[ccp4bb] protein gets trapped
Dear all, I would like to share with you some problems I am experiencing with a protein, in case someone has an idea about what it is going on: I am working with a protein module that gets trapped in Ni-NTA or glutathione beads. If the protein is purified without tag (ion exchange plus gel filtration), the protein can be purified properly. However, when expressing with a His or GST tag, it gets trapped and it cannot be eluted with imidazole/glutathione or even with 8M Urea (in case it is unfolded) Does someone know what can be happening? We do not have any information about an enzimatic activity of the module that might permit the protein bind covalently to the beads Furthermore, we got the crystal structure in complex with another protein and the folding of the module does not resemble anything with enzimatic activity Many thanks in advance for all your suggestions, Maria
Re: [ccp4bb] CCP4 6.3.0 release
Two Windows-specific problems have been reported and fixed yesterday: - cif_mmdic.lib is in %CCP4%\share\ccif, should be in %CCP4%\lib - rapper doesn't work (missing DLL) cif_mmdic.lib can be moved manually, so if you don't use rapper there is no need to update. Installer with fixes is available from ccp4 website and ftp: ftp://ftp.ccp4.ac.uk/ccp4/6.3.0/CCP4-6.3.0.1.msi Additionally ctruncate has been updated, some non-critical issues have been fixed. Marcin -- Scanned by iCritical.
[ccp4bb] resolution limit
Hello CCP4ers, In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & Rsym 224.3 % for multiplicity 7.8 and completeness 98.2 %. I solved the structure by MAD & refined it to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is not twinned. The water content is 68%. I loweredthe multiplicity to 4.1 by excluding few images but still the Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography knowledge makes me believe it's quite Ok to use data upto 2.8 A and report the statistics. Could I request people's views. Thanks very much. Narayan
Re: [ccp4bb] resolution limit
Hi Narayan My only comment would be that P622 is a fairly uncommon space group (currently 43 PDB entries excl homologs), but obviously that doesn't mean it's wrong - just worth double-checking! Just out of interest what's the CC(1/2) statistic for your highest shell? Personally I specify more bins than the default (e.g. 20 instead of 10) so that the highest resolution bin would be somewhat narrower than yours. I would also prefer that the binning is done by equal steps in d*^3 rather than d*^2 as many programs do since this gives a more even spread of reflections in the bins and gives an even narrower binning at the high res end (though it does tend to lump all the low res data into 1 bin!). Cheers -- Ian On 18 July 2012 10:41, narayan viswam wrote: > > > Hello CCP4ers, > > In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & Rsym > 224.3 % for multiplicity 7.8 and completeness 98.2 %. I solved the structure > by MAD & refined it to Rfree 27.3 %. Ths crystal belongs to P622 space group > and it is not twinned. The water content is 68%. I loweredthe multiplicity > to 4.1 by excluding few images but still the Rsym is > 200 % and I/sigmaI > > 2.0. My rudimentary crystallography knowledge makes me believe it's quite Ok > to use data upto 2.8 A and report the statistics. Could I request people's > views. Thanks very much. > Narayan
Re: [ccp4bb] resolution limit
narayan viswam wrote: Hello CCP4ers, In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & Rsym 224.3 % for multiplicity 7.8 and completeness 98.2 %. I solved the structure by MAD & refined it to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is not twinned. The water content is 68%. I loweredthe multiplicity to 4.1 by excluding few images but still the Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography knowledge makes me believe it's quite Ok to use data upto 2.8 A and report the statistics. Could I request people's views. Thanks very much. Narayan After refinement, what is R-free in the last shell? If it is significantly better than random, say around .4 or less, that could be taken as evidence that there is data in the last shell. Also check the error model- Rsym >2 sort of implies the error is greater than the signal, so I/sigI 2 seems surprising. eab
Re: [ccp4bb] resolution limit
