Skip to main content

Research Repository

Advanced Search

Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction.

Drago, VN; Devos, JM; Blakeley, MP; Forsyth, VT; Kovalevsky, AY; Schall, CA; Mueser, TC

Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction. Thumbnail


Authors

VN Drago

JM Devos

MP Blakeley

VT Forsyth

AY Kovalevsky

CA Schall

TC Mueser



Abstract

Biologically active vitamin B6-derivative pyridoxal 5'-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (=0.5?mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1?Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.

Acceptance Date Apr 6, 2022
Publication Date May 4, 2022
Publicly Available Date Mar 29, 2024
Journal NPJ Microgravity
Publisher Nature Publishing Group
Pages 13 - ?
DOI https://doi.org/10.1038/s41526-022-00199-3
Publisher URL https://www.nature.com/articles/s41526-022-00199-3

Files




Downloadable Citations