title: A high-transparency, micro-patternable chip for X-ray diffraction analysis of microcrystals under native growth conditions.
abstract: Microcrystals present a significant impediment to the determination of macromolecular structures by X-ray diffraction methods. Although microfocus synchrotron beamlines and X-ray free-electron lasers (XFELs) can enable the collection of interpretable diffraction data from microcrystals, there is a need for efficient methods of harvesting small volumes (<2 µl) of microcrystals grown under common laboratory formats and delivering them to an X-ray beam source under native growth conditions. One approach that shows promise in overcoming the challenges intrinsic to microcrystal analysis is to pair so-called `fixed-target' sample-delivery devices with microbeam-based X-ray diffraction methods. However, to record weak diffraction patterns it is necessary to fabricate devices from X-ray-transparent materials that minimize background scattering. Presented here is the design of a new micro-diffraction device consisting of three layers fabricated from silicon nitride, photoresist and polyimide film. The chip features low X-ray scattering and X-ray absorption properties, and uses a customizable blend of hydrophobic and hydrophilic surface patterns to help localize microcrystals to defined regions. Microcrystals in their native growth conditions can be loaded into the chips with a standard pipette, allowing data collection at room temperature. Diffraction data collected from hen egg-white lysozyme microcrystals (10-15 µm) loaded into the chips yielded a complete, high-resolution (<1.6 Å) data set sufficient to determine a high-quality structure by molecular replacement. The features of the chip allow the rapid and user-friendly analysis of microcrystals grown under virtually any laboratory format at microfocus synchrotron beamlines and XFELs.
authors.affiliation: Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Murray
authors.affiliation: Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology and Photon Science, and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
authors.forename: Artem Y
authors.initials: AY
authors.lastname: Lyubimov
authors.affiliation: GM/CA@APS, X-Ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Craig M
authors.initials: CM
authors.lastname: Ogata
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Huy
authors.initials: H
authors.lastname: Vo
authors.affiliation: Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology and Photon Science, and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
authors.forename: Monarin
authors.initials: M
authors.lastname: Uervirojnangkoorn
authors.affiliation: Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology and Photon Science, and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
authors.affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
mesh_terms: D000818:Animals; D002645:Chickens; D003460:Crystallization / Q000295:instrumentation; D018360:Crystallography, X-Ray / Q000295:instrumentation*; D004867:Equipment Design; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D009113:Muramidase / Q000737:chemistry*; D017655:Silicon Compounds / Q000737:chemistry*
chemical_list: D017655:Silicon Compounds; C421864:hen egg lysozyme; D009113:Muramidase; C032734:silicon nitride
keywords: X-ray crystallography; XFEL; microcrystals; microfluidics; microfocus beamline; serial data collection; silicon nitride