title: Use of transmission electron microscopy to identify nanocrystals of challenging protein targets.
abstract: The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Hilary P
authors.initials: HP
authors.lastname: Stevenson
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Makhov
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Monica
authors.initials: M
authors.lastname: Calero
authors.affiliation: Departments of Chemical Engineering and Chemistry.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Edwards
authors.affiliation: Department of Structural Biology, andHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305;
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Zeldin
authors.affiliation: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025;
authors.forename: Irimpan I
authors.initials: II
authors.lastname: Mathews
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Guowu
authors.initials: G
authors.lastname: Lin
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Barnes
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Hugo
authors.initials: H
authors.lastname: Santamaria
authors.affiliation: Vaccine and Gene Therapy Institute of Florida, Port St. Lucie, FL 34987; and.
authors.forename: Ted M
authors.initials: TM
authors.lastname: Ross
authors.affiliation: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025;
authors.forename: S Michael
authors.initials: SM
authors.lastname: Soltis
authors.affiliation: Departments of Chemical Engineering and Chemistry.
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.affiliation: JAN Scientific, Inc., Seattle, WA 98105.
authors.forename: V
authors.initials: V
authors.lastname: Nagarajan
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.affiliation: Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025; acohen@slac.stanford.edu guc9@pitt.edu.
authors.forename: Aina E
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; acohen@slac.stanford.edu guc9@pitt.edu.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Calero
mesh_terms: D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry / Q000648:ultrastructure*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure*; D015203:Reproducibility of Results; D061987:Sf9 Cells
chemical_list: D011506:Proteins; D011994:Recombinant Proteins
keywords: crystal characterization; crystal optimization; femtosecond diffraction; nanocrystal detection; structural biology