crystal and that of the drop at the initial mixing stage.
2. Remove crystals from a drop using a Mounted CryoLoop or pipet. Do not
leave the seed exposed to the air for any longer than absolutely necessary.
Macromolecular crystals have a high solvent content and can be damaged or
destroyed by evaporation of water from about the crystal.
3. Place the seed crystals in the microcentrifuge tube containing 50 micro-
liters of stabilizing solution and Seed Bead. Close the microcentrifuge tube.
4. Vortex the microcentrifuge tube containing the seed crystal and the Seed
Bead for 3 minutes. Alternatively, one may choose to sonicate the microcen-
trifuge tube containing the seed crystal and the Seed Bead for 3 minutes. See
Figure 2 below.
5. Pipet 450 microliters of the stabilizing solution into the microcentrifuge
tube containing the vortexed crystal and Seed Bead. This is your seed stock.
Preparing Serial Dilutions for Seeding
• Dropping Solution 1: Undiluted seed stock in stabilizing buffer.
Dilution 1x10 0.
• Dropping Solution 2: 45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 1.
Dilution 1x10 -1.
• Dropping Solution 3: 45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 2.
Dilution 1x10 -2.
• Dropping Solution 4: 45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 3.
Dilution 1x10 -3.
• Dropping Solution 5: 45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 4.
Dilution 1x10 -4.
• Dropping Solution 6: 45 microliters of stabilizing buffer contain-
ing no protein or crystals plus 5 microliters of Dropping Solution 5.
Dilution 1x10 - .
Serial dilution of the seed stock can be performed if seeding experiments us-
ing the seed stock produce too many crystals in the drop. When preparing a
number of serial dilutions of the seed stock, one should reserve a portion of
each serial dilution for future crystallization experiments. What follows is an
example of how to perform a serial dilution to prepare dropping solutions
for seeding. One may prepare fewer or more dilutions depending upon how
many drops are to be set. Also,one may change the dilution from 1:10 to some
other ratio such as 1:2, 1:5, 1:20, and so on. Be certain to mix or vortex the
seed stock prior to performing each dilution. Failure to vortex mix can lead to
inconsistency. See Figure 3.
Setting the Drops - Seeding with the Seed Stock
Set sitting or hanging drops over reservoir solutions of reagent composition
identical to that used to obtain the initial seed crystals. Do not add reservoir
solution to the drops unless one wishes to further dilute the drops (Note: this
may dissolve the seeds). To slow vapor diffusion equilibration one may dilute
the reservoir solution. To speed vapor diffusion equilibration one may use a
more concentrated reservoir solution.
Example 1. Original crystals grown using 2.0 M Ammonium sulfate,
0.1 M HEPES pH 6.8. Stabilizing solution is 2.0 M Ammonium sulfate,
0.1 M HEPES pH 6.8. Seed crystals from step 5 are composed of 500 ml of
2.0 M Ammonium sulfate, 0.1 M HEPES pH 6.8 and crystals, vortexed.
For the crystallization experiment, pipet 2.0 M Ammonium sulfate, 0.1 M
HEPES pH 6.8 into the reagent well (reservoir). For the drop, pipet 1 part
of protein plus 1 part of seed stock. The drop will equilibrate from 1.0 M to
2.0 M Ammonium sulfate.
Example 2. The results of Example 1 produced numerous, small crystals
after only 24 hours. In an effort to reduce the number of crystals, increase
crystal size and slow the experiment one can reduce the concentration of the
reservoir to 70% of the original.
For the crystallization experiment, pipet 1.4 M Ammonium sulfate, 0.07 M
HEPES pH 6.8 into the reagent well (reservoir). For the drop, pipet 1 part of
protein plus 1 part of seed stock. The drop will now equilibrate from 1.0 M to
1.4 M Ammonium sulfate.
Solutions for Crystal Growth
Figure 3
Figure 2
User Guide HR2-320 (pg 3)
Seed BeadTM
Example 3. The results of Example 2 still produced too many, small crystals
after 24 hours. In an effort to reduce the number of crystals and increase
crystal size, one can use a different serial dilution seed stock. From Preparing
Serial Dilutions for Seeding, use Dropping Solution 2 for the seed stock.
For the crystallization experiment, pipet 1.4 M Ammonium sulfate, 0.07 M
HEPES pH 6.8 into the reagent well (reservoir). For the drop, pipet 1 part of
protein plus 1 part of Dropping Solution 2. The drop will now equilibrate
from 1.0 M to 1.4 M Ammonium sulfate, but with fewer seeds.
Microseed Matrix Seeding (MMS)
Microseed Matrix Seeding is the method where seed crystals are systematically
added to a crystallization screen.5-8 By adding seeds instead of protein:
1. It is likely to greatly increase the number of crystallization hits that are
observed;
2. It is likely that good quality crystals will grow, because the crystals often
grow at lower levels of saturation;
3. Reagents can be used where no spontaneous nucleation takes place, so
that the number of crystals can be controlled by determining the num-
ber of nuclei that are added to the well (by diluting the seed stock).
Setting the Drops for Microseed Matrix Seeding (MMS)
Pipet the crystallization screen reagent into the reagent well (reservoir). To
create the drop, pipet 0.2 ml of crystallization screen reagent (reservoir),
0.1 ml of seed stock from step 5 and 0.3 ml of protein solution. As a starting
point, use the same protein concentration here as used to produce the seeds
in a previous screen. Repeat for the remaining reagents.
