[ccp4bb] Postdoctoral Positions of Structural Biology, SUSTech, Shenzhen, China
Dear all, Two postdoctoral positions are currently available in the structural biology laboratory of Dr. Zhiyi Wei (http://bio.sustc.edu.cn/en/?p=189) at Department of Biology, Southern University of Science and Technology (SUSTech, Shenzhen, China). Our research interests is to uncover structural basis of large macromolecular assemblies in neuronal transmembrane signaling and cell-cell/matrix adhesion (Mol Cell, 2011; PNAS, 2013; eLife, 2014; PNAS, 2014; Nature, 2014; PNAS, 2017). We offer an internationally competitive salary and benefits. Shenzhen government provides additional postdoctoral fellowship for Chinese nationals. The research work will be conducted at the Research Center for Life Science, which is equipped with state-of-the-art facilities for X-ray crystallography (Rigaku-007 plus Pilatus detector, crystallization robot), protein chemistry (BiaCore T200, PEAQ-ITC, MALS, etc.), and cell imaging. A cryoEM center is currently under construction, equipped with two Titan-Krios, one Talos and several 120kev EM machines, in the same building with our department. The position can apply research funds as PI for future career development. Candidates should have received (or expect to receive) a Ph.D. in biochemistry or structural biology with a strong publication record. Candidates have experience with mammalian cell culture, cryoEM, or membrane proteins is a plus. Candidates should be highly motivated, have team-work spirit, and be able to work independently. If you are interested in this position, please send your CV, including contact information for 3 references to we...@sustc.edu.cn.
Re: [ccp4bb] manipulation of water molecules in pdb files
Thank you all for quick replies. The issue has been solved by using sortwater and a little manual editing. Using PDBe could be a easier way. Next time I may try it. Best, Zhiyi On 7/31/12, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: The old fashioned water tidy will try to assign matching (non-standard) names to matching H2Os which is useful for analysis of structural waters, but cannot be deposited. I think coot has a tool to move waters to be near the protein but maybe that isn't all you want. Why not start deposition and use PDBe renaming then download the pdb file again to continue refinement? Eleanor On 30 Jul 2012, at 09:27, Zhiyi Wei wrote: Dear all, I have a refine structure with 8 ncs copies and several hundreds of water molecules (which was put in one chain). Now I try to separate these molecules by renaming to the chain id of each adjacent protein molecule. I know RCSB can do this during deposition process. Do anyone know a program can do a similar task? Many thanks! Best, Zhiyi
[ccp4bb] manipulation of water molecules in pdb files
Dear all, I have a refine structure with 8 ncs copies and several hundreds of water molecules (which was put in one chain). Now I try to separate these molecules by renaming to the chain id of each adjacent protein molecule. I know RCSB can do this during deposition process. Do anyone know a program can do a similar task? Many thanks! Best, Zhiyi
Re: [ccp4bb] an ambiguous result of molecular replacement
Hi Leonid, Thank you for your valuable suggestion. It is exactly the case. When I tried P21, it works well. The solution is now very clear. Best, Zhiyi On 3/31/12, Leonid Sazanov saza...@mrc-mbu.cam.ac.uk wrote: Hi, we had the same case in apparent C2221, with many similarly shifted Phaser solutions with high scores. The reason was that crystals were actually nearly perfectly twinned in P21, so indexing and processing indicated C2221. Once data was re-processed in P21, Phaser could easily find two distinct solutions - one for each of twin domains, with LLG scores roughly reflecting twin ratios. Similar case is discussed in detail here: http://www.ncbi.nlm.nih.gov/pubmed/15039553
[ccp4bb] an ambiguous result of molecular replacement
Dear all, I got a weird solution from Phaser. The background is that, space group C2221, resolution ~4A, in complex with a peptide, and having a apo form structure as the search model. Phaser gave two rotation function peaks with Z 7. But when searching translation function peaks, Phaser gave many high Z score peaks listed below rather than a single solution. These peaks share same fraction YZ but with different X. Most of them pasted the packing validation. The when searching the second copy, each solutions have a single translation peak that showed very high Z score ( 20). I check some of these solutions in Coot, and found that the two copies of each solution has the same relative orientation and each solution shifts several angerstroms in X axis. I also tried C222 and did not get better result than C2221. Any comments or suggestion? Thanks a lot! Best, Zhiyi # (#) Frac X Frac Y Frac Z LLG Z-score Split #Groupraw/top 1 1 0.155 0.436 0.287 +219.80 11.17 0 1 272.58/272.58 2 2 0.350 0.436 0.287 +211.92 10.5324 1 267.42/267.42 3 3 0.542 0.436 0.287 +211.31 10.4844 1 266.05/266.05 4 5 0.579 0.436 0.287 +209.66 10.3440 2 260.14/260.14 5 12 0.267 0.436 0.287 +209.11 10.3014 2 256.53/256.53 6 4 0.224 0.435 0.288 +208.41 10.24 8 2 261.39/261.39 7 11 0.485 0.436 0.287 +208.28 10.2340 2 256.95/257.13 8 7 0.191 0.435 0.287 +205.189.97 4 2 258.95/258.95 9 15 0.400 0.436 0.287 +201.909.7030 2 254.20/254.20 109 0.297 0.437 0.287 +201.799.6917 1 257.75/257.75 1116 0.449 0.436 0.287 +198.779.4536 1 252.80/252.80 1217 0.129 0.437 0.287 +195.669.19 3 1 252.41/252.41 1320 0.377 0.436 0.287 +194.959.1327 1 246.37/246.37 1421 0.422 0.436 0.287 +190.728.7833 1 245.42/245.42 1519 0.521 0.437 0.287 +190.228.7445 1 246.46/246.46
