Graphene Oxide Grid Preparation
Today one of the biggest challenges in cryo-EM is grid preparation. Common difficulties lie in ice thickness, too high or too low particle numbers, particle aggregation and a preferred orientation of the particles. In many cases a support layer can be of use. For large proteins like ribosomes an amorphous carbon support layer has proven useful1. But for small proteins amorphous carbon is not an option due to its high background noise in combination with the already low contrast of the protein. Often the contrast is reduced to a point where alignment is no longer possible.
Graphene based support layers are an attractive alternative. They offer similar advantages like amorphous carbon while being almost electron transparent. While the preparation of pure graphene supports can be time consuming, they have been shown to work2. Graphene itself is hydrophobic but through a treatment with hydrogen plasma it is possible to make it hydrophilic and increase particle absorption on the grid2. An alternative graphene based support is graphene oxide, which is hydrophilic on its own3. Graphene-oxide is commercially available as solubilized flakes in sizes ranging from less than 0.5 µm to bigger than 50µm. This allows for a simple grid preparation in a few minutes without expensive equipment.
Areas of criticism are a slight decrease in the electron transparency compared to graphene and a variance in support layer thickness due to the possible deposition of multiple layers of flakes in a random manner. While the decrease in the electron transparency of single sheet graphene oxide compared to single sheet graphen is not relevant in most cases it is necessary to reduce the deposition of multiple flakes on top of each other, which will affect the image quality. Therefore it is critical to optimize graphene oxide preparation conditions and protocols. We started out with the protocol suggested in 3 and adapted it to a point where we are able to obtain mostly uniform deposition of graphene oxide flakes, resulting in mainly monolayer and bilayer areas with minimal amounts of empty holes or multiple-layer flake depositions (see end of the video for examples).
We identified that the type of grid, glow discharge conditions, graphene oxide flake size and concentration and deposition time all play a role in the final outcome. Additionally, a crucial step in the preparation procedure was the extensive washing of the grids directly post graphene oxide deposition. For us, it was possible to adjust for different grids by adjusting the glow discharge conditions while keeping all other parameters the same.
We have successfully applied this protocol of Graphene Oxide grids in several projects 4.
References:
1 R.M. Voorhees, I.S. Fernandez, S.H.W. Scheres & R.S. Hegde (2014). Structure of the mammalian ribosome-Sec61 complex to 3.4 Angstrom resolution". Cell, 157, 1632-1643.
2 C.J. Russo & L.A. Passmore (2014). Robust evaluation of 3D electron cryomicroscopy data using tilt-pairs. J Str Biol, 187, 112-118.
3 R.S. Pantelic, J.C. Meyer, U. Kaiser, W.Baumeister, & J.M. Plitzko (2010). Graphene oxide: a substrate for optimizing preparations of frozen-hydrated samples. Journal of structural biology, 170(1), 152-156.
4 A. Boland, T.G. Martin, Z. Zhang, J. Yang, X.-C. Bai , L. Chang, S.H.W. Scheres, D. Barford (2017). Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution. Nat Struct Mol Biol., 24(4):414-418
Protocol:
Materials: GO Solution Sigma; ddH20; Tweezers (Dumont N5AC); Pipette (3µl and 20µl); Tabletop centrifuge; Parafilm; Whatman filter paper No1
1. Dilute GO solution 10x to 0.2mg/mL with ddH20
2. Spin for 15 seconds at approximately 300 rcf to remove aggregates
3. Glow discharge grids with carbon side up (Quanitfoil Au 300 R1.2/1.3 1minute at 0.2mBar and 40mA; Quanitfoil Cu 300 R1.2/1.3 60 seconds at 0.2mBar and 30mA)
4. Prepare a flat and clean working area (e.g. with parafilm)
5. Prepare 3x 20µl drops of ddH20 for each grid you want to prepare
6. Take up grids with anti-capillary tweezers
7. Place 3µl of the diluted GO solution (1) on the carbon side of the grid
8. Incubate for 1 minute
9. Remove solution with filter paper.
10. Take up first drop of ddH20 with the carbon side and remove it with the filter paper.
11. Same for the second drop.
12. Take up third drop of ddH20 with the back side of the grid.
13. Dry for 5 minutes
Here are some glow discharge conditions that worked in the past for Quantifoil grids. As said before these conditions vary between grids :
Edwards Sputter Coater S150B: 0.2mBar, 40mA, 60 seconds (Au 300 R1.2/1.3)
Edwards Sputter Coater S150B: 0.2mBar, 30mA, 60 seconds (Cu 300 R1.2/1.3)
K100X Glow Discharge System: 0.2mBar, 50mA, 75 seconds
