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On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

ABSTRACT: Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

SUBMITTER: Lieske J

PROVIDER: S-EPMC6608620 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Lieske Julia J Cerv Maximilian M Kreida Stefan S Komadina Dana D Fischer Janine J Barthelmess Miriam M Fischer Pontus P Pakendorf Tim T Yefanov Oleksandr O Mariani Valerio V Seine Thomas T Ross Breyan H BH Crosas Eva E Lorbeer Olga O Burkhardt Anja A Lane Thomas J TJ Guenther Sebastian S Bergtholdt Julian J Schoen Silvan S Törnroth-Horsefield Susanna S Chapman Henry N HN Meents Alke A

IUCrJ 20190619 Pt 4

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for <i>in-sit ...[more]

PMID: 31316815

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Project description:Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens.

Dataset Information

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Publications

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

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