[ccp4bb] Vacancy at Evotec UK (Abingdon)
Dear All, There is an opportunity to join the Structural Biology team at Evotec based in the UK as a Senior Scientist (Protein purification). Please see the advert in the link below. Interested candidates should apply directly through our website. Please do not send applications through to me directly. Closing date is July 31st and applicants must be eligible to work in UK. The position is for a 12 month contract. https://recruitment.evotec.com/VacancyDetails.aspx?FromSearch=True=QSAukis1tlc==649 Best wishes, Matthias To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Crystallographer position at Evotec
Dear All, There is an opportunity to join the Structural Biology team at Evotec based in the U.K as a Senior Scientist (Crystallography, initial 12 month contract). Please see the advert in the link below. Interested candidates should apply directly through our website. Please do not send applications through to me directly. Closing date is June 30th and applicants must be eligible to work in UK. https://recruitment.evotec.com/VacancyDetails.aspx?FromSearch=True=QSAukis1tlc==646 Kind regards, Matthias Zebisch STATEMENT OF CONFIDENTIALITY. This email and any attachments may contain confidential, proprietary, privileged and/or private information. If received in error, please notify us immediately by reply email and then delete this email and any attachments from your system. Thank you! Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered Office: 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Anisotropic diffraction
Dear Roberto, I think you should try different special orientations of your crystals in the loop to find the one in which the measurable reflections stay away from the slow moving region (usually aligned with the beamstop holder, so horizontal in your image), in which reflections cannot be acccurately measured and are discarded by integration programs. This way you wont at least lose more of your precious reflections. Generally Id recommend not to discard any measurable reflection irrespective of very bad overall statistics. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 2/25/2015 10:07 PM, Orru, Roberto wrote: Dear All, My crystals are heavily affected by diffraction anisotropy as you can see in the images attached. I have been using the server at UCLA (http://services.mbi.ucla.edu/anisoscale/) to treat the datasets, but I was wondering if there is any suggestion on how to setup the strategy for the data collections and for the following integration. Thanks in advance, Roberto
Re: [ccp4bb] merging anisotropic datasets
Hi Bob, we have done elliptic truncation before merging/scaling as lined out in 2012 Zebisch (JMB). However, I see no reason not to scale the 3 runs together with aimless (to lets say 3.4A) and then subject the resulting merged file to the sawata server. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 11/12/2014 9:26 PM, Robert Keenan wrote: I have three datasets of varying quality collected from different regions of a single crystal. In each case, the data are anisotropic (from Aimless using CC1/20.5): Dataset 1: 3.5, 3.5 5.5 A Dataset 2: 4.2, 4.3, 4.8 A Dataset 3: 3.7, 3.9, 4.4 A I initially took a simple-minded approach and processed each dataset at the appropriate resolution limit (set 1: 3.5A; set 2: 4.2A; set 3: 3.7A) using XDS/XSCALE as implemented in xia2. The resulting merged dataset seems fine (albeit with ugly stats in the high-res bins because of the anisotropy), and it allowed me to solve the structure (Rfree/Rcryst ~31/26). Now I am wondering if I can improve the maps further by first applying an ellipsoidal truncation (using the UCLA diffraction anisotropy server) and then scaling/merging the three datasets together. However, it seems that the UCLA anisotropy server only allows input of one dataset at a time, and it outputs merged amplitudes. Is there some way to obtain the elliptically truncated but unmerged data for each of the three datasets? More generally, are there preferred strategies for dealing with strongly anisotropic data? Bob Robert Keenan Associate Professor Dept. of Biochemistry Molecular Biology GCIS W238 University of Chicago 929 East 57th Street Chicago, IL 60637 (o) 773.834.2292 (f) 773.834.5416 (e) bkee...@uchicago.edu mailto:bkee...@uchicago.edu http://keenanlab.bsd.uchicago.edu
Re: [ccp4bb] ATP library file in REFMAC