As has been shown recently (and discussed on this board), Rsym is not the best measure of data quality (if any measure at all): http://www.sciencemag.org/content/336/6084/1030.abstract > narayan viswam wrote: >> Hello CCP4ers, >> In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & >> Rsym 224.3 % >> for multiplicity 7.8 and completeness 98.2 %. I solved the structure by >> MAD & refined it >> to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is not >> twinned. The water >> content is 68%. I loweredthe multiplicity to 4.1 by excluding few >> images but still the >> Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography >> knowledge makes me >> believe it's quite Ok to use data upto 2.8 A and report the statistics. >> Could I request >> people's views. Thanks very much. >> Narayan > > After refinement, what is R-free in the last shell? If it is significantly > better > than random, say around .4 or less, that could be taken as evidence that > there > is data in the last shell. > Also check the error model- Rsym >2 sort of implies the error is greater > than > the signal, so I/sigI 2 seems surprising. > eab > -- Edwin Pozharski, PhD University of Maryland, Baltimore
Re: [ccp4bb] recommendations for pH meter
I like the Fisher AB15 meter. Large LCD display for presbyopic eyes, and it will do multipoint calibrations. Most laboratory grade pH meters will do multipoint calibration, however. We typically do a 3 point pH calibration at 4, 7, 10, but you can do more if you like. I pair this with a refillable accuTuph combination pH electrode that has a built-in Ag/AgCl reference electrode. These electrodes have thicker glass membranes than the usual pH electrodes, and are a little more student-resistant. The refillable electrode solution allows you to maintain and use this electrode for many, many years. It is possible to get 5-10 years service out of these electrodes. If you will be calibrating concentrated Tris buffers on a regular basis, consider spending a little extra on a double junction electrode rather than the single junction electrode. Double junction electrodes are more resistant to clogging in general and "Tris effect" in particular. (Tris binds Ag+ in the reference electrode, and can make your pH electrode go "wonky" for a while after exposure.) Gel combination electrodes are worthless, and I would not consider using one in the research laboratory. They have very limited shelf and useful life, and cannot be refurbished when they go bad. And they don't recover gracefully from exposure to Tris. I haven't tried FET pH electrodes, but they seem needlessly expensive considering the glass membrane technology works very well and gives years of service. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 7/18/2012 2:21 AM, Anirban Banerjee wrote: Dear all, May I ask for recommendations for pH meters that work reliably over the range of buffers and components that constitute common crystallization screens ? Thanks very much in advance. Anirban
[ccp4bb] Postdoctoral position in Molecular Neuroscience (Paris)
Postdoc position in Molecular Neuroscience at Ecole Normale Supérieure (Paris) Team: “Glutamate Receptors” ( http://www.biologie.ens.fr/neuronr/ ) Location: Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Paris, France Start date: September 2012. We seek to hire a highly motivated postdoctoral scientist to work on the subunit-specific allosteric regulation of NMDA receptors using a combination of molecular, biophysical and structural approaches (receptor engineering, cellular electrophysiology, 3D modeling, ligand docking etc…). The candidate should have a background in cellular electrophysiology and molecular approaches. The candidate should also show a strong interest in molecular and cellular neuroscience with special focus on receptor/ion channel function and pharmacology. Candidates with a background at the interface between biology and chemistry are welcome. The project is to be developed in the team of Pierre Paoletti at the Institut de Biologie de l’Ecole Normale Supérieure (IBENS) in Paris, a team that has international recognition for its work on the structure, function and pharmacology of NMDA receptors. The position is funded for at least two years. IBENS is a leading biology institute in Europe that gathers several world-class researchers in fields ranging from genetics and genomics, developmental biology, systems biology and neuroscience. IBENS is located in the center of Paris (Latin Quarter) in a highly stimulating and dynamic environment, close by to several other top research institutions (Institut Curie, Collège de France…). Please send a CV and brief statement of research experience to: Pierre Paoletti (pierre.paole...@ens.fr) Selection of publications from the host team: • Hatton and Paoletti (2005) Neuron, 46, 261-274 • Gielen et al. (2008) Neuron, 57, 80-93. • Mony et al. (2009) Molecular Pharmacology, 75, 60-74. • Sensi et al. (2009) Nature Reviews Neuroscience, 10, 780-791. (Review) • Gielen et al. (2009) Nature, 459, 703-707. • Nozaki et al. (2011) Nature Neuroscience, 14, 1017-1082. • Mony et al. (2011) EMBO Journal, 30, 3134-3146. • Riou et al. (2012) PLoS ONE, 7(4):e35134.
