Ultrafast Electron Cryomicroscopy of Single Protein Particles

单个蛋白质颗粒的超快电子冷冻显微镜

• 批准号: 7663192
• 负责人: Ahmed H. Zewail
• 金额: $ 40.5万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663192
• 关键词: Biological; Characteristics; Classification; Complex; Cryoelectron Microscopy; Deposition; Electron Microscope; Electron Microscopy; Electrons; Equilibrium; Filament; Genetic Transcription; Guns; Heating; Ice; Image; Lasers; Lead; Left; Lighting; Macromolecular Complexes; Measurement; Measures; Microscope; Movement; Photons; Physiologic pulse; Population Heterogeneity; Proteins; RNA Polymerase II; Research; Research Personnel; Resolution; Sampling; Source; Specimen; Structure; Surface; Technology; Time; Transcription Factor TFIIB; Travel; Work; improved; macromolecule; multicatalytic endopeptidase complex; novel; particle; programs; promoter; reconstruction; transcription factor TFIIF; voltage

DESCRIPTION (provided by applicant): Recent advances at Caltech have introduced the possibility of Ultrafast Electron Microscopy (UEM), wherein an ultrafast (pico-femtosecond) laser pulse is used to liberate bursts of electrons from the source filament which are then used to image specimens with exposure times 9 or more orders of magnitude faster than normal. Because the principal resolution limitation in modern single particle analysis today is beam-induced specimen movement, we hypothesize that if we record images faster than large macromolecules can move appreciably, the resulting images will be dramatically sharper. Improved image sharpness will lead directly to more precise alignments, more powerful classification of heterogeneous populations, and higher resolution reconstructions. Towards that end, we will characterize beam-induced specimen movement as a function of exposure time through ~13 orders of magnitude (10+1 to 10-12 s). Because electrons repel each other, however, ultrafast electron bursts spread while traveling down the microscope column, degrading coherence. Thus we will also characterize by theoretical calculation and direct experimental measurement how short the bursts can be and still maintain adequate coherence. These two pieces of information should allow us to find that exposure time which optimally balances reduced specimen movement against decreased coherence to deliver the sharpest possible images. Using this optimal exposure time, we will record thousands of "single particle" images of an ideal "control" complex, the 20S proteasome, to demonstrate the merits of biological UEM. The culmination of this project will then be to determine an unknown structure of a complex of major importance. If successful, this advance could make structure determination to near-atomic resolution routine for large macromolecular complexes.

描述（由申请人提供）：CalTech的最新进展引入了超快电子显微镜（UEM）的可能性，其中使用超快（pico-femtosecond）激光脉冲用于从源丝中释放电子爆发的电子爆发，然后将其用于图像带有暴露时间9或更多幅度较高量较高的尺寸较高量的较高尺寸的样本。由于当今现代单个粒子分析中的主要分辨率限制是梁诱导的样品运动，因此我们假设，如果我们比大型大分子更快地记录图像可以显着移动，则将产生的图像将显着更加清晰。改进的图像清晰度将直接导致更精确的比对，异质种群的更强大的分类以及更高的分辨率重建。为此，我们将通过〜13个数量级（10+1至10-12 s）的曝光时间来表征梁诱导的样品运动。但是，由于电子互相排斥，因此超快电子爆发在沿显微镜柱行驶时传播，从而使连贯性降解。因此，我们还将以理论计算和直接实验测量的特征来表征爆发的短距离，并且仍然保持足够的连贯性。这两个信息应该使我们能够找到曝光时间，从而最佳地平衡了标本运动与相干性降低以提供最清晰可能的图像。使用此最佳暴露时间，我们将记录成千上万个理想“控制”复合体的“单个粒子”图像，即20S蛋白酶体，以证明生物UEM的优点。然后，该项目的高潮将是确定重要重要性复合物的未知结构。如果成功，这一进步可以对大型大分子复合物的近原子分辨率进行结构确定。