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Hello Bing,<br>
<br>
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dir="ltr">General strategies: I select COOT for the model
building; using phenix.real_sapce_refine for the real space
refinement with secondary structure restrain and REFMAC for the
reciprocal space refinement.<br>
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<br>
what's the purpose of reciprocal space refinement if you don't have
any measured reflections ? (it's cryo-EM data, which is map!).<br>
<br>
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<div>Few questions are listed here.<br>
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1. The map was too bigger to open it in COOT. The
phenix.map_to_structure_factors was used to obtaine ~120 MB
sized MTZ file (still a little big for my computer). I
manually build up the whole ball-shaped phage with the rigid
body fit in COOT (from two X-ray structures to 120 chains). My
first question will be: In this case, should I crop the map in
Chimera or other software and only focus on a small asymmetric
unit to do the model building and the followed refinement.<br>
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<br>
Sometimes box is way larger than actual model. If that's the case
you can try<br>
<br>
phenix.map_box model.pdb map.mrc<br>
<br>
Also, you can do<br>
<br>
phenix.map_box model.pdb map.mrc selection="chain A"<br>
<br>
that will give you a box with map and selected part of model.<br>
<br>
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<br>
2. I would like to do a real space refinement after the model
building. <br>
Input files:� �<br>
��� A pdb file I just built up from COOT<br>
��� A original MAP file<br>
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<br>
This is all you need for real-space refinement.<br>
<br>
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��� A transfered MTZ file<br>
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<br>
This is fiction, you don't need it.<br>
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��� Two restraint files from two X-ray structures by ProSMART
(TXT formart)<br>
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<br>
Phenix does not recognize ProSMART. You can use Phenix tools to
generate secondary structure restraints, such as
phenix.secondary_structure_restraints.<br>
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The refinement parameters I would like to select in GUI
interface:<br>
��� minimization_global, rigid_body, local_grid_search, adp<br>
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<br>
At 6A resolution you are not going to see sidechains, so no need to
do local_grid_search.<br>
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��� Use secondary structure restraints<br>
��� Reference model restraints: use starting model as
reference, main chain, side chain, fix outliers, secondary
structure only<br>
��� Rotamer restraints<br>
��� Ramachandran restraints<br>
��� Show per residue<br>
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I'd do a default run first to see what happens.<br>
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My second set of questions: Should I select the MAP file (~870
MB) or the MTZ file (~120 MB)? </div>
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<br>
If MTZ file is a FT of the map (full box of reflections, not a
sphere) then both files (map and mtz) contain exact same
information, one in real space and the other in reciprocal space.
I'd use original data (map), not manipulated one (mtz).<br>
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<div>Is that necessary to add the two restraint files from
ProSMART if I use the starting model as reference? </div>
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Phenix does not recognize ProSMART.<br>
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<div>Is the refinement parameters selected properly?<br>
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See above.<br>
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3. I gave a try by phenix.real_space_refine. An first error
showed up:<br>
Number of atoms with unknown nonbonded energy type symbols:
6840<br>
��� "ATOM��� 184� HG1 SER 1� 12 .*.���� H� "<br>
��� "ATOM��� 458� HG1 SER 1� 30 .*.���� H� "<br>
��� "ATOM��� 699� HG1 SER 1� 45 .*.���� H� "<br>
��� "ATOM��� 720� HG1 SER 1� 47 .*.���� H� "<br>
��� "ATOM��� 762� HG1 SER 1� 50 .*.���� H� "<br>
��� "ATOM�� 1241� HG1 SER 1� 81 .*.���� H� "<br>
��� "ATOM�� 1465� HG1 SER 1� 95 .*.���� H� "<br>
��� "ATOM�� 1747� HG1 SER 1 113 .*.���� H� "<br>
��� "ATOM�� 1758� HG1 SER 1 114 .*.���� H� "<br>
��� "ATOM�� 2173� HG1 SER 1 141 .*.���� H� "<br>
��� ... (remaining 6830 not shown)</div>
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<br>
Looks like PDB file is bad. Serine residue does not have HG1, it
should be HG. Get rid of H by using<br>
<br>
phenix.reduce model.pdb -trim > model-no-h.pdb <br>
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<div>I tried phenix.ready_set to fix this problem according to a
previous discussion but it gave me another error: ENDMDL
record missing at end of input.<br>
Thus my third question will be how to fix the first error?<br>
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<br>
Looks like your file contains several models (MODEL-ENDMDL). This is
not supported. Convert it into multi-chain model:<br>
<br>
phenix.models_as_chains model.pdb<br>
<br>
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Thank you for patience! I would really appreciate your help!</div>
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<br>
You are welcome! Let us know should you have any more questions or
need help!<br>
<br>
Pavel<br>
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