Cant you edit the ATP.cif on your computer to have the correct expected bond length? Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 8/27/2014 4:12 PM, Bernard D Santarsiero wrote: I recently refined a structure in CCP4/REFMAC with ATP in the structure. Upon submission to Acta for publication, the wwPDB validation report was run. Several things were flagged, including the C4-C5 bond in the adenosine moiety as being too long. It generally refines to 1.46-1.47A. The ideal distance in the validation report is 1.38A, and the upon review of the ATP.cif file in the REFMAC library, the target distance is 1.49A (and listed as a double bond). Clearly 1.37-1.38A is a reasonable target value. HIC-Up gives the target bond length as 1.404A. Where can I grab a revised ATP.cif file? I guess I'll need to re-refine all of my structures and re-run the validation report. BTW, I also looked at the PDB_REDO structure report for my structure, and can't reproduce the Rcryst and Rfree values with the same model. Bernie -- Bernard D. Santarsiero Research Professor Center for Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy Center for Structural Biology Center for Clinical and Translational Science University of Illinois at Chicago MC870 3070MBRB 900 South Ashland Avenue Chicago, IL 60607-7173 USA (312) 413-0339 (office) (312) 413-9303 (FAX) http://www.uic.edu/labs/bds http://scholar.google.com/citations?user=fGauLBMJ
Re: [ccp4bb] double bonds in oleoyl-CoA
Hi Wei, your double bond is clearly not correctly defined in your cif file. It should be significantly shorter - 1.3A instead of 1.5A. However, it is easy to edit the expected values manually into your cif file. Try to find PAM.cif in your coot directory (palmitoleate) to find the correct value. Be careful that you edit the correct bond. Your numbering might be different. Are you sure that there is no cif file for your ligand already existing? Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 8/20/2014 3:45 PM, Wei Shi wrote: Hi all, I am working on solving a X-ray crystallographic protein-ligand structures. Attached please find initial oleoyl-CoA structure (elbow.001.pdb) and the ligand structure in the final model (ligand in structure.pdb). When open these in pymol and use show as lines and set valence, 0.1 to show double bond, the double bond between C9 and C10 in oleoyl-CoA fatty acid part is only visible in the initial ligand structure (elbow.001.pdb) but not in the ligand in the final model (ligand in structure.pdb). I got the initial ligand using smile string by Phenix. eLBOW and fit the initial ligand to the electron density, and then refine the structure in the presence of cif file. I am wondering whether any of you happen to know why in the final model (ligand in structure.pdb), double bond (C9) is not visible. Is it because the double bond is missing or is it because I didn't display it correctly? Thank you so much! Best, Wei
Re: [ccp4bb] SO4 geometry
Hi Vaheh, might be that you inverted the naming chirality by accident. Simply regularize in coot and then carefully replacing into density should do. If that doesnt do invert yourself by drawing one oxygen through the sulfur center. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/31/2014 8:01 PM, Oganesyan, Vaheh wrote: Colleagues, Sorry to bother for something really minor. The Refmac usually always recognizes tetrahedral SO4 groups and there were no problems related to its geometry. Two attached files demonstrate SO4 geometry before and after refinement. Would you be able to point me to mistake I’m doing? Thank you. Regards, Vaheh Oganesyan, PhD Antibody discovery and protein engineering MedImmune, LLC. www.medimmune.com To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] large domain motion and calculation of flip flop angle
As usual, I'd recommend the program Dyndom which is available from CCP4i. This will identify domain borders + hinge regions as well. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/2/2014 4:52 PM, rajan kumar wrote: Dear all, sorry for off topic question. My protein is composed to two domains connected by a flexible linker and shows large domain motion with large RMSD value of 9.0 A* difference with respect to the initial structure, when simulated for 20 ns. As the first domain doesn't shows any RMSD change when superimposed to initial structure but the other domain moves significantly. so my question is how i could be to calculate the angle of motion in degree of the flexible domain with respect to the initial structure after superimposition. thank you all in advance Regards *Rajan kumar choudhary* *Senior Research Fellow* *Department of Atomic Energy(Govt.Of India)* *ACTREC TATA Memorial Center * *Kharghar Navi-Mumbai* *Mumbai-410210* *India*
[ccp4bb] pymol multiple transparent surfaces
