Dear Robert I have cross checked by running the gel and silver stain that confirms it is only the protein i am targeting.
no exact hit was obtained for this cell parameter and SG, as it previously checked during "balbes" run... thanks and regards pramod On Wed, Jun 19, 2013 at 2:28 PM, Robert Esnouf <[email protected]>wrote: > > Hi Pramod, > > You mention "this is the limitation of my data that came from three month > old crystal". If the crystal took that long to grow (did it?) then a > crystallized (1) proteolytic fragment or (2) low level contaminant is quite > likely. > > 1) If you have the crystal still you could try to redissolve it and run a > gel > > 2) If it is the second case then the unit cell is probably already in the > PDB. You can check with this tool... > > http://www.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi > > Good luck, > Robert > > -- > > Dr. Robert Esnouf, > University Research Lecturer > and Head of Research Computing, > Wellcome Trust Centre for Human Genetics, > Old Road Campus, Roosevelt Drive, > Oxford OX3 7BN, UK > > Emails: [email protected] Tel: (+44) - 1865 - 287783 > and [email protected] Fax: (+44) - 1865 - 287547 > > > ---- Original message ---- > >Date: Wed, 19 Jun 2013 03:20:57 +0530 > >From: CCP4 bulletin board <[email protected]> (on behalf of Pramod > Kumar <[email protected]>) > >Subject: Re: [ccp4bb] str solving problem > >To: [email protected] > > > > Apology for delay > > > > Dear Abhinav.. > > > > Kept the ratio 2 with correlation of completeness > > above 75 > > r factor reduces with under -5 to 6 lees than r > > free. > > > > I am trying different models.. > > > > Dear Herman > > -Is the protein that crystallized the protein you > > think it is? Plasmids do get mixed-up and sometimes > > a more abundant natural protein with about the same > > molecular weight as the protein of interest gets > > purified. > > > > Yes, I have done silver stain that confirms exact > > MW, further related literature and biological > > properties supports, > > > > -How do the diffraction images look like: are there > > strong ice-rings, multiple diffraction patterns or > > other problems? Could there have been misindexing in > > one of the directions? > > > > no ice ring is appeared, diffraction pattern looks > > ok, misindexing in any direction is not conclusive > > to me (plz see the imj attachment) > > > > Dear Eugene > > > > the phaser .sol file states... > > SOLU SET RFZ=6.6 TFZ=8.8 PAK=0 LLG=102 TFZ==8.2 > > LLG=1804 TFZ==22.9 > > SOLU SPAC C 2 2 21 > > SOLU 6DIM ENSE ensemble1 EULER 180.061 0.000 0.000 > > FRAC -0.49950 -0.50021 -0.00006 BFAC 0.00000 > > SOLU ENSE ensemble1 VRMS 0.877 > > > > for building the model I used the 'autorikshaw > > pipeline' and it has given rfact/rfree 0.4645 / > > 0.4888 but back to refmac again starts rising > > > > Dear Edward > > > > Patterson analysis obtained from xtrige... > > > > > > Xtrige summary > > > > Twinning and intensity statistics summary (acentric data): > > > > > > > > Statistics independent of twin laws > > <I^2>/<I>^2 : 2.078 > > <F>^2/<F^2> : 0.777 > > <|E^2-1|> : 0.758 > > <|L|>, <L^2>: 0.507, 0.340 > > Multivariate Z score L-test: 2.003 > > > > > > > > The multivariate Z score is a quality measure of the given > > spread in intensities. Good to reasonable data are expected > > to have a Z score lower than 3.5. > > Large values can indicate twinning, but small values do not > > > > > > necessarily exclude it. > > > > No (pseudo)merohedral twin laws were found. > > > > Patterson analyses > > - Largest peak height : 4.674 > > (corresponding p value : 0.95872) > > > > The largest off-origin peak in the Patterson function is 4.67% of the > > > > > > height of the origin peak. No significant pseudotranslation is detected. > > > > The results of the L-test indicate that the intensity statistics > > behave as expected. No twinning is suspected. > > > > integration in P1 and attempt to carry out > > molecular replacement in P1 from this Zanuda > > concludes as follow > > > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > > > > | >> 5 | C 2 2 21 | 0.5985 | 0.4265 | 0.3918 | 0.4880 | > > > > --------------------------------------------------------------------- > > > > | 1 | P 1 | 0.5308 | 0.4236 | 0.3970 | 0.5024 | > > > > | 2 | P 1 21 1 | 0.6302 | -- | 0.3912 | 0.4989 | > > > > | 5 | C 2 2 21 | 0.7318 | -- | 0.3804 | 0.5009 | > > > > --------------------------------------------------------------------- > > > > | << 5 | C 2 2 21 | 0.7318 | -- | 0.3804 | 0.5009 | > > > > --------------------------------------------------------------------- > > > > R-factor in the original subgroup is NOT the best. > > > > The original spacegroup assignment seems to be incorrect. > > > > thanks and regards > > > > pramod > > > > On Tue, Jun 18, 2013 at 3:32 PM, Edward Lowe > > <[email protected]> wrote: > > > > Dear Pramod, > > It's difficult to be certain based on what you > > have posted but my best guess would be problems > > with symmetry determination it's quite possible > > for pointless to be fooled into assigning higher > > symmetry if some form of pseudo-symmetry is > > present. What does the native Patterson look like? > > Any large off-origin peaks might give you a clue. > > The safest thing to do would be to integrate in P1 > > and attempt to carry out molecular replacement in > > P1 and see how that refines. Hopefully be able to > > determine the correct symmetry from this (Zanuda > > may well be able to help at this point!). > > Ed. > > -- > > Dr. E.D. Lowe > > Department of Biochemistry > > University of Oxford > > South Parks Road > > Oxford, UK > > OX1 3QU > > e:[email protected] > > t: +44 (0) 1865 613288 > > f: +44 (0) 1865 613201 > > From: Pramod Kumar <[email protected]> > > Reply-To: Pramod Kumar <[email protected]> > > Date: Tue, 18 Jun 2013 02:58:36 +0530 > > To: <[email protected]> > > Subject: Re: [ccp4bb] str solving problem > > 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 > > <[email protected]> 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 <[email protected]> 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. > > ************************************************ > > > > -- > > ************************************************ > > Pramod Kumar. > > Graduate Student. > > Crystallography lab. > > Department Of Biotechnology. > > Indian Institute Of Technology Roorkee > > Uttranchal.247667 > > India > > +919359189657. > > ************************************************ > >________________ > >pramod2.jpg (359k bytes) > >________________ > >pramod1.jpg (371k bytes) > -- ************************************************ Pramod Kumar. Graduate Student. Crystallography lab. Department Of Biotechnology. Indian Institute Of Technology Roorkee Uttranchal.247667 India +919359189657. ************************************************
