[ccp4bb] modelling C-terminal COOH in coot
Dear all, I have a structure solved by molecular replacement. The C-terminus at the end of the chain shows as CO, not COOH. How do I make it into COOH in COOT? I tried to place an O in the relevant position, but seems not working this way. Thanks, Hubing
Re: [ccp4bb] Phaser and Molrep gave different solutions
Hi, Thanks for those who replied to this thread. I have been trying all the means that people suggested: search protein alone, DNA alone. However, both not working out. One thing Ray Brown suggested MR works if the molecules have identical sequences. So I just played around with the following way: 1) use the chainsaw editted model in MR; 2) mutate the protein sequence back to my protein and refine the solution in Coot; 3) use the partially refined protein-DNA as the new search model and run Phaser again then I get the following results for the two copies in ASU: RFZ=6.5 TFZ=10.7 LLG=903 RFZ=6.2 TFZ=17.4 LLG=1130. After one round of rigid body and restraint refinement in Refmac5, the Rfac and Rfree drops from 0.50/0.51 to 0.46/0.51. I did not refine the DNA sequences yet. I am not sure if this way I can further refine the structure, or do I bring two much bias into the structure. Please correct me if any. Thanks. Best, Hubing On Thu, Jul 21, 2011 at 11:31 PM, ray-br...@att.net wrote: I have also tried for years to solve a protein-DNA complex without sucess. If you have a lot more DNA than protein in the AU then MR will not work. You always get a good RFZ score but you cannot solve the translation if the DNA molecules are forming long stacks. With a plausible packing you will of course get model phases and a nice map but refinement will not work and you will get stuck at 40-50% Rf. You may have a chancewith MR if you only have a small DNA and a much bigger protein molecule or if the search models and the molecules have identical sequences. To solve this structure you probably have to do Se-labeled protein and SAD etc. or collect anomalous from metal ions if present. Cheers. Ray Brown -- *From:* Hubing Lou louhub...@gmail.com *To:* CCP4BB@JISCMAIL.AC.UK *Sent:* Thu, July 21, 2011 6:39:49 AM *Subject:* Re: [ccp4bb] Phaser and Molrep gave different solutions I was worried as well with the low TFZ score. Usually successful cases with score 8. I am still puzzled why Phaser and Molrep gave different solutions. Does this mean molecular replacement do not work out in this case so more crystals have to be prepared? A little more information might be helpful to dissolve the problem here. The model I used is a protein-DNA complex. The protein was Chainsaw editted but the DNA sequence was directly borrowed from the original model. Best, Hubing On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi, It's not a bad idea to read the Phaser manual for molecular replacement; see http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement Soon after the start, in a table on the right hand side, there is: TFZ score 5, have I solved it ? No. Hence with a TFZ score of 3.8 you do not have a solution using Phaser. Fred. Hubing Lou wrote: Dear all, I am stuck in a molecular replacement case and looking for advices. I have been working on a protein-DNA complex structure. Data was processed by HKL2000 to 2.6Ang and some of the data statistics are shown below: Space group: P21, Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90 Redundancy: 2.8 (2.7) Completeness: 94.8 (93.1) Linear R-fac: 0.051 (0.442) Data quality was checked by Phenix.xtriage and there's no problem. I then prepared a model by Chainsaw. Our protein shares only 30% of sequence similarity with the model, but structurally they are in the same group and almost identical in apo form. Matthrews Coeff indaced two monomers in AU. I then ran Phaser in automated search mode and there's a solution with RFZ score 4.8, TFZ score 3.8. The electron density map was not bad with DNA double helix clearly seen. However Refmac5 couldn't get Rfree lower than 50%. I then changed to MolRep, ran self rotation function first then used the first 10 peaks for translation search. Again there's a solution but it is different from that from Phaser. I attached a picture here. Checking in coot, the packing is the same. But, the refinement couldn't get Rfree lower than 50%. I have tried to include NCS, TLS refinement in Refmac, both not working. Hope someone out there can help. Thanks very much for your time. Hubing --**--**
Re: [ccp4bb] Phaser and Molrep gave different solutions
