I have a few questions relating to 3D Classification and Align 3D jobs.
What does performing 3D classification with 2 map inputs achieve? Does it just redistribute the particles into one of these 2 classes? If I understand correctly, then running it with 1 map does nothing since it would output 1 class.
I was thinking of running 3D classification with 2 input maps of my protein. One of them is an octamer and the other is a nonamer, but they’re both rings that are made of the same repeating subunit.
In this case, a solvent/focus mask covering one would not be a good fit for the other. Where could I take it from here?
I was thinking that since, according to my understanding, I have to run an Align 3D job to align them before doing that, maybe I can align both proteins in a way where one subunit is perfectly aligned and have the focus mask be on that region, but I’m not sure how to achieve this, or if it is reasonable. What would I do/what masks would I use in the Align 3D or the 3D Classification job to achieve this?
I am having trouble deciding what the different types of mask inputs do in the Align 3D job, i.e. What does a reference mask do in comparison with “Mask (map to align, connection 1)” and what does “create mask inputs do”. I read through the tutorial for the job, but I don’t really understand it. Why would I need to use either of these options or both?
I apologize if any of these questions are silly, but I’m really struggling to understand these jobs and what the masks do. Thank you in advance.
As you said, 3D Classification with two maps will redistribute particles among those two classes, with each particle going to the class it matches best. If the classes are very different from each other this can work quite well (as it does in the FaNaC1 case study), but often it’s best to provide more classes to allow for a junk class and perhaps some unforeseen heterogeneity beyond the octamer/nonamer.
And yes, a 3D Classification with one class would do nothing. In fact, you can’t run a job with one class! At least two are required.
Two input maps
If the two rings are centered on the same point, you could use a sphere (or perhaps a disk) centered on that point that’s big enough to cover both. However, I think your idea of using Align 3D to center one subunit makes sense, but in this case I’d probably use a mask covering the opposite side of the ring instead of the subunit you aligned, because the opposite side will have the greatest difference between the two classes.
Align 3D
Each mask is applied to one volume. So the reference mask is applied to the reference volume, “Mask (map to align, connection 1)” is applied to the first volume you dragged into the volumes input, “Mask (map to align, connection 2)” is applied to the second volume you dragged into the volumes input, etc. I’ve made a note to make this clearer in the guide!
“Create mask inputs” controls whether these mask input slots exist at all. By default they are hidden, and the volumes are aligned unmasked.
I hope that helps! Please let me know what questions you still have!
