title: Transmission electron microscopy as a tool for nanocrystal characterization pre- and post-injector.
abstract: Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 1040 Biomedical Science Tower 3, Pittsburgh, PA 15260, USA.
authors.forename: H P
authors.initials: HP
authors.lastname: Stevenson
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: D P
authors.initials: DP
authors.lastname: DePonte
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 1040 Biomedical Science Tower 3, Pittsburgh, PA 15260, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Makhov
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 1040 Biomedical Science Tower 3, Pittsburgh, PA 15260, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.affiliation: Department of Structural Biology and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
authors.forename: O B
authors.initials: OB
authors.lastname: Zeldin
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Stanford University, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: S
authors.initials: S
authors.lastname: Boutet
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 1040 Biomedical Science Tower 3, Pittsburgh, PA 15260, USA gcalero@structbio.pitt.edu.
authors.forename: G
authors.initials: G
authors.lastname: Calero
authors.affiliation: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, 2575 Sand Hill Road, Menlo Park, CA 94025, USA acohen@slac.stanford.edu.
authors.forename: A E
authors.initials: AE
authors.lastname: Cohen
mesh_terms: D004583:Electrons; D005583:Fourier Analysis; D006801:Humans; D007834:Lasers; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008968:Molecular Conformation; D009113:Muramidase / Q000737:chemistry; D053758:Nanoparticles / Q000648:ultrastructure*; D012319:RNA Polymerase II / Q000737:chemistry; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: C473449:PTH1R protein, human; D044168:Receptor, Parathyroid Hormone, Type 1; D012319:RNA Polymerase II; D009113:Muramidase
keywords: crystal characterization; crystal injector; crystallography; femtosecond diffraction; sample delivery; transmission electron microscopy (TEM)