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| eman2:negativestain [2025/06/19 01:12] – created steveludtke | eman2:negativestain [2026/03/05 14:35] (current) – steveludtke |
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| * Negative stain is generally limited to ~20 A resolution due to stain penetration and general staining quality. It is also basically limited to showing you an envelope surrounding the protein, though there may be some residual low contrast density present inside the stain envelope with inverted contrast. | * Negative stain is generally limited to ~20 A resolution due to stain penetration and general staining quality. It is also basically limited to showing you an envelope surrounding the protein, though there may be some residual low contrast density present inside the stain envelope with inverted contrast. |
| * Confusingly for novices, single particle reconstructions of negative stain images can sometimes yield FSC curves which extend to subnanometer resolution, even when they are clearly not exhibiting any secondary structure features. This is due to a misunderstanding of what the FSC curve is actually telling us. In this case, it simply means that the sharpness of the stain envelope is better than 10 A. ie - even though it isn't representing high resolution features, it is doing so very self-consistently, resulting in a sharp edge, which corresponds to "high resolution". Such FSC curves are meaningless (or at least not useful in assessing the features you can expect to see). | * **Resolution is not the same as in cryoEM!** Confusingly for novices, single particle reconstructions of negative stain images can sometimes yield FSC curves which extend to subnanometer resolution, even when they are clearly not exhibiting any secondary structure features. This is due to a misunderstanding of what the FSC curve is actually telling us. In this case, it simply means that the sharpness of the stain envelope is better than 10 A. ie - even though it isn't representing high resolution features, it is doing so very self-consistently, resulting in a sharp edge, which corresponds to "high resolution". Such FSC curves are meaningless (or at least not useful in assessing the features you can expect to see). |
| * There is very little point in collecting large numbers of particles in stain, due to the inherent resolution limit. If your reason to look at large numbers of particles (more than 2-3000, less with symmetry) is to look at variability, just be warned that the conditions in negative stain are quite far from the cryo conditions which are believed to mimic solution conditions. That is, I would be concerned about trusting any 'variability' results in stain. If you are at that point, it is time to move to ice. | * There is very little point in collecting large numbers of particles in stain, due to the inherent resolution limit. If your reason to look at large numbers of particles (more than 2-3000, less with symmetry) is to look at variability, just be warned that the conditions in negative stain are quite far from the cryo conditions which are believed to mimic solution conditions. That is, I would be concerned about trusting any 'variability' results in stain. If you are at that point, it is time to move to ice. |
| * When using the "Import Micrographs & Est. Defocus" item in the project manager (e2rawdata from the command line), do NOT specify the X-ray pixel filter, which eliminates histogram outliers, assuming they are detector artifacts. In negative stain, the noise levels are so low that this detection often fails and zeroes out high contrast pixels which should be retained. | * When using the "Import Micrographs & Est. Defocus" item in the project manager (e2rawdata from the command line), do NOT specify the X-ray pixel filter, which eliminates histogram outliers, assuming they are detector artifacts. In negative stain, the noise levels are so low that this detection often fails and zeroes out high contrast pixels which should be retained. |
