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Laboratory cryo x-ray microscopy for 3D cell imaging.

ABSTRACT: Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposure times and limited up-time. Here we report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Stabilization of the laser-plasma source combined with new optics and sample preparation provide high-resolution cell imaging, both in 2D with ten-second exposures and in 3D with twenty-minute tomography. Examples include monitoring of the distribution of carbon-dense vesicles in starving HEK293T cells and imaging the interaction between natural killer cells and target cells.

SUBMITTER: Fogelqvist E

PROVIDER: S-EPMC5647409 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Laboratory cryo x-ray microscopy for 3D cell imaging.

Fogelqvist Emelie E Kördel Mikael M Carannante Valentina V Önfelt Björn B Hertz Hans M HM

Scientific reports 20171018 1

Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposu ...[more]

PMID: 29044158

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Project description:X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable Rwork/Rfree. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.

Dataset Information

Laboratory cryo x-ray microscopy for 3D cell imaging.

Publications

Laboratory cryo x-ray microscopy for 3D cell imaging.

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