Dear Venkat,
Crystals are
categorized as extremely thin needles that are clustered and protrude around a
single nucleation site, and appear “fuzzy” in nature. I would particularly
recommend three different ways to optimize them, which I prioritized in
descending order:
1. Take the sea urchin crystal cluster and crush them
with the seed bead from Hampton, making different dilutions. Alternatively, you
can try different types of seeding (streak seeding or jab seeding)
2. Take the serial dilutions from the first
recommendation and add them to new crystallization screens.
3. Try an optimization screen with organic solvents
(ETOH, Isopropanol, acetone, dioxane, DMSO, MPD), varying the concentration
from 0-10% added to the already optimized mother liquor.
In essence, seeding is a powerful tool for the
separation of nucleation and growth of the crystal, whereas the addition of the
organic solvents acts like salt to compete for water molecules and changes the
dielectric of the medium.
Refferences:
1. Bergfors, T (2003) Seeds to crystals
2. D’Arcy, A (2007) An
automated microseed matrix screening method for protein crystallization
3. McPherson, A (1999) Crystallization
of biological macromolecules, Cold Spring Harbor
Sincerely, lorenzo
Lorenzo Ihsan Finci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard
Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityCollege
of Life SciencesBeijing, China
Date: Wed, 21 May 2014 15:28:16 +0530
From: [email protected]
Subject: [ccp4bb] Sea Urchin like crystals
To: [email protected]
Dear All,
I'm working on 24.3 KDa protein (pI 4.84) with (Asp + Glu): 36 and (Arg + Lys):
16. Protein is in buffer Tris (10mM), pH 7.5. I got sea urchin like crystal
clusters @ protein conc 38.5 mg/ml in sitting drop method; condition 0.2M NH4I,
20% PEG3350 & pH 6.8 (PEG/ION A12). These crystals are very thin. I tried
different pH values ranging from 6.0 -9.0 with 0.5 units increment, PEG conc.
from 10 -30% and NH4I conc. from 0.1 -0.5 M independently in hanging drop
method and ended up with NO Crystals. Can somebody suggest strategy to improve
crystals. Please find the crystal pic with attachment.
Regards,Venkat.
