Re: [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation
I've had exactly the same with an enzyme from Trypanosoma brucei, native enzyme gives ca. 2 A data, a search around the crystallization conditions for the Se-Met enzyme returns absolutely nothing. What I should do is to search for new crystallisation conditions (which I won't do: trypanosome means no-one will provide any funding for the project nowadays). So if you do not get any crystals from your derivatized protein starting from the crystallisation conditions of your native protein, then you must start a new crystallisation screening process right from the start. Remember that introducing Se-Mets can change the surface of your protein; it can also introduce an equilibrium between several forms of the protein, whereas you only had one form for the native enzyme - so you may need to try to purify (rapidly) further. If this de novo crystallisation fails, then you need to try alternative phasing methods (such as heavy atom soaks or co-crystallisation with heavy atoms). Fred. > Message du 13/06/11 07:53 > De : "atul kumar" > A : CCP4BB@JISCMAIL.AC.UK > Copie à : > Objet : [ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation > > -- Forwarded message -- > From: Dilip Kumar > Date: Mon, Jun 13, 2011 at 11:21 AM > Subject: Fwd: Regarding Sel-Met containing proteing crystallisation > To: atulsingh21...@gmail.com > > > > > -- Forwarded message -- > From: Dilip Kumar > Date: Sat, Jun 11, 2011 at 6:09 PM > Subject: Regarding Sel-Met containing proteing crystallisation > To: CCP4BB@jiscmail.ac.uk > > > Dear all > > I have got protein crystals,crystallisation condition (LiCl, PEG and > HEPES) .Crystals of native protein have been successesfully reproduced but > when i tried to reproduce these crystals with protein having Met replaced by > Sel-Met, i could not get any crystal.I tried crystallisation trials by > varying pH and PEG concentration and diffferent drop ratio but i could not > get any hit.Please suggest me what could be the possible reasons behind it? > And also suggest the other variables that i can try ? > thanks > With Regards > > -- > Dilip Tiwari > Graduate Student > Structural Biology Unit > Institute of Genomics & Integrative Biology > Delhi-07 > > > > -- > Dilip Tiwari > Graduate Student > Structural Biology Unit > Institute of Genomics & Integrative Biology > Delhi-07 >
[ccp4bb] Fwd: Regarding Sel-Met containing proteing crystallisation
-- Forwarded message -- From: Dilip Kumar Date: Mon, Jun 13, 2011 at 11:21 AM Subject: Fwd: Regarding Sel-Met containing proteing crystallisation To: atulsingh21...@gmail.com -- Forwarded message -- From: Dilip Kumar Date: Sat, Jun 11, 2011 at 6:09 PM Subject: Regarding Sel-Met containing proteing crystallisation To: CCP4BB@jiscmail.ac.uk Dear all I have got protein crystals,crystallisation condition (LiCl, PEG and HEPES) .Crystals of native protein have been successesfully reproduced but when i tried to reproduce these crystals with protein having Met replaced by Sel-Met, i could not get any crystal.I tried crystallisation trials by varying pH and PEG concentration and diffferent drop ratio but i could not get any hit.Please suggest me what could be the possible reasons behind it? And also suggest the other variables that i can try ? thanks With Regards -- Dilip Tiwari Graduate Student Structural Biology Unit Institute of Genomics & Integrative Biology Delhi-07 -- Dilip Tiwari Graduate Student Structural Biology Unit Institute of Genomics & Integrative Biology Delhi-07
Re: [ccp4bb] off-topic: Synchrotron look alike
Hi Harry, Apple, GCHQ, what's the difference? http://petewarden.github.com/iPhoneTracker/ /Derek P.S. Apple seem to have done a significantly better job of the car parking issue... On 12 Jun 2011, at 21:17, Harry wrote: > Hi > > Hmmm. I was actually struck by the similarity between Apple's new HQ and > GCHQ, the UK government's "top-secret" communications monitoring station - > > http://www.google.co.uk/searchq=gchq&hl=en&client=safari&rls=en&biw=1175&bih=764&prmd=ivnsm&source=lnms&tbm=isch&ei=hA_1TYeHJYOw8gOC1NSXBw&sa=X&oi=mode_link&ct=mode&cd=2&sqi=2&ved=0CBMQ_AUoAQ > > (all that should be on one line, or just google for "gchq" and look at the > images). > > On 12 Jun 2011, at 19:55, Derek Logan wrote: > >> Hi everyone, >> >> The latest update on the Apple story is that they are actually going to >> build their new headquarters in Southern Sweden: >> >> http://www.maxlab.lu.se/media_press/research/pm_exteriornew.html >> >> You can see that the lawn outside has been severely affected by the reality >> distortion field. Actually Apple seem to have moved away from the monolithic >> design in Fred's mail towards something "greener" (well with grass on the >> roof anyway...) and more compact. Check out another, even more elegant >> earlier design here: >> >> http://www.maxlab.lu.se/maxlab/max4/images/maxiv_buildning_suggestions/Snöhetta/MaxLab_Aerial_FINAL_II.jpg >> >> /Derek >> ___ >> Derek Logantel: +46 46 222 1443 >> Associate Professorfax: +46 46 222 4692 >> Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 >> Centre for Molecular Protein Science www.cmps.lu.se >> Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com >> >> On 9 Jun 2011, at 17:49, Vellieux Frederic wrote: >> >>> Sorry, a bit off topic. But in the news today (check google news for >>> example): Steve Jobs (Apple) has revealed his plans for the new "Apple >>> campus" (Apple headquarters) in Cupertino, California. Should look familiar >>> to macromolecular crystallographers. >>> >>> Fred. >>> > > Harry > -- > Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, > Cambridge, CB2 0QH >
