regarding artcifacts in the 3D reconstruction : particle size and box size?

[Abdul Basit Khan]

Dear Steve and group members,

   Thanks to the tutorial and your help i was bale to generate 3D map/envelope of my protein assembly. though the 3D initial model and refined envelope looked good somehow projecting the expected shape of the complex. But the resolution of the unmasked, masked tight and light rose to 4.8A with wavy CTF curves in the refined models which to my basic knowledge is due to some artificats probably.

one of the reasons I think can be : there are many particles and close to each other. while boxing or picking particle some edges of the adjacent particles gets into the box, will that hamper and add artifacts in making model? so should the box size and particle size be same? usually the box size dimension is kept 1.5 times of particle size cricle

need your suggestions, advice and guidance

Thanks and Regards

Abdul Basit

[Steve Ludtke]

In most cases having a second nearby particle in the same box isn't a problem, but if the particles touch or overlap it can cause issues, particularly if there is any self-consistency in the interaction (eg - 2 hydrophopic patches latching on to each other). As long as there is a gap between the 2 particles it doesn't normally cause an issue. Beyond that you'd have to share your results to offer useful comments. If you prefer not to share publicly, you're welcome to email me privately and I'll give you my opinion. What I'd need to see is:

- a few representative 2-D class averages

- your best isosurface view of your current best map

- x/y/z projections of your current best map ("RngXYZ" button in e2display browser with default options)

- x/y/z projections of one of threed_even_unmasked.hdf from your best refinement folder

- the FSC curve you're concerned with

The box size should be 1.25 - 1.5x the longest axis of the particle to reduce artifacts. If there are particles nearby, making the box smaller doesn't help, because the CTF ringing from the nearby particles overlaps with the particle of interest. Without having the other particle present in the boxed image, accurate ctf correction isn't possible.

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