Dear BB-lers, I want to draw in pymol 2 objects, which are behind each other, as transparent surfaces. The surface at the back gets simply deleted. This would be correct (save some rendering time) if the object at the front was not see-thru. What wheel do I need to turn to get the surface of my object in the back drawn? Thanks a lot, Matthias -- - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk -
[ccp4bb]
Dear Avinash, in addition to Eleanors suggestion you might want to consider using Dyndom in CCP4 or via its web server. It will also identify hinge regions and percent twist motion vs. closure motion. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 2/23/2014 6:57 PM, Eleanor Dodson wrote: One way is this: Align Domain1 of structure1 to Domain1 of structure 2 on domain 1 (using LSQKAB or whatever.) Then align domain2 of the output aligned structure 1 to domain 2 of structure 2 The polar Chi or Kappa or whatever output from that alignment is the angular shift required Eleanor On 22 Feb 2014, at 06:19, avinash singh wrote: Dear CCp4b users, I have a protein which has been crystallized in two different conditions. In one of those conditions, the structure shows the domain shifting. Is there any programme or online server which calculates the angular shift in domain when campared to the other condition crystallized structure which shows no such domain shift. Thanks in advance Avinash Singh
Re: [ccp4bb] cryoprotection condition
Hi Rana, you are almost there. For the first one I'd add (if at all) 5% PEG200 and for the second I'd mix in 10% PEG200. Only very big crystals might need even more cryoprotectant. Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 2/8/2014 12:44 PM, rana ibd wrote: Dear CCP4 Does anyone know a good cryoprotection condition for these two conditions I would be grateful The first condition: 10% PEG 2, 20%PEG MME 550. 0.03M of NPS ( NPS is sodium nitrate, disodium hydrogen phosphate, and ammonium sulfate), 0.1MMES/Imadizole pH 6.5 The second condition: 20% PEG 3350, 0.2M sodium iodide or 0.2M sodium acetate Thank you Rana
Re: [ccp4bb] seeding, reproducibility
Dear Mahesh, cross-seeding into other conditions often works but still the chances are much lower than seeding the same form. Best wishes, Matthias (Please desist from sending so large attachments to half the planet!) - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 1/17/2014 10:24 PM, Mahesh Lingaraju wrote: Hi Folks The protein that I am working on gives several initial hits which are needles. And at random, I picked the needles from a condition (0.2 M cacl2, 0.1 M HEPES pH 7.5 28% PEG 400) and seeded into another screen where I added 1µl protein + 1µl reservoir solution + 0.3 µl seed stock. I prepared the seed stock fresh by adding 36 µl of the reservoir solution to preexisting 2µl drop. One of the conditions from the seeded screen gave me a hit that looks really promising ( see attached). I am sort of positive that these crystals are protein as they are UV active. I tried to reproduce these but with needles from another condition which actually look much nicer than the seeds i used to produce these crystals. However, I failed to do so. While i understand that seed quality is important, I find it interesting that the crystals do not reproduce with similar or better looking seeds. Is it common that the seeds absolutely need to be from the same condition to reproduce hits ? thanks for any suggestions Mahesh
Re: [ccp4bb] Best compounds for heavy atom soaks
free cysteines? - pCMB phosphate-binding? - tungstate Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 1/15/2014 5:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
[ccp4bb] Membrane protein structures from mammalian expression
Dear crystallographers, happy new year to everybody! Can I ask you to send me examples where expression of fully integral membrane proteins in mammalian cells (not insect!) has led to the successfull determination of a crystal structure? Thanks very much, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk -
Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- *Priyank*
Re: [ccp4bb] the f' and f'' of heavy cluster
Dear Lisa, if you have proper anomalous data I rather recommend using the Phaser SAD pipeline and specify cluster search. Worked instantly for me. Good luck! - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 11/21/2013 6:29 AM, LISA wrote: Dear All, I am running autosharp with a single wavelength data soked with Ta6Br12. This data collected at the wavelength of 1.254A. I told the autosharp the f' -20 and f''10.5. The autosharp result said these values are not correct? How can we get the f' and f'' of this cluster? Thank you. Lisa
