Hi Jerry,
 
Thanks a lot for your helpful suggestions. I am a novice in the field of structural biology and all of your suggestions and inputs are very help full to me and I guess it will be super helpful to the others like me as well. I was not aware of this CCPEM mailing list, came to know a month ago. It is a great platform as experts and novices like me can share their experiences here and I think it will help a lot of people want to learn cryoEM.
 
About the 2D classes with both T2 and T8, I got more than 50 good classes (“Ignoring CTFs until the first peaks" were set to No). The only difference between the T2 and T8 is that I got beta-sheet separation in T8. But as you said it may overfit and also it is a good idea to redo 2D classes with subset selection using the T2 parameter for the first and second 2D classification steps (if one needs to see beta-sheet separation in the 2D classes. The initial models were generated using around 50 good 2D classes.   
I generated initial model from the T8 2D classes and it looked fine but in a 3D classification all four classes very bad. There were a lot of places with missing electron density. I did 3D also using T8 parameter and that may cause this problem. I shall try the 3D classification again using T2 parameter and will let you know how it goes.
 
Can anyone please tell me is it okay to have the spearhead like electron density at the second refine map? I mean should I do another round of refinement with this map or this is garbage (even though the resolution is 3.8) and should start over with another 3D model?
 
Also, have a very stupid question, in the second refinement, I kept the symmetry option C1 and just change the twist (179.4) and rise (2.375). I was thinking this is how I am going to impose the pseudo-21 helical symmetry but I am not sure. Should I keep the symmetry parameter empty instead of putting C1 or I need to just write pseudo-21?  
 
Thanks,
Victor

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