Nonlinear Optical Imaging for Guiding Protein Structure Determination

用于指导蛋白质结构测定的非线性光学成像

基本信息

批准号：
8888526
负责人：
GARTH Jason SIMPSON
金额：
$ 26.61万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8888526
关键词：
Adopted Affect Birefringence Caliber Chemicals Communities Crystal Formation Crystallization Crystallography Custom Data Collection Defect Detection Development Dimensions Disease Dose Dyes Equilibrium Failure Feedback Fluorescence Fluorescence Microscopy Foundations Generations Goals Growth Image In Situ Kinetics Laboratories Lasers Legal patent Length Licensing Measurement Methods Microscopy Mothers Optics Peer Review Photons Population Positioning Attribute Preparation Procedures Production Property Protein Analysis Proteins Protocols documentation Registries Resolution Resources Roentgen Rays Sampling Signal Transduction Source Staging Structure Synchrotrons Technology Time Twin Multiple Birth Uncertainty United States National Institutes of Health X ray diffraction analysis X-Ray Diffraction base beamline contrast imaging distilled alcoholic beverage fluorescence microscope free-electron laser high reward high risk image guided imaging platform improved instrument instrumentation intercalation journal article nano nanocrystal optical imaging protein structure public health relevance screening second harmonic structural biology success synchrotron radiation theories two-photon ultraviolet

项目摘要

DESCRIPTION (provided by applicant): High-resolution protein structures determined by X-ray diffraction require the production of high-quality, well-ordered crystals. The generation of such crystals typically involves the identification of initial crystallization conditions, followedby optimization to produce well-ordered crystals, and subsequent X-ray diffraction, most commonly performed at synchrotron facilities. Each step in this pipeline can typically require weeks or months to assess success or failure in iterative optimizations, limited by a combination of slow kinetics for protein nucleation and growth in order to generate large well-ordered single crystals, and limited access to synchrotron facilities for assessing crystal quality and performing data collection. We propose the use of nonlinear optical imaging to rapidly inform the final three key experimental steps of initial crystal formation, optimization of crystal quality prior to diffractin, and synchrotron X-ray diffraction analysis. For the first goal, intercalation of protein crystals wth "SHG-phores" is proposed for enhancing the range and sizes of crystals detectable by SHG, with proof-of-concept measurements suggesting enhancement factors of 1000-fold are achievable. In situ assessment of crystal quality is proposed for rapid optimization based the use of polarization-dependent SHG imaging for identification of multi-domain, twinned, and highly mosaic crystals. Finally, instrumentation to enable rapid serial crystallography of micro- and nano-crystalline showers are proposed based on integration of synchrotron XRD with multi-modal confocal reflectance, brightfield transmittance, birefringence, two-photon excited ultraviolet fluorescence (TPE-UVF), and UV-SHG, all acquired simultaneously at up to video rate and with perfect image registry. Using this combined measurement suite, we aim to enable routine and confident detection of crystals 1-5mm in diameter to enable diffraction measurements with a 1 mm collimated source. The proposed efforts build on a foundation laid during the previous initial cycle of NIH support. SHG and TPE-UVF microscopy first proposed in our previous period of support are now established and widely used methods within the crystallography community. Previous support directly contributed to 35 peer-reviewed journal articles, 66 presentations, and 3 issued patents. Our technology has been licensed, and a commercial SHG/TPE-UVF microscope is now available for protein crystal screening. In addition, a custom first-generation nonlinear optical imaging instrument has been integrated into a macromolecular crystallography beamline at Argonne National Laboratory.

描述（由申请人提供）：通过 X 射线衍射确定的高分辨率蛋白质结构需要生产高质量、有序的晶体。此类晶体的生成通常涉及初始结晶条件的识别，然后进行优化以生产。有序的晶体，以及随后的 X 射线衍射，最常在同步加速器设施中执行，此流程中的每个步骤通常需要数周或数月的时间来评估迭代优化的成功或失败，但受到多种因素的限制。蛋白质成核和生长的缓慢动力学，以产生大的有序单晶，我们建议使用非线性光学成像来快速了解初始晶体形成、衍射前晶体质量优化和同步加速器 X 射线的最后三个关键实验步骤。对于第一个目标，建议用“SHG-phores”插入蛋白质晶体，以增强 SHG 可检测到的晶体的范围和尺寸，并通过概念验证测量表明增强因子。可以实现 1000 倍的晶体质量原位评估，以便基于使用偏振相关的 SHG 成像来识别多域、孪晶和高度镶嵌晶体，从而实现快速优化。 - 基于同步加速器 XRD 与多模态共焦反射率、明场透射率、双折射、双光子激发的集成，提出了纳米晶簇射紫外荧光 (TPE-UVF) 和 UV-SHG，全部以高达视频速率同时采集并具有完美的图像配准，使用此组合测量套件，我们的目标是能够对直径 1-5mm 的晶体进行常规且可靠的检测。所提出的工作建立在 NIH 支持的先前初始周期中奠定的基础上，并且在我们之前的支持期间首次提出了 TPE-UVF 显微镜，现在已在其中建立并广泛使用的方法。先前的支持直接促成了 35 篇同行评审的期刊文章、66 项演示和 3 项已发布的专利，并且商用 SHG/TPE-UVF 显微镜现已可用于蛋白质晶体筛选。定制的第一代非线性光学成像仪器已集成到阿贡国家实验室的高分子晶体学光束线中。