Re: [ccp4bb] off topic
Dear Amr, the gel is some kind of phase separation enriched in your protein (perhaps indeed aggregates). The fact that both the crystals and the gel gives blue tryptophan fluorescence clearly shows that both are protein. I think it is quite frequent that these kind of phase separations serve as nucleation points. Well done, shoot'em! Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 10/19/2013 7:24 PM, amro selem wrote: Dear Cryptographers, I have repeated question , i did screening and with in one day i got crystals, also this is some gelled protein . under UV, the crystals appear blue but also the gelatin like precipitate too . i will attach the photo for some help if the birefrigence say some information if it is salt or protein. the screen condition is 0.2 ammonium sulphate and 15% peg 8000.also have feeling that crystals are still growing . best regards Amr ·
Re: [ccp4bb] High Rsyms between 5-7 A
Dear Ursula, I wouldnt be worried too much abot 4.4% Rsym to be honest. At this high redundancy rather look on Rpim. I once had a case with a low I/sI medium resolution shell where I had strong translational NCS. Is this the case? Go publish! Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 10/14/2013 7:23 PM, Ursula Schulze-Gahmen wrote: I have a data set with high Rsym in the lower resolution ranges, and I don't understand what is going on. The crystal diffracts to about 3.0 A and has large cell dimension ( space group P6522 a= 185., c= 360.) Mosaicity is low. I processed the data in P6522, solved the structure and refined it. The maps look good and the structure refines very well to R/Rfree of 20.5, 23.5%. But the dataset has a total Rsym of 22%, a redundancy of 20, an Rpim of 7.6%, and CC1/2 of 0.55 in the highest resolution shell. The problem seems to be in the lower resolution region around 8-5.5A. The Rsym there is around 20%, it actually has a bump in this resolution region and is slightly lower at 5.0 A before it steadily increases to higher resolution. The high Rsym region between 8-5.5 A correlates with very low I/sig which also increases again around 5.0 A and then steadily decreases. The diffraction image shows very weak spots too. My question is: Is it likely that the data are processed correctly and the crystal packing causes this strange diffraction pattern, or is there something wrong with the data processing or the crystal? Any suggestions would be greatly appreciated. Ursula -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220
Re: [ccp4bb] Off Topic- DNA length
Dear Wei, doesnt sound too special to me. DNA in itself is a very soluble molecule. The part which hangs out of the core complex drives solubility of the whole thing. As a drawback crystallizability may also go down... All the best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 10/2/2013 4:24 PM, Wei Huang wrote: Dear CCP4er, I have an interesting observation for one of my DNA binding protein--when the length of DNA flanking both DNA binding site increase, the solubility of protein-DNA complex increases. And I can get a nice sigmoid curve from protein solubility versus DNA length. The solubility of protein complex saturated at the DNA length with additional 12 base pairs of DNA on both sides. Has anyone had similar observation before? What is the implication for this DNA-binding protein? Thanks for sharing your opinions! Best regards, Wei
Re: [ccp4bb] visualizing G310 helices in PYMOL
Dear Faisal, I usually assign in PYMOL the secondary structure generally obeying DSSP output. You have to use the alter command. eg: alter myprotein and resi 103:106, ss='H' Bytheway, pymol swaps inside and outside color on left handed helices, wich you might also have. Hope this helps, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 9/26/2013 9:02 PM, Faisal Tarique wrote: Dear all Sorry for the off topic question. My protein has few G310 helices. It is clearly visible through STRIDE or DSSP, but when i open the structure in PYMOL it didnt show it. Other visualization graphics like CHIMERA and VMD are able to pick few of them but not all the G310 helices..For manuscript preparation i have drawn the topology diagram taking the output from STRIDE and DSSP while the overall 3D structure is from PYMOL..Will it be O.K to show some helices missing from the output of pymol while they are present in the toplogy diagrma ? or the reviewer will raise the issue? hope you are able to understand what i mean to say. Please suggest me -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Low 280 absorbance imidazole?
