Cryogen sample preparation faces challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in new constructed protein structures. Various experimental approaches are used to overcome uneven particle distribution, including adding detergents or membrane mimics to the sample, modifying the grid support film and tilting specific stage during data collection.
This protocol analyzes a simple practical method to address uneven particle distribution through sample preparation optimization, providing our reference for researchers to efficiently illustrate macromolecule structures using cryogen. To begin, collect the pooled protein fractions containing MJ small heat shock protein 16.5. Use centrifugal filters with a 50 kilodalton molecular weight cutoff to concentrate the fractions at four degrees Celsius to approximately 65 milligrams.
After concentration, centrifuge the sample at 16, 000 G for 10 minutes at four degrees Celsius to remove aggregates. Aliquot 50 microliter volumes of the supernatant into 0.2 milliliter thin wall PCR tubes. For transmission electron microscopy, thaw two frozen protein tubes on ice.
Once thawed, flick the tube several times to mix. Then centrifuge the protein solution at 16, 000 G for 10 minutes at four degrees Celsius to remove aggregates. Next, inject the supernatant onto a size exclusion chromatography column and collect 0.5 milliliter elution fractions.
Collect the elucian fractions corresponding to the peak, exhibiting the highest ultraviolet absorbance at 280 nanometers. For buffer exchange, with a pair of scissors, cut a dialysis membrane into 30 by 30 millimeter pieces. Incubate the pieces in distilled water or buffer solutions to equilibrate them.
Next, add 55 microliters of the protein solution to a chamber with 50 microliter microdialysis buttons. Hold the dialysis membrane vertically using forceps and gently touch one edge of the membrane against tissue paper to drain excess liquid. Carefully cover the microdialysis button with the membrane.
Place the O-ring on top of the dialysis membrane and gently roll it into the groove on the button's edge. Submerge each dialysis button in a separate beaker containing the destination buffer with the membrane side facing upwards. Use a syringe with a fine needle to carefully punch the membrane and recover the protein solution from the microdialysis chamber.
Load five microliters of the sample at a concentration of 20 to 120 nanograms per milliliter onto a glow discharge grid. Prepare three water drops of 50 microliters each on a paraffin film sheet. Rub the grid surface against the water drops to wash the sample.
Now load five microliters of filtered 1%urinal acetate solution onto the grid, and immediately pipette away the urinal acetate solution. Load another five microliters of the urinal acetate solution onto the grid. Gently touch the tweezer side of the grid with filter paper to remove excess staining solution and let the grid dry.
Mount the tweezers and glow discharge grid assembly onto the instrument. Then load four microliters of the sample onto the carbon side of the grid. Initiate the plunge freezing process.
To prepare the grids to load onto the transmission electron microscope, or TEM, first place a grid box containing vitrified grids in the auto-grid assembly station and unscrew the lid of a grid box containing vitrified grids. Place the auto-grid ring in the designated cutout space at the assembly station. Carefully move the vitrified grid from the grid box and fit it inside the auto-grid ring.
Now align the disc to position the circular opening on top of the auto-grid ring and grid assembly. To fix the grid into the auto-grid ring, place the C clip insertion tool on top of the grid and press down gently to secure the C clip inside. Rotate the disc back and move the assembled grids into the auto-grid storage box.
Assemble the cassette loading station and cool it with liquid nitrogen. Place the auto-grid storage box in the loading station. Move the grid assembly from the auto-grid storage box to the cassette.
Use the handle to place the cassette into the auto-loader capsule. Check the pin in the auto-loader to confirm its free movement and ensure it is not frozen. Particle accumulation in arrays was observed on all tested grids regardless of buffer conditions or surface charge modifications.
A higher protein concentration combined with negatively charged grids in nine millimolar of mobstress, 50 millimolar of sodium chloride and 0.1 millimolar of EDTA buffer resulted in the desired distribution of single particles within grid holes, reducing protein array formation. This optimized condition led to a high conical FSC area ratio value of 0.80 and an SCF value of 0.859, confirming a uniform particle distribution.
