Issues With 3D Variability

Each time I use 3D variability the end result is a map with frames that track changes of volume contouring, as opposed to actual conformational changes. I am using default settings in 3D variability, and I am solving 3 different modes. I have tried masking the variable region as well as the entire molecule and still get the same result. I have also tried using maps generated from different ab-initio classes and I’m still geting the same result.

Are there any parameters that I can change that would favor screening for conformational changes?

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@Lucas do you mean that the variability components you get show only increase/decrease in the overall density of the protein, rather than motion or flexing?

How small is the protein, and is it a membrane protein? Is there a lot of detergent or other contaminants in the sample? How many particles do you have, and what resolutions have you got so far in a consensus refinement?

If it’s a small protein, you can try to use the “High-pass filter” option and set that to say 20A. This will force 3DVA to only consider information above 20A in the variability components, and can help with small proteins, or cases where there is so much contamination or per-particle contrast variation that swamps the variability of the protein itself.

Correct. I’m not seeing any conformation changes or flexing, just increases/decreases in density. I have 433,000 particles, and the estimated resolution on my best map is 3.05 A. The particle has D3 symmetry and is a 230 kDa hexamer. It is not a membrane protein. I will try the high-pass filter and let you know what happens. Thanks for your response.

UPDATE: Adjusting the high-pass resolution worked like a charm. Thanks @apunjani for the advice.

@Lucas thanks for reporting - awesome!