Hi everybody, I would just like to contribute that absorption at 280 results from methylated imidazole, a contamination hard to get rid of. Imidazole itself does not absorb at 280. Bye, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 8/21/2013 7:43 PM, Bosch, Juergen wrote: How about low pH elution or EDTA as alternative ? Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Aug 21, 2013, at 14:40, Prof. Dr. Arne Skerra ske...@tum.de mailto:ske...@tum.de wrote: Hi Bernhard, We know this problem since the very early days of the His-tag and have been using high purity imidazole from Merck since then with success, which essentially shows no absorption at 280 nm. If you like I can enquire the current order number in my lab. Cheers, ASk Am 21.08.2013 um 16:33 schrieb Bernhard Rupp: Hi Fellows, could someone please point me towards the source of a known high purity imidazole with low absorbance at 280 nm? I am facing the problem of detecting a low absorption protein in high imidazole background after IMAC gradient elution. In the UV spectra of the 2 imidazoles I checked there is some contaminant that absorbs at 280… Thx, BR Bernhard Rupp Marie Curie Incoming International Fellow Innsbruck Medical University Schöpfstrasse 41 A 6020 Innsbruck – Austria +43 (676) 571-0536 bernhard.r...@i-med.ac.at mailto:bernhard.r...@i-med.ac.at Dept. of Forensic Crystallography k.-k. Hofkristallamt Vista, CA 92084 001 (925) 209-7429 b...@ruppweb.org mailto:b...@ruppweb.org b...@hofkristallamt.org mailto:b...@hofkristallamt.org http://www.ruppweb.org/ --- * Prof. Dr. Arne Skerra* Lehrstuhl f. Biologische Chemie | Technische Universitaet Muenchen Emil-Erlenmeyer-Forum 5 | 85350 Freising-Weihenstephan | Germany Phone: +49 (0)8161 71-4351 | Fax: -4352 http://www.wzw.tum.de/bc | eMail: ske...@tum.de mailto:ske...@tum.de
Re: [ccp4bb] laboratory phage infection [SEC=UNCLASSIFIED]
Dear Aidong, we never had that problem but I heard that only long dry(!) heat treatment of glassware destroys the phages. No need to say that you have to do it with ALL your glassware. Good luck, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/2/2013 12:15 PM, aidong wrote: Many thanks for your rapid inputs. We used to make glyerol stocks but now we do not since the protein expression is not stable. Our lab is about 10 students, who have to make their own proteins so our three large-scale shakers are very crowded every day. We did try a formaldehyde gas treatment one time during our new year leave, however, the situation did not seem to improve. Since that gas is much harder to handle, we only stick to ozone treatment. Intriguingly, our neighbor lab, also a crystallography lab, which constantly uses bacterial cultures although not as much as we do, is just fine. Chloroform/phenol treatment might be a good one because some of our constructs are just very difficult to get any transformed colonies while some others are so easy. We have used gel extraction method to purify our plasmids because we thought some phage DNA is contaminated. This method slightly improved our situations but not complete. But I am thinking the entire phages are simply there in plasmid preps purified through miniprep columns. Cheers Aidong On Mar 2, 2013, at 7:30 PM, DUFF, Anthony wrote: Hi Aidong, some ideas... Do you use glycerol stocks of transformed expression cells (phage risk), or do you do fresh transformations each time? Do you have many people working together, or do you have periods of bacterial growth separated by periods of inactivity? Do you have neighboring labs who may be working with, or infected with phage? Do you have any old stocks, liquid media, antibiotics, etc, that could be contaminated? sincerely, Anthony From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of aidong [a...@xmu.edu.cn] Sent: Saturday, 2 March 2013 7:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] laboratory phage infection Hi All, Really sorry for my non-CCP4 related question. Our lab mainly uses bacterial cultures to produce targets proteins for crystallizations. However, we have been struggling with phage infections to our bacterial cultures for a quite long time. To control its devastating effects, we have regularly been using large-dose ozone treatments on the whole lab space. We have also tried anti-phage BL21/DE3 strains from Sigma USA but found it was still not avoidable. The lab has been maintained well hygienic and its outdoor environment is clean and neat. We keep good ventilations, including windows and central AC systems. However, phages are still eating up our cultures with very low percentage of survivals. Therefore, this has been our big headache. We wonder whether you have the same experience and how to keep a lab free of those bugs. Your suggestions are deeply appreciated. Thanks. Sincerely Aidong Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University 3 South Xiangan Road Xiangan, Xiamen 361102 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/ Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University 3 South Xiangan Road Xiangan, Xiamen 361102 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/
