Hello,<br><br>I am refining a low-resolution structure of a protein complex (4.4 Å).<br>The structure was solved by molecular replacement using structures of individual subunits as search models.  Here are some of the questions that I have (and also points raised by our reviewers.)    Your thoughts and suggestions on how to proceed with this will be very much appreciated.<br>
<br>1) The B factors are very high (300-400).  The Wilson B calculated by phenix.xtriage is ~200.   Is this something I should be concerned about?<br><br>
2) What is the expected (or &#39;acceptable&#39;) gap between R and Rfree at this resolution.  (Current R = 27%, Rfree = 33%)  Does this sound reasonable?<br>
<br>3) It has been suggested to me that I should try adding riding hydrogens during the last round of refinement (to help with geometry).  Is this something I should do?<br>And if so, should I remove them when I deposit the file to the PDB.   (Hydrogens at 4.5A are probably going to raise a lot of eyebrows...<br>
<br>4) What is the &#39;acceptable&#39; coordinate and phase errors for structures at this resolution.  (They are now 1.4Å and 36˚)<br><br>5) I have been using secondary structure restraints during the refinement, which seems to work reasonably well.  I also tried refinement using reference model (the structure of the individual components).  But refining with reference models seems to result in high RMSD bonds/angles.  Is there something I&#39;m missing?<br>
<br>Short of growing better crystals, are there other strategies I should be trying with the existing data?<br><br>Thank you!<br><br>Jenn<br>