A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

Abstract

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.

A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst

“…When diffracted at RT another unit cell population of HEX-1 cyto was detected, differing in the c-axis. The unit cell parameters of HEX-1 cyto collected at RT are enlarged compared to those diffracted at 100 K, however, comparable to recently published structures of HEX-1 cyto, also recorded at RT 17 , 39 . All HEX-1 structures (Fig.…”

“…Collecting X-ray diffraction data from crystals directly within the living cells can mitigate those problems. We previously established in cellulo data collection strategies at room temperature (RT) using silicon chips at XFELs and synchrotron sources 17 , 39 . Here, we extend the methodology to serial cryo-crystallography using MicroMesh mounts (Fig.…”

InCellCryst - A streamlined approach to structure elucidation usingin cellulocrystallized recombinant proteins

“…We previously established in cellulo data collection strategies at room temperature (RT) using silicon chips at XFELs and synchrotron sources 17,36 . Here, we extend the methodology to serial cryo-crystallography using MicroMesh mounts (Fig.…”

A 3D Printer in the Lab: Not Only a Toy

“…But you will want more once you have dipped your toes in 3D printing. Scanning for literature at this point will return plenty of 3D printable objects: molecular structural models, [ 4–;6 ] corals, [ 7 ] crystal structures, and 3D printed chips for obtaining crystals, [ 8 ] which are valuable models for teaching, and give the student a physical model of chemical structures, and so on (Figure 2b). There are, for example, instructions on how to convert crystallographic data to 3D printable models (Figure 2c) [ 9–12 ] or even how to convert NMR spectra to 3D prints (Figure 2d).…”