title: 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 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.|Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Barnes
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Menlo Park, CA 94025, USA.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Kovaleva
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Hilary P
authors.initials: HP
authors.lastname: Stevenson
authors.affiliation: Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Aaron S
authors.initials: AS
authors.lastname: Brewster
authors.affiliation: Linac Coherent Light Source, Menlo Park, CA 94025, USA.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: DePonte
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Menlo Park, CA 94025, USA.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Baxter
authors.affiliation: Stanford Synchrotron Radiation Lightsource, Menlo Park, CA 94025, USA.
authors.forename: Aina E
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
mesh_terms: D003460:Crystallization / Q000379:methods*; D003461:Crystallography / Q000379:methods* / Q000639:trends; D008422:Materials Testing / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D011506:Proteins
keywords: Liquid-jet injector; Serial femtosecond crystallography; Transmission electron microscopy; X-ray free electron laser