[ccp4bb] Regarding refinement in Refmac5
Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi
[ccp4bb] PhD position in Bolzano (Italy)
Dear All (possible candidates!), there is a call for PhD fellowships in the framework of the multidisciplinary PhD school in MOUNTAIN ENVIRONMENTS AND AGRICULTURE http://www.unibz.it/en/sciencetechnology/progs/phd/phdmountainenvironment/default.html A 3 year PhD fellowship is available to a suitable candidate for one of the following projects: Molecular characterization of the bio synthetic pathway of amylovoran, a pathogenicity factor in Erwinia amylovora Biomolecular study of iron uptake by desferrioxamine in the plant pathogen Erwinia amylovora the work will involve cloning of genes, protein expression, purification and crystallization and structure determination by X-ray crystallography. Proteins will be characterized using in solution techniques including NMR available through collaboration with other laboratories. To apply to the public competition please visit the link: http://www.unibz.it/en/public/research/phd/prospectivePhdstudents.html there you will find the link to the official call document: http://www.unibz.it/SiteCollectionDocuments/2012_PhD_Ausschreibung_en.pdf as well as the link to the pre-enrolment page: https://aws.unibz.it/exup/ The applications must be sent by 31 August 2012. It is recommended that applications are completed and sent as soon as possible If you wish to apply and want more information on the project and for informal enquiries please contact: stefano.ben...@unibz.it For information on how to apply please contact our secretary: stefania.falc...@unibz.it Best regards Stefano Benini, Ph.D. Assistant Professor http://pro.unibz.it/staff2/sbenini/ * Laboratory of Bioorganic Chemistry and Crystallography Faculty of Science and Technology Free University of Bolzano Piazza Università, 5 39100 Bolzano, Italy Office (room K2.14): +39 0471 017128 Laboratory (room E.012): +39 0471 017901 Fax: +39 0471 017009
Re: [ccp4bb] Regarding refinement in Refmac5
Refmac takes its spacegroup fro the MTZ file, so if it isn't P32 2 1 then the refinement will be wrong. You may have to change the spacegroup in the file Phil On 18 Jul 2012, at 17:28, Deepthi wrote: > Hi all > > I am working with a small mutant protein which is 56 amino acids long. The > crystal diffracted at 1.4A0 and the space group is p3221. I did molecular > replacement using Phenix software with all the data (1.4A0) and got a > solution. Phenix did auto building with waters and R-free was 0.3123. > > I mutated some residues which don't align with the model protein to > Alanines. When i change the residues back to their respective side chains > Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 > data) but R-free is shooting up to 0.41. It is not accepting any changes to > the Phenix generated model. I have no idea what is going on. Can anyone help > me? > > Thank You in advance > Deepthi > >
Re: [ccp4bb] Regarding refinement in Refmac5
Hi there, Not much information provided. How was the initial model refined ? Phenix ? It could be a problem with the Refmac refinement protocol (difficult to say with so little information) if you switched from Phenix to Refmac. How certain are you 1 - of the space group; 2 - that the crystal wasn't twinned ? You can have both and it can be "annoying". Further, at this resolution I think you could use one of the SHELXes (forgot the terminology) for refinement, that could be more appropriate. F.V. Deepthi wrote: Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi
Re: [ccp4bb] Regarding refinement in Refmac5
I tried opening the model with other spacegroups MTZ file. The map doesn't fit well for other spacegroups. The initial model was refined using Phenix Autobuild software. I tried MR with every spacegroup possible in primitive hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try using other softwares for refinement but this is annoying. I also tried mutating the model to poly alanines and refine but this made it worse. The R-free went up to 0.546. I initially thought it might be a space group problem but trying other space groups doesn't work either. Thank youvery much for the help Deepthi On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic wrote: > Hi there, > > Not much information provided. How was the initial model refined ? Phenix > ? It could be a problem with the Refmac refinement protocol (difficult to > say with so little information) if you switched from Phenix to Refmac. > > How certain are you 1 - of the space group; 2 - that the crystal wasn't > twinned ? You can have both and it can be "annoying". > > Further, at