Cryo-EM grid preparation using gas dynamic virtual nozzles
使用气体动态虚拟喷嘴进行冷冻电镜网格制备
基本信息
- 批准号:10009848
- 负责人:
- 金额:$ 16.81万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-04-01 至 2021-09-30
- 项目状态:已结题
- 来源:
- 关键词:3-DimensionalAddressAirAlgorithmsApoferritinAreaAutomationBiologicalCarbonComplexCoupledCryoelectron MicroscopyCrystallizationDepositionDevelopmentDevicesDiffusionEthaneFeedbackFreezingGasesHeterogeneityIceImmersionLiquid substanceMacromolecular ComplexesMicrofluidic MicrochipsMicrofluidicsMolecular WeightMonitorMotorPreparationProteinsResearch PersonnelResolutionSamplingShapesSpace PerceptionSpeedSpottingsStreamStructureSurfaceSystemTechnologyThickThinnessTimeTracerTranslatingTranslationsVisualizationWateraqueousarmbasecarbon fiberdetectorfluorescence imaginginstrumentinstrumentationmacromoleculemillisecondparticleprotein complexprototypereconstructionstructural biologytoolvirtual
项目摘要
Project Summary
Recent advances in cryo-electron microscopy (cryoEM), such as the development of
direct detectors, automation and 3D particle reconstruction algorithms, have
transformed structural biology by enabling investigators to obtain near atomic resolution
structures without the need to grow crystals. A significant challenge that limits the wider
applicability of cryoEM as a structural tool is the preparation of suitable samples in thin
(< 100 nm) layers of vitreous ice in the approximately micron-sized holes of a substrate
grid. This arises from the large surface-area-to-volume ratio of the thin aqueous layers
prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules
preferentially localize to the air-water interface in the thin liquid layer, giving rise to
preferred orientations or, in some cases, denaturation. A significant development in
overcoming this problem has been the development of droplet based approaches to
depositing samples on the grids. This minimizes the dwell time of the protein in the
water layer, i.e., the time between spotting of the sample on the grid and plunge-
freezing of the liquid layer. The spot-to-plunge time of state-of-the-art instrumentation
(~10 ms), however, is still nearly three orders of magnitude longer than the time for
diffusion of sample to the air-water interface (~ 10-100 microseconds). The proposed
instrument will use thin liquid jets formed using gas dynamic virtual nozzles (GDVN)
together with an ultrarapid (~50 m/s) plunging system to reduce the spot-to-plunge time
to the microsecond regime. By minimizing this time to less than the diffusion time to the
air-water interface, preferential orientation and degradation issues can be minimized.
Additionally, the device has the potential to precisely control the ice layer thickness and
readily adapt to time-resolved studies. Successful development has the potential to
significantly widen the biological macromolecules and scientific questions that can be
addressed by high-resolution structure determination using single particle cryoEM.
项目总结
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Osman Bilsel其他文献
Osman Bilsel的其他文献
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STRUCTURAL CHANGES IN HUMAN SOD1 BY POST-TRANLATIONAL MODIFICATION
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- 批准号:
8168648 - 财政年份:2010
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$ 16.81万 - 项目类别:
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$ 16.81万 - 项目类别:
PROBING THE DENATURED STATES OF TWO BETA-PROTEINS
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$ 16.81万 - 项目类别:
TIME RESOLVED SAXS OF MICROSECOND PROTEIN FOLDING INTERMEDIATES
微秒蛋白质折叠中间体的时间分辨萨克斯
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7182104 - 财政年份:2005
- 资助金额:
$ 16.81万 - 项目类别:
FOLDING AND ASSEMBLY OF DIMERIC BETA-BARREL PROTEINS
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6975526 - 财政年份:2004
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