Leginon

Hello William,

I have been using Quantifoil Copper 200 mesh grids with R2/4 spacing.

These are nice for the automated hole finding because the holes are 4 um apart, which makes picking the correct hole easier.

I think this is what the scripps people use, yes?

Cheers,

Jonny

Got this from Joel:

Rh/Cu 400 Mesh grid with 2um hole and 4 um spacing is his favorite.

In recent published work (the recent Cheng et al and Stagg et al 2006 JSB papers), C-flat grids were used. Were these the standard C-flat grids which have 2 micron diameter holes with 2 microns edge-edge spacing (i.e. like the Quantifoil 2/2 grids) or did you use customized grids (e.g. like the Quantifoil 2/4 grids - with 4 microns edge-edge spacing - which were recommended before)?

William

C-Flat Holey Carbon Film defines edge-to-edge distance while Quantifoil defines center-to-center distance. Therefore, C-Flat that is 2 um hole and 2 um edge-to-edge distance is equivalent to the layout of Quantifoil 2/4 grids.

Anchi

"anchi" wrote: C-Flat Holey Carbon Film defines edge-to-edge distance while Quantifoil defines center-to-center distance. Therefore, C-Flat that is 2 um hole and 2 um edge-to-edge distance is equivalent to the layout of Quantifoil 2/4 grids.

Anchi

I'm still confused. In measurements from our own recent images obtained from Quantifoil R 2/4 grids, the holes are about 2 um in diameter with a 4 um edge-to-edge distance (and 6 um center-to-center distance). The data set collected at Scripps also has 2 um diameter holes with 4 um edge-to-edge distance. I also have images from old data sets in Leeds where Quantifoil R 2/2 grids were used with 2 um diameter holes and 2 um edge-to-edge distance. Also, looking at the Stagg et al paper and making manual measurements from an image of a grid square, it actually looks like there is a 1 um edge-to-edge distance (assuming 2 um diameter holes as I don't think there is a scale bar in the image). The Cheng et al paper has a similar type of image and appears to have 2 um diameter holes with 4 um edge-to-edge distance.

I had a great deal of difficulties with aligning the presets to give sufficient targeting accuracy when I used R 2/2 grids in the past. Even when iterating over the alignment of the different presets a large number of times, it still generally was not accurate enough for the subsequent targeting,

William

Hi. William,

O.K., you are probably right since you did measure it. Quantifoil space is edge-to-edge, too, then.

In addition, the square shown in The 280k GroEL paper did in a way have a customed hole but not on purpose. It was one of the early prototype C-Flat grid Protochip made for us while we worked with them to develop it. Its spacing, according to Joel Quispe was not purposely made into any standard distance, just what the manufacturers thought would be good to cram as many holes in without weakening the film. Whatever you measured out from that has nothing to do with the commercial final product.

The figure in my paper came from a Quantifoil grid that Joel gave me from his stack, so it was likely R 2/4 in whichever way it is defined. According to Joel, the hole sizes from Quantifoil is quite consistent but the spacing is not always spot on.

Therefore, you probably want to order C-Flate at 4 um edge-to-edge distance if you want it to behave more like Quantifoil R2/4 then.

Anchi

The new paper on automation of random conical tilt, currently available on the in press section of the Journal of Structural Biology website, mentions that you have started using a new type grid called Cryo-Mesh. Do you have any more information about these grids - what sort of material they are and also when they might become generally available? I think the paper says that there is a forthcoming paper specifically about the Cryo-Mesh grids and that they will be available from Protochip? By the way, do the Cryo-Mesh grids still have ordered arrays of circular holes (like the Quantifoil and C-flat grids)?

William

Cryo-Mesh grid is still a research project so you will have to wait to get details on that.

(posting for Joel)