[ccp4bb] Superpose, SSM
Dear CCP4 users, I am using the ccp4i version 6.2.0 under windows 7. I've come across a problem with superpose. The outputfile appears to have additional line feeds (see picture) which, however are not seen in the windows notepad. The structure can also be opened in coot and pymol. However, it is not possible to use it within CCP4, eg. for a subsequent superposition. Is this problem known to anybody and is there a simple workaround available? I need to compare hell of a lot of relative domain orientations... I did not have this problem on a second computer with ccp4 6.1.2. When I updated to 6.2.0, the situation was as described above. Any help will be highly appreciated, Thanks, Matthias attachment: superpose.png
Re: [ccp4bb] Superpose, SSM
Hi again, Thanks for your quick replies but I think I made myself not clear. here is what I'm doing: 1) superpose proteinA.pdb onto proteinB.pdb : works, but gives out proteinA_lsq1.pdb with extra empty lines (not the anisou lines ;o) ) 2) superpose proteinA_lsq1.pdb onto proteinC.pdb : doesnt work because proteinA_lsq1.pdb cannot be read Any ideas? Even if there is some compatibility issue between CCP4 and windows, I guess superpose should be able to read its own files, shouldnt it? Thanks, Matthias On 9/26/2011 9:13 PM, Jacob Keller wrote: I vaguely recall notepad doing something wacky with files in certain cases...why don't you get the excellent text editor NoteTab Light [sic] (I use it all the time--free and works great), then take a look at your files and see whether MS notepad altered the files. JPK On Mon, Sep 26, 2011 at 2:42 PM, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: Dear CCP4 users, I am using the ccp4i version 6.2.0 under windows 7. I've come across a problem with superpose. The outputfile appears to have additional line feeds (see picture) which, however are not seen in the windows notepad. The structure can also be opened in coot and pymol. However, it is not possible to use it within CCP4, eg. for a subsequent superposition. Is this problem known to anybody and is there a simple workaround available? I need to compare hell of a lot of relative domain orientations... I did not have this problem on a second computer with ccp4 6.1.2. When I updated to 6.2.0, the situation was as described above. Any help will be highly appreciated, Thanks, Matthias
Re: [ccp4bb] large R-Rfree difference in final structure
Hi Careina, in our lab we once had the problem, that the asymmetric unit contained 8 molecules, whereas 7 had only been modeled. Somehow the 8th monomer had evaded detection. So be careful not to miss density. Matthias On 7/13/2011 7:54 PM, Robbie Joosten wrote: Hi Careina, Assuming you don't suffer from a very poor data parameter ratio that would lead to such a large R-free/R, you need to improve your refinement. If you have NCS you should use local NCS restraints. You could also try jelly-body restraints, although they may not work at your resolution. Cheers, Robbie Date: Wed, 13 Jul 2011 08:38:38 -0700 From: careinaedgo...@yahoo.com Subject: [ccp4bb] large R-Rfree difference in final structure To: CCP4BB@JISCMAIL.AC.UK Dear ccp4 bulletin board I just have a slight concern regarding my Rwork Rfree difference. I have a structure that I have solved. I am reasonably content that it is complete because it has refined well, it no longer has bad geometries and contacts and all the rotamers, ramachandra, bond lengths etc are good. It gives favourable scores on molprobity and procheck. My only concern is the R factor difference. The resolution of the structure is 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33 which seems to me to be rather high. Should I be concerned? During refinement Rfree only drops from about 0.36 to 0.33 while the R factor drops from 0.31 to 0.24.. I have removed automatic weighting in refmac in order to constrain my bond lengths and angles during a couple of rounds of refinement. This did not have any effect on the R factors, however. I am fairly content that the space group I have chosen is correct so I am not sure what else could cause the big difference in R factors? There is no twinning. Can I be satisfied that my structure is correct despite the high R free or should I be doing other checks/ trying other things before I can submit this structure? Thank you for any help Careina
Re: [ccp4bb] Question about movie making
VirtualDub is the (absolutely free) program of choice, I'd say. Good luck, Matthias Am 07/03/2011 21:21, schrieb mjvdwo...@netscape.net: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
