Non-uniform refinement strategy on 100kDa protein with no symmetry

3D Refinement
1

Hi,
I am working on 100kDa protein having no symmetry. I have collected data in Talos Artica. While running Non-uniform refinement New with default parameter it is showing resolution (4-5 ) angstrom and end up in 3 iteration only.But map is not looking good.
But then by changing these following parameter
No.of passes 2
initial resolution 15
do symmetry alignment off
minimise over per particle scale on
The resolution it is showing in (6-7) angstrom and map is also looking better.But it is not improving.I have noticed one thing that at 3 iteration it is showing resolution in between 4-5 angstrom but after 6 iteration it is showing resolution in between 6-7 angstrom.
Can you please comment on this if I am running job with wrong parameter.
Even by using mask it is not improving.

Thanks,

Regards,
Priyanka

2

Can you post more information? 2D classes before running ab initio/3D refinements, the NU refined maps with each parameter set to explain what you mean by “bad” and “good”.

3

Hi,
This is 2d class averages I am using for refinement.
while running abinito with more than 1 classes.Is it possible that one of orientation of map is split as a one of a class? As I faced this problem which make me difficult to understand that it is different orientation or different conformation.

P1_J222_selected_36_classes
P1_J222_selected_36_classes999×398 41.6 KB

Thanks,

Regards,
Priyanka