Re: [ccp4bb] off-topic: Synchrotron look alike
Hi Hmmm. I was actually struck by the similarity between Apple's new HQ and GCHQ, the UK government's "top-secret" communications monitoring station - http://www.google.co.uk/searchq=gchq&hl=en&client=safari&rls=en&biw=1175&bih=764&prmd=ivnsm&source=lnms&tbm=isch&ei=hA_1TYeHJYOw8gOC1NSXBw&sa=X&oi=mode_link&ct=mode&cd=2&sqi=2&ved=0CBMQ_AUoAQ (all that should be on one line, or just google for "gchq" and look at the images). On 12 Jun 2011, at 19:55, Derek Logan wrote: Hi everyone, The latest update on the Apple story is that they are actually going to build their new headquarters in Southern Sweden: http://www.maxlab.lu.se/media_press/research/pm_exteriornew.html You can see that the lawn outside has been severely affected by the reality distortion field. Actually Apple seem to have moved away from the monolithic design in Fred's mail towards something "greener" (well with grass on the roof anyway...) and more compact. Check out another, even more elegant earlier design here: http://www.maxlab.lu.se/maxlab/max4/images/maxiv_buildning_suggestions/Snöhetta/MaxLab_Aerial_FINAL_II.jpg /Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com On 9 Jun 2011, at 17:49, Vellieux Frederic wrote: Sorry, a bit off topic. But in the news today (check google news for example): Steve Jobs (Apple) has revealed his plans for the new "Apple campus" (Apple headquarters) in Cupertino, California. Should look familiar to macromolecular crystallographers. Fred. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] off-topic: Synchrotron look alike
Hi everyone, The latest update on the Apple story is that they are actually going to build their new headquarters in Southern Sweden: http://www.maxlab.lu.se/media_press/research/pm_exteriornew.html You can see that the lawn outside has been severely affected by the reality distortion field. Actually Apple seem to have moved away from the monolithic design in Fred's mail towards something "greener" (well with grass on the roof anyway...) and more compact. Check out another, even more elegant earlier design here: http://www.maxlab.lu.se/maxlab/max4/images/maxiv_buildning_suggestions/Snöhetta/MaxLab_Aerial_FINAL_II.jpg /Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com On 9 Jun 2011, at 17:49, Vellieux Frederic wrote: > Sorry, a bit off topic. But in the news today (check google news for > example): Steve Jobs (Apple) has revealed his plans for the new "Apple > campus" (Apple headquarters) in Cupertino, California. Should look familiar > to macromolecular crystallographers. > > Fred. >
Re: [ccp4bb] XDS question
Dear Marco, in terms of using XDS, STRONG_PIXEL=99 is _highly_ non-standard (I personally try to stick with the defaults ... although at synchrotrons I do use STRONG_PIXEL=6 instead of the default of 3). I realize this is just a way to pick the strong reflection for indexing (since STRONG_PIXEL is used by COLSPOT , but not by INTEGRATE), but in the long run it would be important to figure out the relation between the strong lattice and the weak lattice (you could post the cell parameters here, or even better upload a few frames to some Internet service so that people can take a look themselves). You seem to be able to solve the structure using the lattice corresponding to the strong spots, if I understand correctly. The lattice that covers both the weak and the strong spots certainly has longer cell axes, and I'd guess that there are maybe twice as many molecules in the ASU. Taking account of this, you may be able to solve the structure using all reflections, and you might get some important insight concerning the binding mode(s) of the substrate. best, Kay Am 20:59, schrieb Marco Lolicato: Dear all, I have a particular problem... so, I have a beautiful crystal with nice diffraction pattern at 2.7A. The diffraction images are composed by very strong spots and weak spots. With XDS, if I collect all spots I get good map, but it is impossible to solve the structure by molecular replacement. If I collect only the strongest spots (STRONG_PIXEL=9), I'm able to solve a very good structure... My problem is: I was trying to get the apo-structure of my protein. I obtained nice crystals of the "apo-protein", but using the method above, in the structure I have found also the ligand!! (probably incorporated during the overexpression). My protein is a multimer and, biochemically, I found that the endogenus ligand bond to the protein is in the ratio 1:6. ...and I got a crystal in this way. So, is there a way to analyze all spots in the diffraction pattern to have a structure of the apo-protein? Is a good idea discard the strongest spots and try to analyze only the weak spots? If yes, how I can do it? All the best, Marco
