ジャーナル: J Struct Biol / 年: 2008 タイトル: A test-bed for optimizing high-resolution single particle reconstructions. 著者: Scott M Stagg / Gabriel C Lander / Joel Quispe / Neil R Voss / Anchi Cheng / Henry Bradlow / Steven Bradlow / Bridget Carragher / Clinton S Potter / 要旨: It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still ...It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still celebrated events. The electron microscope has a resolving power to better than 2A, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4A (FSC(0.5)) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined.
詳細: Protochips C-flat grid: holey carbon with 2um holes and 2um spacing 400 mesh copper grid
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 93 K / 装置: OTHER 詳細: Vitrification instrument: Vitrobot. Grid plasma cleaned for 20s with Fischione 1020 plasma cleaner using 75% Argon 25% Oxygen mix. 手法: Temperature of chamber was 4 degrees C. 0 seconds drain time. Single blot. 0 mm offset. 4 ul sample applied to grid. Blot for 3.5 seconds before plunging.
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電子顕微鏡法
顕微鏡
FEI TECNAI F20
温度
平均: 102 K
アライメント法
Legacy - 非点収差: objective lens astigmatism was corrected automatically using Leginon