Cryo-EM Structural Analysis of Holliday Junctions

The purified Holliday junction complex was checked for homogeneity by screening negatively-stained samples on a Morgagni electron microscope (FEI, Hillsboro, Oregon). For cryo preparation, we applied 3.0 μL of complex solution to a C-flat 1.2/1.3 Cu grid (400 mesh) (Protochips, Raleigh, NC), which had been glow discharged at 20 mA for 30 s. Grids were plunge-frozen with a Vitrobot Mark II (FEI) at 85% humidity, offset -2, blot time 7 s. Images were recorded on a Tecnai F30 electron microscope (FEI) operated at 300 kV and using a liquid-nitrogen cooled 626 cryo-specimen holder (Gatan Inc., Pleasanton, CA). We used the semi-automated acquisition program SerialEM (Mastronarde, 2005 (link)) to record 1359 movies with a Falcon II direct detector (FEI) at an underfocus set between 2.7 – 4.2 μm. Each movie consisted of 25 frames, collected in a 2 s exposure of 35.5 electrons/Å². The nominal magnification was 78,000x, corresponding to a magnification of 100,000x on the detector and 1.4 Å pixel size on the specimen. Movie frames were aligned and summed using motioncorr (Li et al., 2013 (link)).

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Laxmikanthan G., Xu C., Brilot A.F., Warren D., Steele L., Seah N., Tong W., Grigorieff N., Landy A, & Van Duyne G.D. (2016). Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction. eLife, 5, e14313.

Publication 2016

Morgagni electron microscope Vitrobot Mark II Tecnai F30 electron microscope 626 cryo-specimen holder Falcon II direct detector

Electrons Frames Frozen Holliday junction Humidity Mark ii Microscope electron Nitrogen

Corresponding Organization :

Other organizations : Brown University, Providence College, Brandeis University, Janelia Research Campus, Howard Hughes Medical Institute, University of Pennsylvania

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Variable analysis

independent variables

Glow discharge conditions (20 mA for 30 s)
Plunge-freezing conditions (85% humidity, offset -2, blot time 7 s)

dependent variables

Homogeneity of the purified Holliday junction complex
Cryo-EM image quality

control variables

Concentration of the complex solution applied to the grid
Grid type (C-flat 1.2/1.3 Cu grid, 400 mesh)
Electron microscope (Tecnai F30, 300 kV)
Cryo-specimen holder (liquid-nitrogen cooled 626)
Acquisition software (SerialEM)
Direct detector (Falcon II)
Magnification (78,000x nominal, 100,000x on the detector)
Pixel size (1.4 Å on the specimen)
Exposure conditions (25 frames, 2 s exposure, 35.5 electrons/Å^2)
Image processing (motioncorr for movie frame alignment and summation)

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