this resolution I think you could use one of the SHELXes > (forgot the terminology) for refinement, that could be more appropriate. > > F.V. > > > Deepthi wrote: > >> Hi all >> >> I am working with a small mutant protein which is 56 amino acids long. >> The crystal diffracted at 1.4A0 and the space group is p3221. I did >> molecular replacement using Phenix software with all the data (1.4A0) and >> got a solution. Phenix did auto building with waters and R-free was 0.3123. >> >> I mutated some residues which don't align with the model protein to >> Alanines. When i change the residues back to their respective side chains >> Refmac5 won't refine it well. The maps looks clear( you can guess its >> 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any >> changes to the Phenix generated model. I have no idea what is going on. Can >> anyone help me? >> >> Thank You in advance >> Deepthi >> >> >> -- Deepthi
Re: [ccp4bb] strict structure based alignment
Dear all, thanks a lot for all your comments and suggestions for the alignment. I tested already the pdb server, which works great and I am currently installing a few other programs mentioned (Chimera, Prosmart.) for comparison. Best Regards Christian Am Freitag 13 Juli 2012 16:30:57 schrieb Christian Roth: > Dear all, > > I want align a couple or protein structures by secondary structure matching > to one target and want get a kind of aminoacid alignment file e.g. what > residue fit the other, without adjustments due to sequence based > alignments. > I tried Strap, but as far as I understood it, it takes also the sequence > into account. I tried also Rapido, but this does only a pairwise > comparison. Superpose does align it nicely (ccp4 based or Coot based) but > there seems to be no option to print the sequence alignment in a file and > it is again just a pairwise comparison . > Is there an other program which does something similar? > > Best Regards > > Christian >
Re: [ccp4bb] Regarding refinement in Refmac5
What happens if you do a round of refinement in phenix.refine after you have done the mutations? Note: Phenix Autobuild is a tool to build your model, not refine it (though it does some refinement internally but may not be as fine tuned as your data/model may require). Pavel On Wed, Jul 18, 2012 at 9:50 AM, Deepthi wrote: > I tried opening the model with other spacegroups MTZ file. The map doesn't > fit well for other spacegroups. The initial model was refined using Phenix > Autobuild software. I tried MR with every spacegroup possible in primitive > hexagonal. Only p3221 worked. There is no twinning in the crystal. I will > try using other softwares for refinement but this is annoying. I also tried > mutating the model to poly alanines and refine but this made it worse. The > R-free went up to 0.546. > I initially thought it might be a space group problem but trying other > space groups doesn't work either. > > Thank youvery much for the help > Deepthi > > > On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic < > frederic.velli...@ibs.fr> wrote: > >> Hi there, >> >> Not much information provided. How was the initial model refined ? Phenix >> ? It could be a problem with the Refmac refinement protocol (difficult to >> say with so little information) if you switched from Phenix to Refmac. >> >> How certain are you 1 - of the space group; 2 - that the crystal wasn't >> twinned ? You can have both and it can be "annoying". >> >> Further, at this resolution I think you could use one of the SHELXes >> (forgot the terminology) for refinement, that could be more appropriate. >> >> F.V. >> >> >> Deepthi wrote: >> >>> Hi all >>> >>> I am working with a small mutant protein which is 56 amino acids long. >>> The crystal diffracted at 1.4A0 and the space group is p3221. I did >>> molecular replacement using Phenix software with all the data (1.4A0) and >>> got a solution. Phenix did auto building with waters and R-free was 0.3123. >>> >>> I mutated some residues which don't align with the model protein to >>> Alanines. When i change the residues back to their respective side chains >>> Refmac5 won't refine it well. The maps looks clear( you can guess its >>> 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any >>> changes to the Phenix generated model. I have no idea what is going on. Can >>> anyone help me? >>> >>> Thank You in advance >>> Deepthi >>> >>> >>> > > > -- > Deepthi >
Re: [ccp4bb] Regarding refinement in Refmac5
