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Hi Pramod, Refined LLG value of 1800 and R-factor of 46% at the end of the Phaser run does indicate a good MR solution. Your maps also look quite good and some positive density is visible near side chains. It would be helpful to see your translational Z-scores for both molecules placed, which can be found in the .sol file that Phaser outputs. It is puzzling that your model does not refine. Have your tried feeding your MR model and phases into an automated building program like ArpWarp, Buccaneer or AutoBuild (in Phenix suite)? Hope this helps. Eugene On 17 June 2013 22:28, Pramod Kumar <[log in to unmask]> wrote: > Dear Abhinav > > * I would suggest you integrate in p1 and run pointless.* > after integrating in p1 and running the pointless its still concluding the > SG c2221. > > *How do you determine the resolution cut off of 2.9A?* > the basis of resolution at 2.9A* was the completeness and I/Sigma value. > > Two molecule in ASU further supported by Matthews coefficient and solvent > percentage content. > > *Is this a homology model? or at least side chains replaced by correct > ones, like with chainsaw? > Have you trimmed loops and floppy regions in the model?* > > its not homology model, I have used chainsaw to make the model for > molecular replacement, > and trimmed the model for floppy, loopy ambiguousness regions. > > plz see the snap shot of the model. > > > thanks and regards... > > pramod > > > > > > On Tue, Jun 18, 2013 at 1:16 AM, Abhinav Kumar <[log in to unmask] > > wrote: > >> Hi Pramod, >> I would suggest you integrate in p1 and run pointless. >> If your Rfree is above 0.45, I wouldn't trust the model. >> >> How do you determine the resolution cut off of 2.9A? >> >> In MR, the score should improve when the program places the second copy >> to the first molecule. >> >> Is this a homology model? or at least side chains replaced by correct >> ones, like with chainsaw? >> Have you trimmed loops and floppy regions in the model? >> >> >> Thanks, >> Abhinav >> >> JCSG@SSRL, SLAC >> (650) 926-2992 >> On 06/17/2013 12:36 PM, Pramod Kumar wrote: >> >> *Dear Abhinav Kumar >> * >> *thanks for kind suggestions >> >> * >> * >> I have tried as follow. >> >> >> 1. You should try to identify the correct space group first. >> >> *integration in p21 given the following statics in pointless >> * >> >> * Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation* >> >> * [h,k,l] 0.566 0.957 0.094 25580 0.00* >> >> * [l,-k,h] 0.434 0.853 0.195 25580 0.20* >> >> * >> *integration in c2221 given the following statics in pointless >> * >> >> * Spacegroup TotProb SysAbsProb Reindex Conditions* >> >> ** >> >> * ( 20) 0.997 0.997 00l: l=2n (zone 1) >> * >> >> * moreover Zanuda and molrep's chekall space group option also >> suggests c2221, >> >> 2. A template with 31% identity is not a great model. The number of >> molecules in ASU will affect your chances of success.Hopefully it's not >> large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. >> Did you try phaser? >> >> * >> * * this is the limitation of my data that came from three month >> old crystal, there are two molecules in ASU (what is the extant to say >> "reasonably good" please ?), initially phaser was failed, with the balbes >> output as ensemble it worked with following profile .. >> >> * >> >> *** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 **** >> >> *************************************************************************************** >> >> ** >> >> * Current is Best Solution (first)* >> >> * New Best LLG : 101.7 (8.70)* >> >> * Best Search Component so far: ensemble1 * >> >> ** >> >> *************************************************************************************** >> >> **** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING 2.5.2 **** >> >> *************************************************************************************** >> >> ** >> >> * Resolution of All Data (Number): 2.91 47.64 (15877)* >> >> * Resolution of Selected Data (Number): 2.91 47.64 (15877)* >> >> ** >> >> ** >> >> * Refinement Table (Unsorted)* >> >> * ---------------------------* >> >> * #+ = input