Re: [ccp4bb] Structure Determination combining X-ray Data and NMR
Hi Yuan, Bad geometry is a general issue for most low resolution structure refinement. There are quite a lot papers discussing it. I think you can try to set a reference structure or set high restrain in refinement, which should be easily achieved in Phenix. How did you know the B-factors are too high? There is no standard for a single case. B-factors varies from crystal to crystal even them have the same molecular content. Wilson-B factor may be a good indicator. I am a little bit worried about your R and R-free. Them are too close (Rfree is even lower than R!). What is your space group? If you want to reduce refining parameters to increase data/para ratio, you can try group B-factor refinement in Phenix. For combining NMR data, you should have NOE assignment. Best, Zhiyi On 1/6/12, 商元 shangyuan5...@gmail.com wrote: Dear All, I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326. The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan
Re: [ccp4bb] From non-twinned to twinned?
Thanks. Your suggestion remind me that I forgot to mention one more information. Actually, I did try to shot different regions of crystal to reduce radiation damage. Say, the sca1 set used one region and the sca2 set used another. Is it possible that this two regions has different crystal domain arrangement (one is normal and another is twinned)? The crystal looks like a nice single crystal. Zhiyi On 1/5/12, Carlos Frazao fra...@itqb.unl.pt wrote: Hi If the beam cross section is significantly smaller then the crystal dimensions, when you rotate the crystal you will be constantly illuminating new volumes of the crystal. If the crystal contains differently oriented single crystal domains (that is indeed a twinned crystal) then you may have orientations where only one of those single crystal domains are being measured, and other orientations where multiple single crystal domains are collected. The same applies if you apply translations at the crystal (to collect data from fresh crystal regions to decrease the radiation damage effect). Hope it helps, cheers, Carlos Zhiyi Wei wrote: Thank you for all the quick reply. Here are the additional information about the crystal. The crystal was frozen and shot in synchrotron. The collection was 1degree/frame. The space group and unit cell are P21 and a=70 b=165 c=170 alpha=90 beta=90.1 gamma=90. I processed the data in P1 first (I did not see any abnormal changes during integration), and then merge them using P21 or P2221. P21 gives reasonable Rmerge of ~10% while P2221 has much higher Rmerge of ~30%. I reprocessed the data with mosflm using P1. And Pointless also suggested the space group of P21. Zhiyi On 1/5/12, Jens Kaiser kai...@caltech.edu wrote: What are your cell constants and space group? It sounds to me you misindexed and then artificially twinned your structure by integrating/merging in too high of a symmetry. I've seen that happen for primitive hexagonal which was actually C-centered monoclinic. Also, in my experience this is more likely to happen with denzo/scalepack as it refines every image and does postrefinement in scalepack. XDS will decide for you on a symmetry after integration, and in MOSFLM you should see earlier that something is wrong. HTH Jens On Wed, 2012-01-04 at 21:42 +0800, Zhiyi Wei wrote: Dear all, I recently collected a dataset (~2000 frames) from a single crystal. If merge first 600 frames (sca1) or last 600 frames (sca2), Rmerge values from scalepack seem to be ok (~10%) though rejection ratios are high (~5%). But if I merge all frames together, Rmerge value goes up to ~20% and rejection is extremely high (~20%). Then, I checked sca1 and sca2 by xtriage in phenix. Surprisingly, the logfiles told me that sca1 is no twining while sca2 is very likely to be twinned. I never met this case before. So, I am wondering if it is possible from a non-twinned structure to a twinned structure just due to radiation damage. If the answer is yes, does it mean that I should not collect a large number of frames to amplify anomalous signals by using this crystal? Thanks a lot! Best, Zhiyi -- ** Dr. Carlos Frazao Structural Biology Laboratory - Macromolecular Crystallography Unit ITQB-UNL, Av Republica, Apartado 127 2781-901 Oeiras, Portugal Phone: (351)-214469666 FAX:(351)-214433644 e-mail: fra...@itqb.unl.pt www.itqb.unl.pt
[ccp4bb] From non-twinned to twinned?