Thank You so much . I am trying to process the data again in all space groups possible for primitive hexagonal and try refinement again. And i didn't use Phenix after i got the MR solution. Probably refining the mutated model again would give some more information. Thank You once again. Appreciated Deepthi On Wed, Jul 18, 2012 at 10:02 AM, Garib N Murshudov wrote: > Can you check space group in your mtz and pdb? I have seen this happening > when they disagree. > It is annoying and I would like it to be sorted out. If you want you can > send your data and I can try to sort it out. > > > Garib > > On 18 Jul 2012, at 17:50, Deepthi wrote: > > I tried opening the model with other spacegroups MTZ file. The map doesn't > fit well for other spacegroups. The initial model was refined using Phenix > Autobuild software. I tried MR with every spacegroup possible in primitive > hexagonal. Only p3221 worked. There is no twinning in the crystal. I will > try using other softwares for refinement but this is annoying. I also tried > mutating the model to poly alanines and refine but this made it worse. The > R-free went up to 0.546. > I initially thought it might be a space group problem but trying other > space groups doesn't work either. > > Thank youvery much for the help > Deepthi > > On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic < > frederic.velli...@ibs.fr> wrote: > >> Hi there, >> >> Not much information provided. How was the initial model refined ? Phenix >> ? It could be a problem with the Refmac refinement protocol (difficult to >> say with so little information) if you switched from Phenix to Refmac. >> >> How certain are you 1 - of the space group; 2 - that the crystal wasn't >> twinned ? You can have both and it can be "annoying". >> >> Further, at this resolution I think you could use one of the SHELXes >> (forgot the terminology) for refinement, that could be more appropriate. >> >> F.V. >> >> >> Deepthi wrote: >> >>> Hi all >>> >>> I am working with a small mutant protein which is 56 amino acids long. >>> The crystal diffracted at 1.4A0 and the space group is p3221. I did >>> molecular replacement using Phenix software with all the data (1.4A0) and >>> got a solution. Phenix did auto building with waters and R-free was 0.3123. >>> >>> I mutated some residues which don't align with the model protein to >>> Alanines. When i change the residues back to their respective side chains >>> Refmac5 won't refine it well. The maps looks clear( you can guess its >>> 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any >>> changes to the Phenix generated model. I have no idea what is going on. Can >>> anyone help me? >>> >>> Thank You in advance >>> Deepthi >>> >>> >>> > > > -- > Deepthi > > > Dr Garib N Murshudov > Group Leader, MRC Laboratory of Molecular Biology > Hills Road > Cambridge > CB2 0QH UK > Email: ga...@mrc-lmb.cam.ac.uk > Web http://www.mrc-lmb.cam.ac.uk > > > > > -- Deepthi
Re: [ccp4bb] Regarding refinement in Refmac5
How about trying some ARP/WARP? JPK On Wed, Jul 18, 2012 at 11:54 AM, Pavel Afonine wrote: > What happens if you do a round of refinement in phenix.refine after you > have done the mutations? Note: Phenix Autobuild is a tool to build your > model, not refine it (though it does some refinement internally but may not > be as fine tuned as your data/model may require). > > Pavel > > > On Wed, Jul 18, 2012 at 9:50 AM, Deepthi wrote: > >> I tried opening the model with other spacegroups MTZ file. The map >> doesn't fit well for other spacegroups. The initial model was refined using >> Phenix Autobuild software. I tried MR with every spacegroup possible in >> primitive hexagonal. Only p3221 worked. There is no twinning in the >> crystal. I will try using other softwares for refinement but this is >> annoying. I also tried mutating the model to poly alanines and refine but >> this made it worse. The R-free went up to 0.546. >> I initially thought it might be a space group problem but trying other >> space groups doesn't work either. >> >> Thank youvery much for the help >> Deepthi >> >> >> On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic < >> frederic.velli...@ibs.fr> wrote: >> >>> Hi there, >>> >>> Not much information provided. How was the initial model refined ? >>> Phenix ? It could be a problem with the Refmac refinement protocol >>> (difficult to say with so little information) if you switched from Phenix >>> to Refmac. >>> >>> How certain are you 1 - of the space group; 2 - that the crystal wasn't >>> twinned ? You can have both and it can be "annoying". >>> >>> Further, at this resolution I think you could use one of the SHELXes >>> (forgot the terminology) for refinement, that could be more appropriate. >>> >>> F.V. >>> >>> >>> Deepthi wrote: >>> Hi all I am working with a small mutant protein which is 56 amino acids long. The crystal diffracted at 1.4A0 and the space group is p3221. I did molecular replacement using Phenix software with all the data (1.4A0) and got a solution. Phenix did auto building with waters and R-free was 0.3123. I mutated some residues which don't align with the model protein to Alanines. When i change the residues back to their respective side chains Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any changes to the Phenix generated model. I have no idea what is going on. Can anyone help me? Thank You in advance Deepthi >> >> >> -- >> Deepthi >> > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Regarding refinement in Refmac5
