Cryo-EM: Preparation, acquisition, and processing of data
本实验方法提取自研究论文:
Sequence-programmable covalent bonding of designed DNA assemblies
Sci Adv, Aug 17, 2018; DOI: 10.1126/sciadv.aau1157

For the brick-like object with TT motifs 1 to 3, concentrations between 700 and 850 nM were used. The samples were applied to C-Flat 1.2/1.3, 1.2/1.3, 2/1, or 2/2 thick grids (Protochips). Plunge freezing was performed with an FEI Vitrobot Mark V instrument with a blot time of 3 s, a blot force of −1, and a drain time of 0 s under 95% humidity and 22°C.

For the brick-like object with TT motifs 1 to 4, concentrations between 560 and 800 nM were used. The samples were applied to C-Flat 1.2/1.3, 2/1, or 2/2 thick grids. Plunge freezing was performed with an FEI Vitrobot Mark V instrument with a blot time of 3 s, a blot force of −1, and a drain time of 0 s under 95% humidity and 22°C.

Automated data collection was performed on a Titan Krios G2 electron microscope (FEI) operated at 300 kV and equipped with a Falcon III direct detector (FEI). We used EPU for single particle and FEI tomography for tilt series acquisition. For all brick-like objects under different conditions, movies comprising 15 frames, 1.5- to 2-s exposure time, and a total dose of 60 e−/Å2 were recorded on a Falcon III (FEI) direct electron detection camera in fractioning mode at a calibrated magnification of ×29,000 with a magnified pixel size of 2.3 Å. Defocus values ranging from −1 to −3 μm were used.

The recorded movies were subjected to motion correction with MotionCor2 (66), and subsequently, contrast transfer function parameters were estimated with CTFFIND4.1 (67). All subsequent processing steps were performed in RELION-2.1 (65, 68). For each data set, references for automated picking were calculated from about 5000 manually selected particles. With the picked particles, multiple rounds of reference-free 2D classification were performed. The best 2D class averages, as judged by visual inspection, were selected. An initial model was produced from a bild file generated by CanDo. After multiple rounds of 3D classification, the classes showing the most features were selected for 3D autorefinement, and subsequently, post-processing for sharpening of the refined map was performed with different manually selected B factors.

A cryo-tomogram for validation of the twist direction was acquired with FEI tomography, with a defocus of −3 μm at a calibrated magnification of ×29,000, corresponding to a magnified pixel size of 2.3 Å. The session was set up as bidirectional tilting in increments of 2° up to 50°, and the dose per image was set to ~2 e2. The resulting tilt series was processed with the IMOD 4.9 routine (69).

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