I also processed with Imosflm and ran Pointless, it was P21. Also it was indicated by the Intensity systematic absences in HKL2000 scale.log file. Best, Hubing On Thu, Jul 21, 2011 at 8:44 PM, herman.schreu...@sanofi-aventis.comwrote: ** Dear Hubing, One maybe stupid question: Your are sure the space group is P21 and not P2 or even something else? Did you test other possible space groups? Choosing the wrong space group could exactly lead to the results you observe. Best, Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Hubing Lou *Sent:* Thursday, July 21, 2011 6:46 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Phaser and Molrep gave different solutions Dear all, I am stuck in a molecular replacement case and looking for advices. I have been working on a protein-DNA complex structure. Data was processed by HKL2000 to 2.6Ang and some of the data statistics are shown below: Space group: P21, Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90 Redundancy: 2.8 (2.7) Completeness: 94.8 (93.1) Linear R-fac: 0.051 (0.442) Data quality was checked by Phenix.xtriage and there's no problem. I then prepared a model by Chainsaw. Our protein shares only 30% of sequence similarity with the model, but structurally they are in the same group and almost identical in apo form. Matthrews Coeff indaced two monomers in AU. I then ran Phaser in automated search mode and there's a solution with RFZ score 4.8, TFZ score 3.8. The electron density map was not bad with DNA double helix clearly seen. However Refmac5 couldn't get Rfree lower than 50%. I then changed to MolRep, ran self rotation function first then used the first 10 peaks for translation search. Again there's a solution but it is different from that from Phaser. I attached a picture here. Checking in coot, the packing is the same. But, the refinement couldn't get Rfree lower than 50%. I have tried to include NCS, TLS refinement in Refmac, both not working. Hope someone out there can help. Thanks very much for your time. Hubing
Re: [ccp4bb] unusual diffraction spots
Hi, The ears extended to other images but not all of them, some images do not show any signs like this. Thanks for replying, Liz. Best, Hubing On Wed, Nov 24, 2010 at 10:26 PM, elizabeth.d...@diamond.ac.uk wrote: I think there may be two effects going on here: I think the “ears” on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics. I think the change in spot shape from round to rod shaped is due to the crystal quality. Do the “ears” only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn’t a beamline optic effect. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE UK Tel. 01235 778057 Mob. 07920 138148 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Hubing Lou *Sent:* 24 November 2010 14:09 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] unusual diffraction spots
To further clarify things, the data was collected at a synchrotron beamline with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The detector type was MarCCD. The crystal was multiple-faced trigonal (space group P3121) the size was about 0.1*0.1*0.15mm. The exposure time was 2s for each image. I am currently refining the structure, however the Rfree stays above 30%. A close inspection shows at high resolution shell the spots become rod shaped. As I said we are preparing new constructs with N-terminal his-tag cleaved. But any other good suggestions out there might be helpful to avoid future frustration. Thanks, Hubing On Wed, Nov 24, 2010 at 10:26 PM, elizabeth.d...@diamond.ac.uk wrote: I think there may be two effects going on here: I think the “ears” on the round spots which also feature on the more rod shaped spots if you look closely could be related to a misalignment of the beamline optics. I think the change in spot shape from round to rod shaped is due to the crystal quality. Do the “ears” only feature on this image of this crystal or do they appear on other images? If the ear effect is a one off then that would tend to suggest it isn’t a beamline optic effect. Liz Dr. Liz Duke Principal Beamline Scientist Diamond Light Source Harwell Science and Innovation Campus Chilton OX11 0DE UK Tel. 01235 778057 Mob. 07920 138148 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Hubing Lou *Sent:* 24 November 2010 14:09 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] unusual diffraction spots Dear CCP4BBer, I recently collected a dataset at synchrotron. The diffraction was quite anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What unusual is in the diffraction image (see the attached file), clearly at low resolution there were some spots with tails (two ears) and at the high resolution shell the spots turned to be rod-shaped. Please, can anyone explain how this could be? Is this related to the anisotropy? The protein was N-terminal his6-tagged, we are currently preparing new samples with the His-tag removed. But any other suggestions are also very welcomed. Regards, Hubing -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] Optimization of needle crystals?