Can you check space group in your mtz and pdb? I have seen this happening when they disagree. It is annoying and I would like it to be sorted out. If you want you can send your data and I can try to sort it out. Garib On 18 Jul 2012, at 17:50, Deepthi wrote: > I tried opening the model with other spacegroups MTZ file. The map doesn't > fit well for other spacegroups. The initial model was refined using Phenix > Autobuild software. I tried MR with every spacegroup possible in primitive > hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try > using other softwares for refinement but this is annoying. I also tried > mutating the model to poly alanines and refine but this made it worse. The > R-free went up to 0.546. > I initially thought it might be a space group problem but trying other space > groups doesn't work either. > > Thank youvery much for the help > Deepthi > > On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic > wrote: > Hi there, > > Not much information provided. How was the initial model refined ? Phenix ? > It could be a problem with the Refmac refinement protocol (difficult to say > with so little information) if you switched from Phenix to Refmac. > > How certain are you 1 - of the space group; 2 - that the crystal wasn't > twinned ? You can have both and it can be "annoying". > > Further, at this resolution I think you could use one of the SHELXes (forgot > the terminology) for refinement, that could be more appropriate. > > F.V. > > > Deepthi wrote: > Hi all > > I am working with a small mutant protein which is 56 amino acids long. The > crystal diffracted at 1.4A0 and the space group is p3221. I did molecular > replacement using Phenix software with all the data (1.4A0) and got a > solution. Phenix did auto building with waters and R-free was 0.3123. > > I mutated some residues which don't align with the model protein to > Alanines. When i change the residues back to their respective side chains > Refmac5 won't refine it well. The maps looks clear( you can guess its 1.4A0 > data) but R-free is shooting up to 0.41. It is not accepting any changes to > the Phenix generated model. I have no idea what is going on. Can anyone help > me? > > Thank You in advance > Deepthi > > > > > > -- > Deepthi Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb] Stereo Microscope
Hello, I am planning to purchase a stereo microscope for visualizing crystallization drops. I would be very grateful if someone let me know the “objective” and “binocular eyepiece” specifications for SZX-7 or SZ-61 (Olympus) to get good magnification. Thank you. Regards, Prasenjit
Re: [ccp4bb] resolution limit
Rsym...what's that? JPK On Wed, Jul 18, 2012 at 9:12 AM, Edwin Pozharski wrote: > As has been shown recently (and discussed on this board), Rsym is not the > best measure of data quality (if any measure at all): > > http://www.sciencemag.org/content/336/6084/1030.abstract > > > > > narayan viswam wrote: > >> Hello CCP4ers, > >> In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & > >> Rsym 224.3 % > >> for multiplicity 7.8 and completeness 98.2 %. I solved the structure by > >> MAD & refined it > >> to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is not > >> twinned. The water > >> content is 68%. I loweredthe multiplicity to 4.1 by excluding few > >> images but still the > >> Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography > >> knowledge makes me > >> believe it's quite Ok to use data upto 2.8 A and report the statistics. > >> Could I request > >> people's views. Thanks very much. > >> Narayan > > > > After refinement, what is R-free in the last shell? If it is > significantly > > better > > than random, say around .4 or less, that could be taken as evidence that > > there > > is data in the last shell. > > Also check the error model- Rsym >2 sort of implies the error is greater > > than > > the signal, so I/sigI 2 seems surprising. > > eab > > > > > -- > Edwin Pozharski, PhD > University of Maryland, Baltimore -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] resolution limit
