University of California, Los Angeles
Grant: $259,569 - National Institutes of Health - Sep. 16, 2009
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Award Description: X-ray protein crystallography is currently the primary methodology used for determining the 3D structure of protein molecules at near-atomic or atomic resolution. However, many biological specimens such as whole cells, cellular organelles, viruses and many important protein molecules are difficult or impossible to crystallize and hence their structures are not accessible by crystallography. Overcoming these limitations requires the employment of different techniques such as nuclear magnetic resonance and cryo-electron microscopy. A very promising approach currently under rapid development is X-ray diffraction microscopy (i.e. extending X-ray crystallography to allow imaging of non-crystalline specimens) in which the X-ray diffraction pattern of a non-crystalline specimen is measured and then directly phased by an iterative algorithm. Since its first experimental demonstration in 1999, X-ray diffraction microscopy has been successfully applied to the 2-D and 3-D imaging of non-crystalline specimens such as whole cells. While the highest resolution achieved thus far is 7 nm, the ultimate resolution is only limited by the X-ray wavelengths and radiation damage to the specimens. Because of its ability to image thick biological specimens and to achieve high spatial resolutions, X-ray diffraction microscopy can be used to bridge the resolution gap between light and electron microscopy. The goals of this proposal are 1) to develop a cryo X-ray diffraction microscope and study the ultimate 3D resolution attainable from frozen-hydrated whole cells; and 2) to test the hypothesis that X-ray diffraction microscopy can be used to image the 3D intracellular structure of frozen-hydrated cells at 10 nm resolution.
Project Description: The overall purpose of this proposal is to develop a cryo X-ray diffraction microscope and test the hypothesis that cryo X-ray diffraction microscopy can be used to image the 3D intracellular structure of frozen-hydrated cells at 10 nm resolution. Our first goal is to develop a cryo X-ray diffraction microscope to be mounted on beamline BL29XUL at SPring-8. The microscope will consist of two chambers: a sample chamber and a detector chamber. The sample chamber includes a pinhole, a pair of corners, a sample assembly and an optical microscope. The chamber will be maintained at helium ambiance during data acquisition to reduce X-ray absorption. The detector chamber includes a vacuum pipe, an attenuator, a photodiode, a beamstop, a CCD camera and an MDC vacuum chamber. Using this cryo X-ray diffraction microscope, we will address the fundamental question - what is the ultimate 3D resolution attainable from whole frozen-hydrated cells? Since the resolution of the X-ray diffraction microscope is ultimately limited by radiation damage, we will analyze the diffraction intensity fade-out as a function of the spatial frequencies and the radiation dose to quantify the resolution attainable. Our second goal is to test the hypothesis that X-ray diffraction microscopy can provide quantitative 3D imaging of frozen-hydrated cells at 10 nm resolution. The X-ray diffraction patterns will be acquired by using the cryo X-ray diffraction microscope. The 3D image reconstruction will be carried out using the guided hybrid input-output algorithm, where we will pay special attention to minimizing the mean phase error by optimizing the experimental parameters. We will obtain two independent data sets from the same frozen-hydrated yeast cell and perform quantitative analysis of the image quality and the spatial resolution achieved. From the quantified reconstructed images, we will identify the 3D intracellular structure inside yeast cells.
Jobs Summary: N/A (Total jobs reported: 0)
Project Status: Not Started
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