Dear all, I recently collected a dataset (~2000 frames) from a single crystal. If merge first 600 frames (sca1) or last 600 frames (sca2), Rmerge values from scalepack seem to be ok (~10%) though rejection ratios are high (~5%). But if I merge all frames together, Rmerge value goes up to ~20% and rejection is extremely high (~20%). Then, I checked sca1 and sca2 by xtriage in phenix. Surprisingly, the logfiles told me that sca1 is no twining while sca2 is very likely to be twinned. I never met this case before. So, I am wondering if it is possible from a non-twinned structure to a twinned structure just due to radiation damage. If the answer is yes, does it mean that I should not collect a large number of frames to amplify anomalous signals by using this crystal? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] From non-twinned to twinned?
Thank you for all the quick reply. Here are the additional information about the crystal. The crystal was frozen and shot in synchrotron. The collection was 1degree/frame. The space group and unit cell are P21 and a=70 b=165 c=170 alpha=90 beta=90.1 gamma=90. I processed the data in P1 first (I did not see any abnormal changes during integration), and then merge them using P21 or P2221. P21 gives reasonable Rmerge of ~10% while P2221 has much higher Rmerge of ~30%. I reprocessed the data with mosflm using P1. And Pointless also suggested the space group of P21. Zhiyi On 1/5/12, Jens Kaiser kai...@caltech.edu wrote: What are your cell constants and space group? It sounds to me you misindexed and then artificially twinned your structure by integrating/merging in too high of a symmetry. I've seen that happen for primitive hexagonal which was actually C-centered monoclinic. Also, in my experience this is more likely to happen with denzo/scalepack as it refines every image and does postrefinement in scalepack. XDS will decide for you on a symmetry after integration, and in MOSFLM you should see earlier that something is wrong. HTH Jens On Wed, 2012-01-04 at 21:42 +0800, Zhiyi Wei wrote: Dear all, I recently collected a dataset (~2000 frames) from a single crystal. If merge first 600 frames (sca1) or last 600 frames (sca2), Rmerge values from scalepack seem to be ok (~10%) though rejection ratios are high (~5%). But if I merge all frames together, Rmerge value goes up to ~20% and rejection is extremely high (~20%). Then, I checked sca1 and sca2 by xtriage in phenix. Surprisingly, the logfiles told me that sca1 is no twining while sca2 is very likely to be twinned. I never met this case before. So, I am wondering if it is possible from a non-twinned structure to a twinned structure just due to radiation damage. If the answer is yes, does it mean that I should not collect a large number of frames to amplify anomalous signals by using this crystal? Thanks a lot! Best, Zhiyi
[ccp4bb] Local refinement strategy
Dear all, I am trying to refine a structure with two domains. The electron density of one domain is reasonable, but that of the other domain is poor. So, I am wondering whether the refinement by Phenix or Refmac can be done locally with two parts, the first domain is refined with normal restrains, the second domain is refined by rigid body and group B? Thank you so much! Best, Zhiyi
[ccp4bb] Change cell parameter
Dear all, I have a P2 derivative dataset with beta=89.6. I try to change the beta to 90.4 to be consistent with the native dataset. Should I do sth with the HKL, like applying a matrix? Thanks a million! Best, Zhiyi
Re: [ccp4bb] Change cell parameter
Dear all, Thank you all! There are so many useful replies. The problem has been solved by using the way introduced by Clemens (see below, I dont have an access to SCALEPACK). BTW, although beta is close to 90, it is not a orthorhombic crystal (Rmerge goes up to ~0.3 after scaling). Thanks again! Zhiyi On Wed, Jun 8, 2011 at 11:20 AM, Clemens Vonrhein vonrh...@globalphasing.com wrote: Hi, try reindex hklin in.mtz hklout out.mtz e REIN HKL -h,-k,l END e Cheers Clemens On Wed, Jun 08, 2011 at 11:14:03AM -0400, Zhiyi Wei wrote: Dear all, I have a P2 derivative dataset with beta=89.6. I try to change the beta to 90.4 to be consistent with the native dataset. Should I do sth with the HKL, like applying a matrix? Thanks a million! Best, Zhiyi -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] Off topic: Bruker Microstar
