Understanding how proteins crystallize remains a long-standing challenge in structural biology. While the final crystals are essential for techniques like X-ray diffraction, the early stages of crystallization—where molecules begin to organize—remain largely invisible. Over the past decade, our team has tackled this challenge through a sustained, collaborative effort across the PSB network, advancing both our scientific understanding and the experimental tools needed to probe these hidden processes.
As model systems, we investigated various globular proteins in the presence of multivalent salts. The overall phase behavior in these systems can be described by a reentrant phase diagram and may additionally feature liquid–liquid phase separation (LLPS), cluster formation, or crystallization. Using kinetic small-angle scattering
studies, we have previously revealed several nonclassical pathways for salt-induced protein crystallization [1]. However, the underlying reasons for these different pathways remained unclear. To gain complementary insights, we investigated the diffusive properties during the kinetic process using different spectroscopic techniques (NSE and NBS). In an initial study [2], we demonstrated the feasibility of this approach by examining the crystallization process of β-lactoglobulin in the presence of zinc chloride, which follows a classical crystallization pathway.
The development of new acquisition modes and analysis frameworks significantly reduced the acquisition time for neutron backscattering measurements to just a few minutes. In our most recent study [3], this allowed us to compare different crystallization processes using complementary techniques (NSE, NBS, DLS, SANS, microscopy) and to characterize multiple distinct pathways. These pathways included initial gel phases with metastable intermediates, the continuous presence of protein assemblies, and crystal growth directly from monomeric solutions.
Scattering experiments were essential for investigating these different pathways. The larger neutron beam size ensures spatial averaging, enabling the detection of rare crystallization events. The use of neutrons further allows continuous observation without the risk of radiation damage and makes it possible to study turbid samples. For samples that remain clear during the process, light scattering provides additional insights. Combining these techniques revealed information about the lifetime of different particles in solution, their individual and collective diffusive behavior, and the nature of particle interactions.
In summary, we have advanced the understanding of protein crystallization by employing a combination of neutron and light scattering techniques to uncover distinct crystallization pathways, including gel-like intermediates and direct crystal growth from monomeric solutions. Our work highlights the importance of complementary methods for probing early-stage dynamics and particle interactions in these complex processes.
C. Beck (ILL, University of Tübingen)
[1] Sauter A, Roosen-Runge F, Zhang F, Lotze G et al. (2015) Faraday Discuss., 179, 41-58
[2] Beck C, Grimaldo M, Roosen-Runge F, Maier R et al. (2019) Cryst. Growth Des., 19, 7036-7045
[3] Beck C, Mosca I, Miñarro LM, Sohmen B et al. (2025) J. Appl. Cryst., 58, 845-858
Figure 1: Time- and length-scales investigated with the different techniques. The kinetically observed time dependencies of the crystallization process fraction shows the nice reproducibility of the system.