Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography

Abstract

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ~16 minutes of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

Strategies for sample delivery for femtosecond crystallography

“…However, improvements in data-processing software and detector technologies are overcoming these problems. In cases where conditions for the growth of large crystals are known, microcrystal suspensions for injector studies (typically ranging between 0.5 and 10 mm in size) have been obtained by crushing larger crystals (often by centrifugation with microbeads), or by modifying the crystallization conditions to promote nucleation through seeding or increasing the protein and/or precipitant concentrations Barnes et al, 2016;Martin-Garcia et al, 2016). While crystals may be combined from multiple crystallization drops, modifying conditions for larger scale batch crystallization simplifies this repetitious process.…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…TEM has been used to simultaneously provide information on crystal size distribution, diffraction quality, and monodispersity [88,89].…”

A Bright Future for Serial Femtosecond Crystallography with XFELs

“…The size distribution, density and quality of microcrystals prepared for SFX data collection are typically characterized using the following techniques: SONICC (Second-Order Nonlinear Imaging of Chiral Crystals), DLS (Dynamic Light Scattering) and TEM (Transmission Electron Microscopy) [31, 76, 77]. …”