Observations, Notes, and Suggestions
1. The 3.0 mm PTFE Seed Bead has a density of 2.2 g/cc.
2. When seeding, one would prefer to have an initial sample / reagent
composition in the drop that will not produce crystals without the addi-
tion of a seed. This will prevent nucleation secondary to the introduced
seeds as well as excessive nucleation.
3. If, after performing the seeding experiment with a particular set of dilu-
tions, one still observes excessive nucleation and small crystals, repeat
the seeding experiment with further dilution of the Seed Stock/Dropping
Solutions.
4. Use a new, clean Seed Bead and microcentrifuge tube each time one
is generating a new seed stock. This will prevent contamination and
carryover.
5. Vortexing the Seed Bead in the presence of detergents and/or other
chemical additives which can foam is not recommended. In the pres-
ence of detergents or other chemical additives which can foam, it is rec-
ommended one use sonication to disrupt the seed using the Seed Bead.
6. Sonication using the Seed Bead allows one to use smaller volumes than
the vortex method.
7. If using sonication do not leave the sample in the ultrasonic bath too
long since this can heat the sample.
8. To prevent splashing when vortexing, grasp the tube in the middle while
vortexing. Should drops of the sample appear on the upper sides of the
tube or in the lid, place the tube in a centrifuge for 3 to 5 seconds to sedi-
ment the liquid. Do not centrifuge for any longer since this will “pellet”
the seeds.
9. Any crystalline protein material can be used for microseeding, including
fine needles, spherulites, microcrystals, irregular poorly-formed crystals
and even granular looking precipitate. Seed anything that might be
crystalline. Skins do not seem to work for seeding.
10. When performing iterative seeding during optimization, be more se-
lective about which seeds to include in the seed stock, identifying and
choosing the best crystals; do not mix the good, bad and the ugly, leave
that to Sergio Leone.
11. Microcrystals in the seed stock are not stable because of the lower protein
concentration in solution. The seed stock should be kept on ice and
frozen as soon as possible, preferably at -80ºC when not being used.
12. It has been observed that, for some proteins, only fresh crystals work for
seeding. Crystals that have been in the lab for a few weeks may not work
for seeding, even though the crystals still diffract. Make a seed stock as
soon as possible after the crystals stop growing.
13. One may consider combining as many hits as possible to make the seed
stock. But be careful to avoid creating solutions that could result in
salt crystals or phase separation. One could try to combine the drops
from all the hits in PEG based conditions to make one seed-stock, and
the drops from all the hits in salt based reagent to make another seed
stock. Avoid mixing metals and salts such as calcium and phosphate
as this can produce salt crystals. Avoid mixing high salt and high PEG
concentration as this can produce phase separation.
14. MMS experiments can be dispensed by manually. The volumes dis-
pensed will be increased slightly to approximately and in the following
order, 1.5 µl of protein, 1 µl of reservoir solution, and 0.5 µl of seed stock.
15. When using automation for MMS, contact dispensing appears to be fa-
vored as non-contact dispensing can be prone to clogging.
16. Be careful not to optimize salt crystals. Salt crystals are a side effect of
MMS due to the random mixing of reagents.
17. The potential of seeding and MMS. Increase hit rate in crystallization
screens. More reagents to choose from for ligand soaking and heavy
atom derivatives. New space groups. Use apo form to seed for ligands
and inhibitors. Avoid twinning. Larger, more ordered, better diffracting
crystals. Cross seeding between complexes.
Solutions for Crystal Growth
User Guide HR2-320 (pg 4)
Seed BeadTM
References and Readings
1. Stura, E.A., Wilson, I.A., Methods: A Companion to Methods in Enzymol-
ogy, 1, pages 38-49 (1990).
2. Nucleic Acids and Proteins: A Practical Approach, Oxford University
Press, pages 99-126 (1992).
3. Luft, J.R., DeTitta, G.T. Acta Cryst. (1999). D55, 988-993.
4. J.R. Luft and G.T. DeTitta, Methods in Enzymology, 276, 110-131 (1997).
5. Gregory Ireton and Barry Stoddard. Microseed matrix screening to im-
prove crystals of yeast cytosine deaminase. Acta Crystallographica sec-
tion D60 (2004) 601–605.
6. Allan D’Arcy, Frederic Villard, May Marsh. An automated microseed ma-
trix-screening method for protein crystallization. Acta Crystallographica
section D63 (2007) 550–554.
7. Galina Obmolova, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet
and Gary L. Gilliland. Promoting crystallization of antibody–antigen
complexes via microseed matrix screening. Acta Crystallographica Sec-
tion D66 (2010) 927–933.
8. Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E.
Chayen and Peter F.M. Baldock. Random Microseeding: A Theoretical
and Practical Exploration of Seed Stability and Seeding Techniques for
Successful Protein Crystallization. Cryst. Growth Des., 2011, 11 (8), pp
3432–3441.
9. Terese Bergfors. Seeds to Crystals. Journal of Structural Biology, 142
(2003), 66 - 76.
10. Transmission electron microscopy for the evaluation and optimi-
zation of crystal growth. Hilary P. Stevenson & Guillermo Calero
et al. Acta Cryst. (2016) D72, 603-615.
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Technical Support
Inquiries regarding the Seed Bead kit and general inquiries regarding crystal-
Solutions for Crystal Growth
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