You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. -- Hubing Lou Centre for Biomolecular Sciences University of St Andrews North Haugh St Andrews Fife KY16 9ST Scotland Quoting Ngo Duc Tri [EMAIL PROTECTED]: Dear ccp4 users, I crystallized a membrane protein and got some conditions showing the small needle crystals. All condition contained Mg2+ and high buffer pH. I learn that it is difficult to optimize the needle crystal. However I would like to ask your experience how to optimize in this case. Here is the attached picture of my crystal. Thank you very much for your advices! My best regards, TriNgo PhD Student - Sungkyunkwan University, Korea -- University of St Andrews Webmail: https://webmail.st-andrews.ac.uk
Re: [ccp4bb] Progress in membrane protein cryo-protect
First of all, thanks very much for those who replied to my question. For those who do not follow this thread, I was asking a cryo question about membrane protein crystals grown at 4C with 0.05-0.2M AS (ammonium sulfate) and 15-26%PEG400. Some of you suggested directly freeze the crystal in LN from the condition of 0.2M AS, 26% PEG400. In practice, it's my experience that PEG400 30% in cold room readily get ice. Perhaps it is due to the air flow, the humidity etc in the cold room. I've also tried 20%PEG400 plus 15% glycerol, still there's ice. So I just sticked to 35% of PEG400 when freeze crystal in cold room. However, in room temperature (20C), 30%PEG400 works fine. I've got crystal in similar conditions at 20C and tried sequential soaking by gradually increase the PEG400 from 18% to 30%. I did four steps, 18%,22%,26% then 30%, each within 3-4 seconds. The crystal diffracted to 3.6A in house. Longer soaking caused cracks. Cold room cryo conditions is still in progress. I'll post it once I get good results. Hubing Quoting Peter Adrian Meyer [EMAIL PROTECTED]: Hi Tim, sorry if I didn't follow this thread carefully enough and this has been mentioned before: To my experience, 20-25% PEG400 by itself is enough for cryo protection. Did you actually test whether you need to change anything at all? Maybe you can just dip your crystals straight from the tray into liquid nitrogen. You can easily test this by preparing the buffer and freezing it without crystals and taking an image. Well, apparently I wasn't following as closely as I could've been either: I just notice a big change in cryo concentrations and thought of suggesting a gradual change. I haven't ever tested PEG400 alone for cryo-protection (usually mixed with glycerol, peg4000, or propandiol). Pete On Fri, 2 Feb 2007, Peter Adrian Meyer wrote: Hi Hubing, I have crystallized a membrane protein at cold room temperature (4°C). The protein was purified in 20mM Tris, pH8 with 1% bOG. The reservoir solution contains 0.1M HEPES pH7.5, 0.05-0.2M (NH4)2SO4 and 15%-26% PEG400. The cryoprotectant was made in such a way that all the other ingredients remained the same except for the PEG400 increased to 35%. The crystal was looped from the well and directly dipped into the cryo, however, the crystal cracked within seconds of soaking. Speedy soaking and transferring of the crytal into liquid N2 resulted 6Å diffraction in ESRF. Not strictly related to membrane proteins, but one approach would be to a series of transfers with gradually increasing percentages of PEG400 (instead of 26% - 35% - LN2;try 26% - 30% (wait a while) - 35% (wait again) - LN2 ). The idea is to avoid drastic changes to the crystal's environment (similar to what Michael Garavito was talking about regarding detergents). You don't mention what temperature you're doing your cryo-soaking at; but if you grew the crystal at 4 C it's probably a good idea to soak, equilibrate and freeze at 4 C as well (you're probably doing this already anyhow). Good luck, Pete Pete Meyer Fu Lab BMCB grad student Cornell University Pete Meyer Fu Lab BMCB grad student Cornell University -- University of St Andrews Webmail: https://webmail.st-andrews.ac.uk
