(January 2001)
I am using Bobscript to generate image files with electron densitiy maps. Is it possible to save them in postcript format? If this is not possible what is the best way to submit rgb files to publication??
Bobscript outputs postscript files by default (without any flags), i.e. bobscript &lgt; input.inp > output.ps.
Also, you can put labels etc. within Bobscript itself; no need to take it elsewhere for that purpose. The area command on top of the file can set the exact size of the output for printing or including in any documents. To help with this, there is a grid (in the O distribution): edit.ps. Usage: print or copy onto an overhead, overlay on your plot and read off postscript coordinates. Displaying a file with `ghostview' or `gv' and reading the mouse coordinates is another easy way to determine PostScript coordinates.
The preferred format(s) is (are) in most cases explicitely mentioned in the instructions for authors. Most journals will like TIFF and EPS. On a related issue: If a journal requests 400 dpi (dots per inch) pictures and you plan the reproduction (print) size to be i.e. 8x4 inches, that means that you need 3200 dots on x and 1600 dots (pixels) on y. So if you make an RGB or TIFF file make sure it is 3200x1600 pixels in the first place. Importing a standard 'render' output of 1200x1200 pixels and then 'set resolution to 400 dpi' in Photoshop is not nearly a cure for good quality pictures …... and, talking about photoshop: Do not forget that TheGimp is out there!
'Hardware' (and some Software)
Oils and cryo-protection
(January 2001)
This started off as a question about low-temperature data collection:
How do you collect a low-temperature dataset with a deoxyhemoglobin crystal without exposing the crystal to atmospheric air?
The discussion evolved into one about oils used for cryo-protection. Summary from a helpful bulletin board member:
It is clear from the responses that oil is no panacea, but it seems to work very well in many cases. We've had good luck so far, but organic solvents in the drop may pose problems. We do see diffuse scattering due to Si, but not enough to be concerned. Some suggest drying the oil as an aid in removing the water layer on the surface of the crystal. We suspect technique is very important here, and oil composition less important. We tried a silcone-based diffusion pump oil from Dow (750). It is thermally stable and claims to be radiation and oxidation resistant. References:
• S. Parkin and H. Hope, J. Appl. Cryst. (1998) pp945-953. Section 2.1 of this paper recommends Paratone-N, possibly saturated with water. Recommends against Si- or F-containing oils due to higher scattering power. Half the xtals they have tried survive oil treatment. Main problems are mechanical strength, loss of water by xtal resulting in cracks, or difficulty removing water layer. They are advocates of quick- dunk cryoprotection when oil does not work.
• H. Hope, Annu. Rev. Biophys. Chem. 1990 19:107-126. More details of oil/cryo handling (covering hanging drop with oil and dragging xtal through oil-water phase, wicking etc.)
• Riboldi-Tunnicliffe and Hilgenfeld, J. Appl. Cryst. (1999) 32, 1003-1005
• "The structures of deoxy human haemoglobin and the mutant Hb Tyrosine a42->His at 120K" Tame and Vallone, Acta Cryst D56, 805-811. It is possible to protect the crystals from oxygen using dithionite, at least long enough to cryo-cool them.
Then some accounts from users, both positive and negative:
Using oil is an excellent method and has been used for many years by small molecule crystallographers for freezing extremely air-sensitive crystals. I've used it successfully with macromolecular crystals too. I've used a perfluoropolyether oil for this (used to be Riedel- de-Hahn RS3000, but this hasn't been manufactured for many years. I haven't needed any since '95 so haven't looked into it seriously, but new sources have been discussed on this BB in the last year or so). For the small molecule case, it works by providing a physical barrier - the amount of oxygen that can diffuse through the oil is actually quite small. Also, something I didn't mention before - most air-sensitive compounds are actually sensitive towards hydrolysis, so it isn't the oxygen that reacts directly with them. Water, of course, is not terribly soluble in perfluoropolyethers. However, nothing which isn't pfpe is soluble in pfpe oils. For macromolecules, it stops evaporation of water from the crystal, giving you time to cool to create a vitreous phase. But the migration of oxygen through the oil is also limited, so that helps too.
We have used MO (mineral oil) only occasionally and with indifferent results. that is, sometimes we get useful freezing but never better diffraction. We purchased a 'panjelly kit' and tried their suggested protocols. Nothing (including lysozyme) diffracted any better than we had obtained by conventional means and in no way did we find any help annealing crystals. Add to this that the stuff does not perform well in the cold room we let it languish on the shelf for some months.
I tried 3 different oils and their mixtures - all successful so far and now I always use it by default. The first oil was the machine oil from the workshop, the latest - Paratone N. No special preparations were required.
Our laboratory has used oil, in place of a cryprotectant, for cubic lipid phase bacteriorhodopsin crystals successfully...
We've tried oil once so far, on crystals of a rather large protein-DNA complex grown from Ammonium sulphate. At room T, they diffracted to 13Å, and frozen in propane, 13Å, but the ones we tried in oil didn't yield a single spot (at a synchrotron). (And we did have help from someone who swears by oil). Now granted, these crystals seem to be useless no matter what we do, but oil-freezing certainly didn't improve things!
The oil method has worked very well with four different crystals in my hands, and it is now the first thing I try. It decreased mosaicity with regard to other cryos in one case, and proved essential in freezing one extremely fragile crystal without damaging it. The other advantage I find is that you do not need a artificial mother liquor. I have also had one crystal that it did not work with, so it is not always a sure thing. I have a feeling that in that latter case it may have had something to due with high solvent content. Briefly the technique I employ is as follows (for hanging drops):
1. cover the drop on the coverslip with a small amount of oil (20-40 ul). When I first read of this technique, I was eager to try it a a troublesome crystal and actually used fresh vacuum pump oil. it worked like a charm, and I have used it since with no trouble.
2. with a loop, fish the crystal out. I like to use a loop smaller than the crystal (spoon it). I get less of the mother liquor sticking to the crystal/loop that way. I also find that it is not to difficult to ge rid of any risidual mother liquor by passing the crystal back and forth through the mother liquor/oil interface. I had trouble with this and loops big enough to hold the entire crystal. The oil "glues" the crystal to the loop.
3. plunge in liguid N2 or freeze in a stream. I usually plunge myself.
Oils are great. We use perfluoropolyether, paratone-N, and 75:25 or 50:50 paratone- N:mineral oil. At least in one case where 100% paratone-N cracks the crystals, the 75:25 mixture worked.
I frequently use oils when using high salt precipitants as the phase difference traps the salt in the crystal and stops diffusion between the cryo protectant and the crystal. I have found it usually works for most high salt crystals and some PEG grown crystals as well.
The problem is the oils diffract and give diffraction rings. I have always found parrafin oil (Hampton) works fine. It gives rings at ~4 and 2.3Å so a normal data set has only two rings. The rings are usually quite small so I don't loose much data. If it wasn't for the rings I'd use oil as first choise as it usually works first time and therefore saves time fiddeling with cryoconditions. Recently I got three to work from: 4M NaFormate, 2.5M A.S. and 24%PEG grown crystals