Dear all, Our group plan to buy a X-ray diffraction system. We found Bruker provide a new X-ray generator called Microstar, which they claim is better than traditional rotating anode tube. Because we did not use Microstar before, we do not know whether this new generator is indeed good choice in regard of both price and reliability. Any suggestions will be greatly appreciated. Thank you in advance! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
I forgot to mention a phenomenon when I did crystal mounting. I found that if open a coverslip, phase separation will appear in drops in minutes and damage to the crystals. And I have tried additive screen. The optimized crystals are really big (a few hundred microns) with sharp edge but diffraction pattern is similar. On Tue, Jan 26, 2010 at 10:42 AM, Zhiyi Wei ustcwebri...@gmail.com wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while 8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
Re: [ccp4bb] Crystal rescue
Thanks for so many quick responses! Actually, I have test several different cryo-protectants, including glycerol, EG, and PEG400. I did not see much differences between these cryo conditions. So, I choose glycerol. I would like to test my crystals in RT. But I don't know how to do this. Just mount crystal to the X-ray machine without cryo stream? Or I should use capillaries? Zhiyi On Wed, Jan 27, 2010 at 5:45 AM, Kelly Daughtry kddau...@bu.edu wrote: Tascimate can be used as the cryo as well. I have had experience with crystals in similar condition and moved the crystals to a 20% increased Tascimate solution and they froze well. I agree with Ezra, room temperature mount your crystal before freezing. It is the only way to know the true problem. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Tue, Jan 26, 2010 at 1:25 PM, Marcus Winter marcus.win...@oxford-diffraction.com wrote: Dear Zhiyi, Ezra is exactly right, of course. The Oxford Diffraction PX Scanner system can assess the diffraction qualities of (putative) protein crystals in situ - in the crystallisation plate. So, directly, you would discover if your 'big and beautiful' crystals actually diffract well... in their mother liquor under ambient conditions and before the addition of any cryo-protect. Do you have a friend or neighbour with a PX Scanner ? If not, please feel most welcome to contact Oxford Diffraction: we would be pleased to assist if at all possible. Good Luck and Best Wishes, Marcus Winter. www.oxford-diffraction.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ezra Peisach Sent: 26 January 2010 16:01 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal rescue First you need to establish if it is your cryo conditions or the crystals. Depending where you are - they might have the equipment to do a wet mount - without freezing. Yes the crystal will not last - but then you know if the problem is in the crystal. If it is - you need better crystals. If it is the cryo - you need to work on that. Tacsimate is mixture of alot of different compounds - but the smears are too close together to be a small salt crystal on top... Good luck, Ezra On 1/26/2010 10:42 AM, Zhiyi Wei wrote: Dear all, I got a problem with my crystals. I have two total different proteins that both can be crystallized in the condition with PEG3350 and Tacsimate (although the concentrations are different) with different shapes. The crystals look big and beautiful. However, when I test them in synchrotron, both of these two types of crystals showed poor diffractions. As showed in the attached diffraction image, the diffraction is up to ~4 A but smear in one direction while8 A in the other direction. The interesting thing is that the diffraction pattern is similar for all crystals (from two different proteins) that I tested without exception although they belong to different space groups. So, I wonder whether these kind of pattern is caused by Tacsimate (I don't know what it is) and how to rescue these crystals. Any suggestions or comments? Thanks a lot! Best, Zhiyi
