Structural information on how base excision repair enzymes remove DNA lesions in the context of chromatin has been scarce. This constitutes a significant gap in our understanding of the mechanisms involved in etiology of human diseases. The main advantage of our protocol is that we have optimized sample preparation using standard laboratory equipment.
This will allow other biochemists in the field to complement their biochemical work with technically feasible cryo-EM studies. Our protocol can additionally be used to investigate how other factors, including pioneer transcription factors, engage the nucleosome. This is an area where structural information has also been limited.
Optimizing the sample preparation before moving onto the freezing conditions was key to moving our project forward, and this is best done with standard biochemical assays in substrate design, binding, and cross-linking conditions. To begin, incubate the NCP and the DNA repair factor of interest on ice for 15 minutes using an optimal buffer for the specific complex. Then, add glutaraldehyde to a final concentration of 0.005%After mixing it well, incubate at room temperature for 13 minutes.
Quench with one molar Tris-CL to a final concentration of 20 millimolar Tris-CL with pH 7.5. Then, concentrate to approximately 50 microliters and exchange the buffer with freezing buffer using a desalting column. Next, determine the absorbances at optical density 280 and 260 nanometers and concentrate further if needed to reach 1.3 to 3 milligrams per milliliters based on optical density 280 nanometers.
After preparing the dialysis button, place the button containing the sample on top of a polyethylene glycol bed with the membrane facing down. To perform the purification by size exclusion chromatography, wash and equilibrate a size exclusion column with 60 milliliters of distilled water, followed by 80 milliliters of freezing buffer overnight at a rate of 0.4 milliliters per minute. Then, immediately analyze the peak fractions and concentrate those fractions containing the histone's infusion protein MBP-PolBeta-APE1.
Demonstrating the following procedures will be Elizabeth Viverette, a post-baccalaureate trainee from the cryo-EM core here in NIH. After turning on the plunge freezer and filling the humidifier with 50 milliliters of distilled water, set the chamber temperature to 22 degrees Celsius and humidity to 98%Then place the ethane cup and liquid nitrogen cup in the respective space holders. Afterward, cover the ethane cup with the ethane lid dispenser and carefully pour liquid nitrogen over it while also filling the nitrogen cup with liquid nitrogen.
When the liquid nitrogen level has stabilized and reaches 100%and the temperature reaches minus 180 degrees Celsius, carefully open the ethane valve and fill the ethane cup until it forms a bubble on the clear lid. Then remove the ethane dispenser. Place two filter papers onto the blotting device and secure them with a metal ring.
Go to the setup and set the blotting parameters as described in the manuscript, then select A-Plunge and click on OK.On the main screen, click on Load Forceps and load them with a grid with the carbon application side facing toward the left. Calibrate the forceps, adjusting the Z-axis to ensure a solid blot. Click on Lower Chamber and apply three microliters of the complex.
Then click on Blot A-Plunge, which will rotate the grid to blot from the front and will plunge-freeze it. After transferring, store the grid in the grid box in the liquid nitrogen chamber. When all four grids have been frozen and placed in the grid box, rotate the lid to neutral position where all the grids are covered by the lid and tighten the screw.
Before loading the samples in the microscope, place the vitrified grids on a ring and secure it using a C-clip under liquid nitrogen in a humidity-controlled room to avoid ice contamination. When loading the microscope, insert the grids in a 12-slot cassette. Shuffle the cassette into a nanocab capsule and load it into the autoloader.
Then, transfer the grid from the cassette to the microscope stage. Adjust the stage to eucentric height by wobbling the stage 10 degrees and simultaneously moving the Z-height until minimal planar shift is observed in the images. Once the eucentric height is reached, start imaging by taking a 3-by-3 montage image of the grid in which each montage is taken at 62x magnification to obtain the atlas.
After picking three squares of varying sizes and taking the eucentric height, image at 210x magnification. Once a square is imaged, pick one hole each from the edge, center, and in between within the squares. Then, image each hole at 2, 600x magnification.
Take the high magnification image from the center of the hole at 36, 000x magnification and at 7.1 seconds exposure time, 60 frames, 1.18 pixel size, and three-micrometer defocus. See representative images of screening. To determine the ratio of NCP to MBP-PolBeta-APE1 to form stable complex, electrophoretic mobility shift assays were performed, which showed a singly-shifted band of the NCP with five-fold molar excess of MBP-PolBeta-APE1.
In smaller volume, assembly of the complex of NCP-PolBeta-APE1 demonstrated that the sample was overly cross-linked and resulted in aggregates without discernible individual complexes. However, ten-fold dilution in NCPs resulted in significantly reduced aggregation and improved particle stability. The preparative gel and size exclusion methods can be combined where preparative gel improves the quality of the NCPs when they contain a significant amount of high molecular weight aggregates, followed by the purification of the complex via size exclusion.
The results show that both methods can be used independently to generate stable complexes. Further, the data collected from both grids show almost identical two-dimensional classes and three dimensional maps at approximately 3.2-angstrom resolution. An optimal ratio of NCP to DNA repair factor should be determined first, then cross-linking of the complex with glutaraldehyde the should be performed at at least 10 times more dilute than the concentration needed for freezing.
After obtaining a cryo-EM 3D map of the complex, further structural refinement will be needed to determine how the DNA repair factor is found and identify what regions are important for these interactions.