http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00383.html > Rsym...what's that? > > JPK > > On Wed, Jul 18, 2012 at 9:12 AM, Edwin Pozharski > wrote: > >> As has been shown recently (and discussed on this board), Rsym is not >> the >> best measure of data quality (if any measure at all): >> >> http://www.sciencemag.org/content/336/6084/1030.abstract >> >> >> >> > narayan viswam wrote: >> >> Hello CCP4ers, >> >> In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & >> >> Rsym 224.3 % >> >> for multiplicity 7.8 and completeness 98.2 %. I solved the structure >> by >> >> MAD & refined it >> >> to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is >> not >> >> twinned. The water >> >> content is 68%. I loweredthe multiplicity to 4.1 by excluding few >> >> images but still the >> >> Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography >> >> knowledge makes me >> >> believe it's quite Ok to use data upto 2.8 A and report the >> statistics. >> >> Could I request >> >> people's views. Thanks very much. >> >> Narayan >> > >> > After refinement, what is R-free in the last shell? If it is >> significantly >> > better >> > than random, say around .4 or less, that could be taken as evidence >> that >> > there >> > is data in the last shell. >> > Also check the error model- Rsym >2 sort of implies the error is >> greater >> > than >> > the signal, so I/sigI 2 seems surprising. >> > eab >> > >> >> >> -- >> Edwin Pozharski, PhD >> University of Maryland, Baltimore > > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *** > -- Edwin Pozharski, PhD University of Maryland, Baltimore
Re: [ccp4bb] resolution limit
I was [too] obliquely alluding to this thread... http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg27056.html JPK On Wed, Jul 18, 2012 at 12:32 PM, Edwin Pozharski wrote: > http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00383.html > > > > > Rsym...what's that? > > > > JPK > > > > On Wed, Jul 18, 2012 at 9:12 AM, Edwin Pozharski > > wrote: > > > >> As has been shown recently (and discussed on this board), Rsym is not > >> the > >> best measure of data quality (if any measure at all): > >> > >> http://www.sciencemag.org/content/336/6084/1030.abstract > >> > >> > >> > >> > narayan viswam wrote: > >> >> Hello CCP4ers, > >> >> In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & > >> >> Rsym 224.3 % > >> >> for multiplicity 7.8 and completeness 98.2 %. I solved the structure > >> by > >> >> MAD & refined it > >> >> to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is > >> not > >> >> twinned. The water > >> >> content is 68%. I loweredthe multiplicity to 4.1 by excluding few > >> >> images but still the > >> >> Rsym is > 200 % and I/sigmaI > 2.0. My rudimentary crystallography > >> >> knowledge makes me > >> >> believe it's quite Ok to use data upto 2.8 A and report the > >> statistics. > >> >> Could I request > >> >> people's views. Thanks very much. > >> >> Narayan > >> > > >> > After refinement, what is R-free in the last shell? If it is > >> significantly > >> > better > >> > than random, say around .4 or less, that could be taken as evidence > >> that > >> > there > >> > is data in the last shell. > >> > Also check the error model- Rsym >2 sort of implies the error is > >> greater > >> > than > >> > the signal, so I/sigI 2 seems surprising. > >> > eab > >> > > >> > >> > >> -- > >> Edwin Pozharski, PhD > >> University of Maryland, Baltimore > > > > > > > > > > -- > > *** > > Jacob Pearson Keller > > Northwestern University > > Medical Scientist Training Program > > email: j-kell...@northwestern.edu > > *** > > > > > -- > Edwin Pozharski, PhD > University of Maryland, Baltimore > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Stereo Microscope
We have an Olympus SZX-12 microscope and are running a 1X objective (with polarizer) and a 10 X eyepiece. The scope will zoom from approximately 10X-90X magnification. At 90X a 400 nL drop in a 96-well plate will nearly fill the field. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 7/18/2012 1:03 PM, PRASENJIT BHAUMIK wrote: Hello, I am planning to purchase a stereo microscope for visualizing crystallization drops. I would be very grateful if someone let me know the “objective” and “binocular eyepiece” specifications for SZX-7 or SZ-61 (Olympus) to get good magnification. Thank you. Regards, Prasenjit
[ccp4bb] 2D and 3D profile in integration
Dear crystallographers What does 2D (Mosflm?) and 3D (XDS?) profile fitting means for data integration? What is the guideline for using either one? References to any literature is highly appreciated. Thank you.
