title: Transmission electron microscopy for the evaluation and optimization of crystal growth.
abstract: The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Hilary P
authors.initials: HP
authors.lastname: Stevenson
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Guowu
authors.initials: G
authors.lastname: Lin
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Barnes
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, M240 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Ieva
authors.initials: I
authors.lastname: Sutkeviciute
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Troy
authors.initials: T
authors.lastname: Krzysiak
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Simon C
authors.initials: SC
authors.lastname: Weiss
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Shelley
authors.initials: S
authors.lastname: Reynolds
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: JAN Scientific Inc., 4726 11th Avenue NE, Suite 101, Seattle, WA 98105, USA.
authors.forename: Veeranagu
authors.initials: V
authors.lastname: Nagarajan
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Makhov
authors.affiliation: School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Lawrence
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Lamm
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Clark
authors.affiliation: Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Gardella
authors.affiliation: School of Life Sciences, Arizona State University, PO Box 874501, Tempe, AZ 85287, USA.
authors.forename: Brenda G
authors.initials: BG
authors.lastname: Hogue
authors.affiliation: Biosciences Division, Argonne National Laboratory, 9700 South Cass Ave, Lemont, IL 60439, USA.
authors.forename: Craig M
authors.initials: CM
authors.lastname: Ogata
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, M240 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Jean Pierre
authors.initials: JP
authors.lastname: Vilardaga
authors.affiliation: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, 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, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
mesh_terms: D003460:Crystallization / Q000379:methods*; D004583:Electrons; D007834:Lasers; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D011506:Proteins
keywords: X-ray free-electron lasers; XFELs; crystal optimization; micro-electron diffraction; nanocrystallography; structural biology; transmission electron microscopy