In situ counter‐diffusion crystallization and long‐term crystal preservation in microfluidic fixed targets for serial crystallography

• Published in: Journal of applied crystallography 2024-10, Vol.57 (5), p.1539-1550
• Main Authors: Liu, Zhongrui, Gu, Kevin, Shelby, Megan, Roy, Debdyuti, Muniyappan, Srinivasan, Schmidt, Marius, Narayanasamy, Sankar Raju, Coleman, Matthew, Frank, Matthias, Kuhl, Tonya L.
• Format: Article
• Language: English
• Subjects: Capillaries; Chambers; counter diffusion; Crystal growth; Crystallization; Crystallography; Crystals; Diffraction; Diffusion; Diffusion chambers; Elongated structure; Fabrication; in situ; Kapton (trademark); long‐term crystal preservation; microfluidic fixed targets; Microfluidics; Mylar; Nucleation; Photoactive yellow protein; Polyimide resins; Polymers; Propylene; protein diffraction; Proteins; Research Papers; Room temperature; serial crystallography; Thin films; vacuum operation; XFELs; X‐ray scattering; Yellow protein
• ISSN: 1600-5767; 0021-8898; 1600-5767
• DOI: 10.1107/S1600576724007544

Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher‐quality protein crystals yielding improved diffraction data and higher‐resolution structures. Typically, counter‐diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X‐ray beamline. Despite the advantages of counter‐diffusion protein crystallization, there are few fixed‐target devices that utilize counter diffusion for crystallization. In this article, different designs of user‐friendly counter‐diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed‐target chip. Methods for rapid chip fabrication using commercially available thin‐film materials such as Mylar, propylene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X‐ray beam time. Importantly, the fixed‐target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed‐target utilization by crystallographers. User‐friendly in situ counter‐diffusion crystallization and long‐term storage setups in microfluidic fixed targets provide a reliable approach to forming large crystals and maintaining their hydration for weeks, allowing ample time to grow, select and preserve the best crystal batches before X‐ray beam time.