Re: [ccp4bb] 2D and 3D profile in integration
Hi Theresa I'd read Jim Pflugrath's 1999 paper in Acta D - "The finer things in X- ray diffraction data collection" Pflugrath, J.W. (1999) Acta Cryst D55, 1718-1725 http://journals.iucr.org/d/issues/1999/10/00/ba0030/ba0030bdy.html To my mind one of the best and most accessible explanations and comparisons available. I think it's on open access On 18 Jul 2012, at 20:11, Theresa Hsu wrote: Dear crystallographers What does 2D (Mosflm?) and 3D (XDS?) profile fitting means for data integration? What is the guideline for using either one? References to any literature is highly appreciated. Thank you. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] peptide fragment
Hello All, I have been working on a structure for one of the research groups here and have come across a peptide fragment of about 10 residues. The protein forms a tetramer and the peptide is found between two of the monomers. The protein itself does not require a peptide for function or formation for that matter. Has anyone come across similar things? and have a reference. We are trying to figure out where it may have come from. My best guess so far is from somewhere in the purification though I have no idea how long the protein sat around before crystallization. Cheers, Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center 101 Stephenson Parkway Norman, OK 73019-5251 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571
[ccp4bb] Postdoc in structural biochemistry - University of Oxford, SGC
We seek a post-doctoral scientist with strong background in protein biochemistry and structural biology, and interest in novel drug discovery to address the unmet medical need in rare diseases. The Metabolism & Organelle Biogenesis group (MOB) at the Structural Genomics Consortium, University of Oxford combines structural, biochemical, and chemical biology methods to dissect the molecular mechanisms underlying inherited enzyme disorders. The post-holder will drive a Wellcome Trust-funded project involving close collaborations with the pharmaceutical industry. Experience with protein expression, purification, crystallization and biochemical characterization is required. Prior training in X-ray structure determination will be an advantage. Dynamic and innovative, you will be a self-starter who enjoys teamwork, and a challenge. Interested candidates please apply via the university vacancy page (vacancy ID 103585): http://www.recruit.ox.ac.uk Informal enquiries can be sent to: wyatt@sgc.ox.ac.uk For information about the MOB group visit: http://www.thesgc.org/scientists/groups/oxford/metabolism-organelle-biogenesis - Dr Wyatt W. Yue PI, Metabolism & Organelle Biogenesis Structural Genomics Consortium University of Oxford UK OX3 7DQ +44 (0)1865 617757
[ccp4bb] postdoctoral positions available for experimental and computational methods for XFEL-based crystallography
Postdoctoral Positions Available for Experimental and Computational Methods Developments for XFEL-based Crystallography Faculty and staff of the SLAC Accelerator National Laboratory and Stanford University are leading an effort to develop methods for structure determination of challenging biological systems with the LCLS X-ray free electron laser. The effort includes collaborations with Lawrence Berkeley National Laboratory, Univ. of California at Berkeley, Univ. of California at Los Angeles, and California Institute of Technology. Particular emphasis is on small crystal characterization, novel sample delivery systems, data processing, experimental phasing, and refinement at low resolution. Extensive experience in computational or experimental crystallography is desirable. Candidates should submit a resume and a list of three references to Axel Brunger, Chair of the Bioimaging Working Group at SLAC, brun...@stanford.edu. Axel T. Brunger Investigator, Howard Hughes Medical Institute Professor of Molecular and Cellular Physiology Stanford University Web:http://atbweb.stanford.edu Email: brun...@stanford.edu Phone: +1 650-736-1031 Fax:+1 650-745-1463
[ccp4bb] refmac wouldn't process dT
hello everyone Does anybody knows why refmac5 will not process any model that contains thymine in its structure? This problem only happens on my PC which supports a 2.1.0 version of ccp4i. The error in the log file says its due to bad value during floating point read. However, this problem never happens on a linux based computer in our lab which supports an older version of ccp4i. Version 2.0.4 specifically. This problem extends over to coot as well. It would not process any mtz files if the model used contains any thymine. Again, this problem only happens on my PC but not on the Linux based computer. Coot's log file says the reason it failed is because it encountered a new ligand and which caused the refmac to shut down. Any feed back on this problem would be much appreciated. Thanks. Norman Zhu Department of Chemistry San Diego State College zhunor...@gmail.com
