A crystallization apparatus for temperature-controlled flow-cell dialysis with real-time visualization.

Niels Junius,Christophe Berzin,Jean-Luc Ferrer,Maxime Terrien,Monika Budayova-Spano,Esko Oksanen

doi:10.1107/s1600576716004635

Abstract

Many instrumentation developments in crystallization have concentrated on massive parallelization assays and reduction of sample volume per experiment to find initial crystallization conditions. Yet improving the size and diffraction quality of the crystals for diffraction studies often requires decoupling of crystal nucleation and growth. This in turn requires the control of variables such as precipitant and protein concentration, equilibration rate, and temperature, which are all difficult parameters to control in the existing setups. The success of the temperature-controlled batch method, originally developed to grow very large crystals for neutron crystallography, demonstrated that the rational optimization of crystal growth has potential in structural biology. A temperature-controlled dialysis button has been developed for our previous device, and a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature-precipitant concentration phase diagrams has been constructed. The presented approach differs from the current paradigm, since it involves serial instead of parallel experiments, exploring multiple crystallization conditions with the same protein sample. The sample is not consumed in the experiment and the conditions can be changed in a reversible fashion, using dialysis with a flowing precipitant reservoir as well as precise temperature control. The control software allows visualization of the crystals, as well as control of the temperature and composition of the crystallization solution. The rational crystallization optimization strategies presented here allow tailoring of crystal size, morphology and diffraction quality, significantly reducing the time, effort and amount of expensive protein material required for structure determination.

Highlights

In X-ray protein crystallography, obtaining crystals of a protein is a delicate step, but it is considerably more laborious to improve these crystals up to a sufficient size and quality for diffraction
It consists of a crystal growth bench that, in addition to accurate temperature control, allows the chemical composition of the crystallization solution to be varied in an automated manner thanks to a dialysis cell equipped with a flowing reservoir
The knowledge of the phase diagram and the specific control of the crystallization parameters such as the temperature and the concentration of crystallization agents and/or additives will allow the number of crystals and their macroscopic defects to be reduced, as well as the selection of the nucleation and/or growth of the desired phase

Summary

Introduction

In X-ray protein crystallography, obtaining crystals of a protein is a delicate step, but it is considerably more laborious to improve these crystals up to a sufficient size and quality for diffraction. The availability of powerful radiation sources, fast detectors and cryocooling techniques has not removed the need for larger and better ordered crystals. The quality of data obtained is often lower than it could be with larger crystals, which may be crucial in experimental phasing, for example (Rice et al, 2000). A major challenge in fast (sub-millisecond) time-resolved X-ray crystallography where the reaction is initiated by light is the uniform initiation across the whole crystal volume (Levantino et al, 2015). Varying activation fractions across a crystal, or a crystal population in serial

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Conclusion