We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.

Crystal structure determination of macromolecules is often hampered by the lack of crystals suitable for diffraction experiments. This article describes a protocol to crystallize the acidic protein bovine β‐lactoglobulin in the presence of yttrium to yield high‐quality crystals that belong to a new space group. The yttrium ions not only are used to engineer the crystallization, but are an integral part of the crystal lattice and can therefore be used to solve the phase problem using anomalous dispersion methods. Protein crystallization conditions were first optimized using an experimental phase diagram in the protein and salt concentration plane. Crystal growth strongly depends on the position in the phase diagram, and the best crystals grow near the phase transition boundaries. The structure analysis demonstrates the specific binding of yttrium ions to surface‐exposed glutamate and aspartate side chains contributed by different molecules in the crystal lattice. By bridging molecules in this manner, contacts between molecules are formed that enable the formation of a stable crystal lattice. The potential application of this strategy to the crystallization of other acidic proteins is discussed on the basis of the universal features of the phase behavior of these proteins and the interactions induced by multivalent ions.

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We report a real-time study on protein crystallization in the presence of multivalent salts using small angle X-ray scattering (SAXS) and optical microscopy, focusing particularly on the nucleation mechanism as well as on the role of the metastable intermediate phase (MIP). Using bovine beta-lactoglobulin as a model system in the presence of the divalent salt CdCl2, we have monitored the early stage of crystallization kinetics which demonstrates a two-step nucleation mechanism: protein aggregates form a MIP, which is followed by the nucleation of crystals within the MIP. Here we focus on characterizing and tuning the structure of the MIP using salt and the related effects on the two-step nucleation kinetics. The results suggest that increasing the salt concentration near the transition zone pseudo-c** enhances the energy barrier for both MIPs and crystal nucleation, leading to slow growth. The structural evolution of the MIP and its effect on subsequent nucleation is discussed based on the growth kinetics. The observed kinetics can be well described, using a rate-equation model based on a clear physical two-step picture. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization, but also elucidates the role and the structural signature of the MIPs in the nonclassical process of protein crystallization.