<div dir="ltr">Dear community,<div><br></div><div>This is my first adventure in crystallography and it seems a rather challenging one.</div><div>I obtained at dataset at 5.06 A resolution of a 600 residue protein.<br></div>
<div><br></div><div>3 domains of the protein are already solved by earlier x-ray crystallographic studies.<br></div><div><br></div><div>While trying to grow better diffracting crystals and waiting for the next chance to shoot crystals at the synchrotron, I am trying to fit the already solved domains into the electron density of the low data set.</div>
<div><br></div><div>Although I tried phaser using the solved structures, I never got a TFZ higher than ~ 6.4.</div><div>Interestingly, Balbes constructed a model which does not contain any of the already solved domains, but, when using the Balbes output in Phaser, I obtain a TFZ score of 11 and an LLG of 330. (4 molecules / ASU; P2221)</div>
<div><br></div><div>If this solution is correct, then in principle it should be possible to fit the already known domains into the structure (I am dreaming of something like a electron-microscopy-type fitting or the early ribosome structures). Phenix refinement however raises the Rfree, instead of lowering it. From my limited knowledge in the field I figured this may be that a) this kind of refinement doesnt work at this resolution or 2) the MR-solution was not really a solution.</div>
<div><br></div><div>So I was very excited, when I found the recent papers about DEN refinement and Phenix-Rosetta.�</div><div><br></div><div>I am trying DEN refinement first, using the already solved structures as a reference. The den_refine program nicely creates 2760 DEN restraints. However, after 1 cycle (about 6 h of CPU time at 30 processors), the Rfree goes up (Initial: 0.49; final: 0.57). Indeed, the grid search seems not to be successful, since in the grid search for gamma/w, all Rfree values are rather similar and the lowest Rfree is 0.54.�</div>
<div><br></div><div>So I would like to ask the community, if ...</div><div><br></div><div>1) I shouldn't trust the phaser TFZ score too much and maybe it is just no solution after all?</div><div><br></div><div>2) the DEN parameters of phenix.den_refine can be optimized to allow to refine a super-low resolution structure? (I am using the standard parameters from the example:�<a href="https://www.phenix-online.org/documentation/den_refine.htm">https://www.phenix-online.org/documentation/den_refine.htm</a>)�</div>
<div><br></div><div>3) 1 cycle is simply not enough and I should let the computation run for a few days to see, if the Rfree drops down later in the run?</div><div><br></div><div>4) Even though Phaser gave me the solution in P2221, may it be that I am trying to refine it in the wrong spacegroup and that's why I fail?</div>
<div><br></div><div>Sincerely,</div><div>Erik</div><div><br></div><div><br></div></div>