number #* = results number* >> >> * #+ #* (Initial LLG & Rval) (Refined LLG & Rval) Unique =# Tmplt SpaceGroup * >> >> * 1 1 908.3 48.8 1804.3 46.4 YES C 2 2 21 * >> >> ** >> >> ** >> >> * Refinement Table (Sorted)* >> >> * -------------------------* >> >> * #+ = input number #* = results number* >> >> * #+ #* (Initial LLG & Rval) (Refined LLG & Rval) Unique =# Tmplt SpaceGroup * >> >> * 1 1 908.3 48.8 1804.3 46.4 YES C 2 2 21 >> >> **but again the refmac is showing >> >> >> >> >> >> * >> >> * *R factor 0.4404 0.4203 >> >> R free 0.4955 0.5027 >> >> Rms BondLength 0.0254 0.0093 >> >> Rms BondAngle 3.1234 1.4581 >> >> Rms ChirVolume 0.1741 0.0706 >> >> * >> * >> >> * >> >> * >> >> * >> * >> >> *3. Did you check for twinning? >> >> * >> >> *TWINNING SUMMARY* >> >> * >> * >> >> *Twinning fraction from H-test: 0.00* >> >> *L-statistic from L-Test: 0.48* >> >> * >> * >> >> * Relation between L statistics and twinning fraction:* >> >> * Twinning fraction = 0.000 L statistics = 0.500:* >> >> * Twinning fraction = 0.100 L statistics = 0.440:* >> >> * Twinning fraction = 0.500 L statistics = 0.375:* >> >> *NO Twinning detected* >> >> * >> * >> >> ** >> >> *thanks and regards >> >> * >> * pramod... >> * >> >> * >> * >> >> ** >> >> * >> * >> * >> >> * >> *On Mon, Jun 17, 2013 at 10:15 PM, Abhinav Kumar < >> [log in to unmask]> wrote: >> * >>> >>> *Hi Pramod, >>> >>> 1. You should try to identify the correct space group first. Did you >>> integrate in p1 and run pointless? ** >>> 2. A template with 31% identity is not a great model. The number of >>> molecules in ASU will affect your chances of success. Hopefully it's not >>> large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. >>> Did you try phaser? >>> 3. Did you check for twinning? >>> >>> * >>> * Thanks, >>> Abhinav * >>> >>> * JCSG@SSRL, SLAC >>> (650) 926-2992 >>> * >>> * On 06/17/2013 09:35 AM, Pramod Kumar wrote: >>> * >>> >>> *Dear group >>> >>> * >>> * I have a crystal data diffracted around 2.9 A*, >>> during the data reduction HKL2000 not convincingly showed the space >>> group (indexed in lower symmetry p1), while the mosflm given C-centered >>> Orthorhombic, and again with little play around HKL2000 given CO, now the >>> model for molecular replacement with closest identity of 31 given a >>> contrast of 2, score 0.30 and wrfac 0.60. but "balbes" uses different >>> models with lesser identity, >>> >>> no matter which way I am going the rFree keep on increasing during >>> refinement in refmac, when I build the model in coot with deletion and >>> addition of residue it starts with relatively low but gradually rises >>> through almost all cycles although model fits to the density well and >>> residue are building, coot validation parameters are also reasonable OK >>> for geometry, rotamer, density fit,.. ** >>> >>> * >>> *now my question.... >>> >>> * >>> ** where should i first check for possible correction? >>> >>> * >>> ** In molecular replacement what should be the red line for identity >>> and related criteria? >>> >>> * >>> ** if initial rFree starts around 50, how likely that its not the right >>> way? >>> >>> * >>> ** my rms bond angle is close to 1 while the bond length is 0.01 and >>> chiral 0.1 concludes what is serious? >>> >>> * >>> * >>> >>> * *sincere apology for amateur query if any... >>> >>> * >>> *thanks in advance >>> * >>> * >>> >>> * >>> *pramod >>> * >>> * >>> * >>> * >>> * >>> * >>> -- >>> ************************************************** >>> Pramod Kumar. >>> Graduate Student. >>> Crystallography lab. >>> Department Of Biotechnology. >>> Indian Institute Of Technology Roorkee >>> Uttranchal.247667 >>> India >>> +919359189657. >>> ************************************************** * >>> >>> * >>> * >>> >> * >> * >> >> >> > > > -- > ************************************************ > Pramod Kumar. > Graduate Student. > Crystallography lab. > Department Of Biotechnology. > Indian Institute Of Technology Roorkee > Uttranchal.247667 > India > +919359189657. > ************************************************ > -- Dr Eugene Valkov MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: [log in to unmask] Tel: +44 (0) 1223 407840