Negative Stain Data: Tutorials, Thon rings, and CTFs?

[Christopher Bottoms]

Dear EMAN2 users and developers,

I've noticed a few things that make working with negative stain data different. For example, I've seen suggestions to set AC to anything between 40 and 80%. I've been using 70%. However, is there an entire tutorial specific to using EMAN2 with negative stain data?

Also, how much weight should I put on the number of Thon rings and the quality of an images CTF when working with negative stain data? I'm currently trying to work with some images that only have one or two Thon rings, for example.

Thanks,

Christopher Bottoms 

University of Missouri

[Steven Ludtke]

Sorry, there is no negative-stain specific tutorial.  Negative stain is generally limited to ~20-25 Å "practical" resolution. Note that I say "practical" here, because at times the standard resolution assessments will give higher resolutions than this. In most cases this is just a sharpening of the stain envelope, and does not actually mean you are seeing all of the molecular features at that lengthscale. The "resolution" with staining is generally limited by the even distribution and penetration of the stain. There have been examples of cryo-negative staining where alpha helices could be resolved, but this A) isn't the same technique and B) isn't what I'd consider a typical result.

General tips:

1) Even if you won't be able to resolve high resolution features, it isn't a bad idea to do some CTF correction for neg. stain, since it will improve the appearance of the model and reduce artifacts. 

2) Yes, likely you will want to use a large % amplitude contrast. I don't have a single good value to give you.

3) As with any other single particle analysis in EMAN, you will need to make sure the images are inverted (or not) such that the particles are white on a darker background.

4) the very strong and irregular stain envelope around the particles can sometimes cause problems with centering and alignment. There are a few tricks you can use for specific situations, but nothing general for all situations. If you run into alignment problems send me a screenshot of the problem and I may be able to suggest some sort of trick.

I should also say that, aside from some rare cases where we add a bit of negative stain to the buffer in a cryo-specimen to improve contrast, we basically never use it (at the NCMI). I'm not saying there is no point in doing it, simply that I view it largely as a technique for A) initial screening, to be replaced by cryo as soon as the specimen is ready or B) very small particles with insufficient contrast for cryo imaging. Even for case B, there are other approaches becoming available.  

Of course, I realize that not everyone has a cryo-equipped scope, but much like x-ray crystallographers go to beamlines as soon as their in-house tabletop xray machines show they have good crystals, if you have a good specimen, there are a number of resources around the country (and the world) where you can pretty easily arrange for a project to do high resolution cryo work, and many many local institutions equipped with decent cryo-scopes for intermediate resolution.

Having said all of that, I'll point out that the EMAN2 documentation is a Wiki, meaning that if someone is interested, they are welcome to start some pages about negative stain...  or extend this discussion on the Google Group.

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Steven Ludtke, Ph.D.

Professor, Dept of Biochemistry and Mol. Biol.         (www.bcm.edu/biochem)

Co-Director National Center For Macromolecular Imaging        (ncmi.bcm.edu)

Co-Director CIBR Center                          (www.bcm.edu/research/cibr)

Baylor College of Medicine                             

slu...@bcm.edu

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[Christopher Bottoms]

Thanks so much! That is very helpful.