Journal:Acta Cryst D:S2059798320008475

Getting the best of ID30B for RT data collection in microchips

Jose A. Gavira, Isaac Rodriguez-Ruiz, Sergio Martinez-Rodriguez, Shibom Basu, Sébastien Teychené, Andrew A. McCarthy and Christoph Mueller-Dieckmann ^[1]

Molecular Tour
We have developed a low-cost approach to optimally collect room temperature (RT) data from multiple crystals grown in microchips, using the counter-diffusion technique. We have shown the feasibility of obtaining 3D structural models at the highest attainable resolution, from crystals grown in microfluidic systems, and diffracted at RT (i.e. without harvesting individual crystals). Since sample handling and manipulation for cryo-protection are critical factors for X-ray structural determination, RT data collection of the three model proteins, glucose isomerase, lysozyme and thaumatin allow us to determine their corresponding models at atomic resolution, near 1.0 Å.

Furthermore, RT diffraction data can reveal motions crucial for catalysis, ligand binding, and allosteric regulation, not always accessible under standard cryogenic data collection. The chips are fabricated by a combination of either OSTEMER-Kapton or OSTEMER-Mylar materials, both produce a sufficiently low scattering background to permit atomic resolution diffraction data collection at room temperature. The proposed system can be easily incorporated into a fully automatized workflow at any synchrotron beamline facilitating the collection of users' data with no-intervention required of the end-user.

• Glucose isomerase, Kapton glucose isomerase (6ybo) is in yellow green; Mylar glucose isomerase (6ybr) is in cyan.

• Lysozyme, Kapton lysozyme (6ybf) is in magenta; Mylar lysozyme (6ybi) is in yellow.

• Thaumatin, Kapton thaumatin (6yc5) is in salmon; Mylar thaumatin (6ybx) is in lavender.

Setup mounted for diffraction experiments at the ID30B beamline of the ESRF with two microchips (Kapton and Mylar) hold in a standard crystallization microplate mount in the plate-gripper goniometer head. Insert are images of thaumatin crystals.

PDB references: RT structure of HEW Lysozyme obtained at 1.13 A resolution from crystal grown in a Kapton microchip 6ybf; RT structure of HEW Lysozyme obtained at 1.12 A resolution from crystal grown in a Mylar microchip 6ybi; RT structure of Glucose Isomerase obtained at 1.06 A resolution from crystal grown in a Kapton microchip 6ybo; RT structure of Glucose Isomerase obtained at 1.20 A resolution from crystal grown in a Mylar microchip 6ybr; RT structure of Thaumatin obtained at 1.14 A resolution from crystal grown in a Mylar microchip 6ybx; RT structure of Thaumatin obtained at 1.35 A resolution from crystal grown in a Kapton microchip 6yc5.

References

1. ↑ Gavira JA, Rodriguez-Ruiz I, Martinez-Rodriguez S, Basu S, Teychene S, McCarthy AA, Mueller-Dieckman C. Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips. Acta Crystallogr D Struct Biol. 2020 Aug 1;76(Pt 8):751-758. doi:, 10.1107/S2059798320008475. Epub 2020 Jul 27. PMID:32744257 doi:http://dx.doi.org/10.1107/S2059798320008475