Re: [ccp4bb] A quick question - monomer lib cif
Dear Stephen, a cif from CCDC is something totally different from a refmac CIF. You need to convert to PDB (you can also use MOE software if available). When you have built in your small molecule PDB into your whole model you just run refmac. Refmac will fail but write out a suggestion for a molecular description cif file. You simply edit this cif file using bond lengths and angles from the CCDC structure (i use pymol for analysis). Through out wrong or weird info (often torsion restraints and stereoisomer info) and use the edited cif file as library input in a new Refmac run. Good luck, Matthias Am 06.01.2011 04:30, schrieb Dr. STEPHEN SIN-YIN, CHUI: Dear all experts, Just a simple question, how can I obtain a monomer library CIF (_lib.cif) of a new small molecule that could be recognized by Refmac5? If i have a CCDC cif file. many thanks stephen -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
[ccp4bb] CCP4
Hi Yahui! I am having this problem as well again and agin. Most problematic is it, if you have non-standard atoms in your compound. I don't really know whrere the problem lies, but here is what I do: Do not use sketcher! Simply generate your ligand using coot by placing atoms into the density (you may start from standard compounds if available). Save your ligand in a pdb file and make sure (text editor), that all atoms belong to the same compound indicated by the same 3 letter identfier (e.g. LIG). Merge this PDB file with your protein in Coot and save. Run Refmac. Refmac will abort but before stopping it will put out a library file with recommended bonds and angles and so on. This file you should manually edit putting in your chemical knowlege of the ligand. Use this cif file in a second run for refmac and for all coot real space refinements. Have fun, Matthias PS: sometimes (no non-standard atoms) a simpler way is to leave the field Regularize with Refmac unchecked, when you create your final library file. Am 6/9/2010 12:23 PM, schrieb Yahui Yan: Hello, Could you please help me with the sketcher? I'm trying to use ccp4 sketcher to generate a new ligand and then complex it with a protein in coot. I've drawn the ligand, numbered each atom and defined each bond type. Then I ran save file, create library description. The pdb file was loaded to coot and worked fine. Then I imported cif file. However, when I tried to refine the ligand, a message popped out, saying 'No restrains'. I double checked the numbering, I think it's Ok. Did I do anything wrong? I'm really struggling on this. If you need more information, please let me know. Thanks very much. Best regards, Yahui -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
Re: [ccp4bb] sketcher
Hi Yahui! I am having this problem as well again and agin. Most problematic is it, if you have non-standard atoms in your compound. I don't really know whrere the problem lies, but here is what I do: Do not use sketcher! Simply generate your ligand using coot by placing atoms into the density (you may start from standard compounds if available). Save your ligand in a pdb file and make sure (text editor), that all atoms belong to the same compound indicated by the same 3 letter identfier (e.g. LIG). Merge this PDB file with your protein in Coot and save. Run Refmac. Refmac will abort but before stopping it will put out a library file with recommended bonds and angles and so on. This file you should manually edit putting in your chemical knowlege of the ligand. Use this cif file in a second run for refmac and for all coot real space refinements. Have fun, Matthias PS: sometimes (no non-standard atoms) a simpler way is to leave the field Regularize with Refmac unchecked, when you create your final library file. Am 6/9/2010 12:23 PM, schrieb Yahui Yan: Hello, Could you please help me with the sketcher? I'm trying to use ccp4 sketcher to generate a new ligand and then complex it with a protein in coot. I've drawn the ligand, numbered each atom and defined each bond type. Then I ran save file, create library description. The pdb file was loaded to coot and worked fine. Then I imported cif file. However, when I tried to refine the ligand, a message popped out, saying 'No restrains'. I double checked the numbering, I think it's Ok. Did I do anything wrong? I'm really struggling on this. If you need more information, please let me know. Thanks very much. Best regards, Yahui -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
[ccp4bb] Anisotropic Diffraction Examples
Dear everybody! I am currently having an issue with anisotropic diffraction and would like to cite some references. I am sure there are plenty of examples outthere. So, if you are having an own published example at hand, can you please send me the reference and maybe the diffraction limits along good and bad directions as well? Thank you a lot, Matthias -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
