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.
3
The 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 microcen trifuge
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
