National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)
5T32DK007680
米国
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
5F31NS096782
米国
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
5R01NS038631
米国
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
5F32MH115595
米国
引用
ジャーナル: J Struct Biol / 年: 2020 タイトル: Light-coupled cryo-plunger for time-resolved cryo-EM. 著者: Nate Yoder / Farzad Jalali-Yazdi / Sigrid Noreng / Alexandra Houser / Isabelle Baconguis / Eric Gouaux / 要旨: Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time- ...Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time-dependent conformational landscapes has been a long-standing goal of structural biology. To harness the power of single particle cryo-EM methods to enable 'time-resolved' structure determination, we have developed a light-coupled cryo-plunger that pairs flash-photolysis of caged ligands with rapid sample vitrification. The 'flash-plunger' consists of a high-power ultraviolet LED coupled with focusing optics and a motorized linear actuator, enabling the user to immobilize protein targets in vitreous ice within a programmable time window - as short as tens of milliseconds - after stimulus delivery. The flash-plunger is a simple, inexpensive and flexible tool to explore short-lived conformational states previously unobtainable by conventional sample preparation methods.
凍結剤: ETHANE-PROPANE / チャンバー内湿度: 70 % / チャンバー内温度: 277.15 K / 装置: HOMEMADE PLUNGER 詳細: Specimen was frozen on a custom-made manual plunge apparatus in a cold room environment maintained at high humidity. Vitrification occurred following 25 ms of exposure to low power UV irradiation..