Re: [ccp4bb] TCEP effect on protein
Hi Ganesh and Mega! I do not agree with Ganesh. I assume, Megha, that truly reversed phaseHPLC was used. This is a denaturing method and the natural disulfide should not form again during the run. Also pH 4 can not be described oxidizing. Actually, reduced proteins are often dialyzed against acidic buffers to prevent disulfide formation via the thiolate anion. Still, a reducing agent may be used during the run? Sorry that I can not offer a solution to the real problem. More experimental details may be necessary. Bets regards, Matthias Am 3/26/2010 8:37 AM, schrieb Ganesh Natrajan: Hi Megha, The two peaks on the HPLC indicate that your protein is existing in a monomer-dimer equilibrium in solution. The dimerisation is most probably caused by disulphide bridges. The use of TCEP is breaking those disulphides and that is causing the equilibrium to move towards the monomeric state. However, when the TCEP is dialysed out, the disulphides start forming again and this is causing the equilibrum to move towards the dimeric state, a process clearly hastened by the strongly oxidising pH 4 of the dialysis buffer. Now it all depends on what you want to do. If you want to use the protein in a (largely) monomeric form, I would recommend that you don't dialyse out the TCEP. regards Ganesh ** Blow, blow, thou winter wind Thou art not so unkind As man's ingratitude; Thy tooth is not so keen, Because thou art not seen, Although thy breath be rude. -William Shakespeare ** On Thu, 25 Mar 2010 22:51:30 -0800, megha goyal mgbio...@gmail.com wrote: Hi all, My protein is relatively pure except the dimer. i used 10 mM TCEP to reduce it (directly added 1 M TCEP to make final volume of 10mM and kept at room temperature for 10 mins] then i dialysed the protein sample to remove TCEP [dialysis buffer is Na acetate pH 4.0]. On performing SDS PAGE analysis after dialysis of the protein we are getting no dimer band, only band of our protein is observed and same is the case with UV reading there is no change in it. But the HPLC analysis of the protein shows two peaks instead of one peak as observed before TCEP treatment. what can be the reason for this. Kindly guide. i need the protein to formulate and conduct stability studies on the sample. the protein we obtain after IEX is pure except the dimer and i do not want to go for SEC as it greatly reduces protein content and also is quite time consuming. any light on what is happening will bevery useful. thanks and regards -- -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
[ccp4bb] sulfur sad phasing
Dear bb! What is the optimal wavelength for Sulfur SAD phasing? Is it 1.9A or should one go below that to reduce absorption/damage. Also, would the same wavelength be appropriate to maximize anomalous scattering to position chlorides, calcium, sulfate in already phased structures? Thanks in advance, Matthias
[ccp4bb] Problem with TLS
Dear BB! I have a strange problem with TLS refinement and refmac. In my AU there are 4 monomers of the same protein. Based on structural subdomains I defined 3 TLS groups for each of those monomers so that the overall TLS group number is 12. When I do TLS and restrained refinement from the GUI using my TLSIN file which has only the group definitions, everything seems fine. I only get quite a few atoms with residul B factor with the strangely exact value of 2.00. Is this normal? I had this also for waters but could overcome this with the keyword TLSD waters exclude. R factors go down to 18%/22.5% (mild anisiotropic diffraction to 1.8A in the best direction). Now, my real problem is, that when I continue with model building and refinement (restrained with fixed TLS or TLS+restrained), I would like to use the original TLSOUT as the new TLSIN file. When I do so, the R factors rather go up ending with values higher then when leaving out TLS completely (~25%/30%). Can someone explain to me why this happens? I would rather like to avoid rerunning TLS everytime again as it is very time consuming. Any help is greatly appericiated. Matthias PS: I have this issue also for other proteins.
[ccp4bb] statistics
Dear everybody! I recently tried out the anisotropy server at http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/ I also see that there is much discussion going on about the correctness of this method. In any case, does anyone know a tool that gives me the datacollection statistics that I normally record from the SCALA log file? I am talking about multiplicity, completeness, I/sI etc all _AFTER_ ellipsoidal truncation and anisotropic scaling. My crystal diffracts in one direction to at least 1.8A whereas in the other directions it is rather 2.0 to 2.1. The spacegroup is C2. Thank you a lot, Matthias
