1. References: Márquez, José A., and Florent Cipriani. "CrystalDirect™: A Novel Approach for Automated Crystal
Harvesting Based on Photoablation of Thin Films." Structural Genomics. Humana Press, 2014. 197-203.
Saridakis, Emmanuel EG, et al. "Phase diagram and dilution experiments in the crystallization of
carboxypeptidase G2." Acta Crystallographica Section D: Biological Crystallography 50.3 (1994): 293-297.
Developing Novel Techniques
for Improving Protein Crystal
Growth
Participants: Sergio Rodriguez Labra,
Jose A. Marquez, Ph.D.
Introduction
Participants: Sergio Rodriguez Labra,
Jose A. Marquez, Ph.D.
X-Ray Protein crystallography is by far the
most common method by which the
molecular structure of most proteins are
resolved today. The resolution of these
structures is the first step to uncovering the
mechanism by which they work inside living
organisms and critical to practical
applications like drug design. In this process,
the growth of quality protein crystals is in
general the most important and challenging
step; most relevant proteins today are
extremely hard to crystallize. For this reason,
it is key to develop new tools and techniques
that allow us to grow higher quality protein
crystals to increase their chances of leading to
successful data to resolve their structures.
Non-optimal batches of Lysozyme and Thaumatin protein crystals were specifically grown
for the project. Taking advantage of the Cdplate’s material, after protein crystals had started
growing, a micro-sized hole was opened on the base of the protein drop with the laser in
order to add a new drop, the CDdrop, with either more protein solution to keep driving
forward the growth of the protein or with more buffer to dilute smaller defective crystals as
to redirect that protein into a single larger higher quality crystal.
Controls were selected and a diverse series of experiments were carried out to optimize our
parameters and the method’s protocol.
Protein is concentrated by the vapor-liquid diffusion method, in
which, by the use of a reservoir with a more highly concentrated
solution of a substance called precipitant, water slowly leaves the
protein drop as vapor toward the reservoir, which concentrates
the protein until it reaches the nucleation point in which it starts
rearranging to form one or more crystals. However, if the
conditions, currently impossible to predict, are not perfect, the protein
will form an amorphous precipitate or crystallize with defects,
internal disorder, or stop growing before it reaches a usable size.
Results were promising and particular
characteristics were discovered.
For instance, a limit on the original
drop’s volume of extra water due to the
drop’s surface tension was found and estimated. Regarding
the addition of protein solution, all attempted experimented
led to the formation of a heavy precipitate forming without
visible crystal growth probably due to the crystal’s surface
poisoning.
Crystal growth Results
Objectives
This project was meant to be a proof-of-
concept to take advantage of the properties of
EMBL-created CrystalDirect technologyto
study the feasibility of changing the dynamic
equilibrium inside the crystallization chamber
to improve the quality of the final crystals.
More specifically, the goal was to proof if
existing crystals could be:
1) grown larger with the addition of more
protein solution,
2) grown better by partially dissolving them
and allowing them to grow back
Conclusions
Methodology
Factors and Challenges involved:
• Surface Poisoning
• Rate of crystal dilution
• Rate of diffusion throughout drop
• Rate of evaporation in chamber
• Measurement limitations
• Seed’s resilience to dissolving
CrystalDirect
Made out of a thin polymer film, it allows the
use of a laser beam (Class 4) that ablates the
material to cut through the base of the chamber
for crystal harvesting automation purposes.
Parameters considered:
• Hole size/shape
• Hole location and distance from crystals
• Volume of CDdrop added
• Crystal’s age at time of experiment
• Number of dilutions
• Extent of dilution
The dilution experiments showed great potential for further
development as the rate of evaporation can be slowed down
through other methods like the use of an oil top layer which
would give time for the diluted solution to dissolve unusable
crystals and redistribute the protein into larger better crystals.
Proving this use would open the doors for X-ray crystallography
to reach quality crystals to solve the structure of much more
complex macromolecules critical for the study of the most
challenging biological processes and diseases of today.
4 hours
About 10-15% growth
11 days after
For the dilution experiments, it was found that
crystals with defects were much more likely to
be affected, and twin crystals were separated.
However, it was very challenging to completely
dissolve any crystal for they would reduce in
size but quickly grow back to their original size
for it was found that the vapor-liquid diffusion
inside the chamber concentrated the drop much
faster after dilution, before allowing crystals to
dissolve.
11 days
5 days
70 minutes afterRight before experiment