[ccp4bb] Postdoctoral position at the European Institute of Oncology, Milano
Postdoctoral research position in Structural Biology of Asymmetric Cell Division A postdoctoral research position is available in the group of Marina Mapelli at the European Institute of Oncology in Milano. The main interest of the group focuses on the molecular mechanisms underlying asymmetric cell divisions, with emphasis on the role of mitotic spindle coupling to cortical polarity (http://www.ieo.it/Italiano/Ricerca/Experimental_Oncology/research/Basic/Pages/MarinaMapelli.aspx). The orientation of the mitotic spindle is fundamental in determining the relative position of daughter cells within the tissue. In several stem cell systems, spindle alignment ensures that only one of the two daughters will retain niche contacts and stem-like properties. The central players of the spindle orientation pathway are the conserved NuMA/LGN/Gαi complexes. In the past years, we started characterizing the biochemical properties and the organizational principles of the NuMA/LGN/Gαi network from purified components (Culurgioni et al. 2011 PNAS, Mapelli Gonzalez. 2012 Open Biol.). Now we are addressing the issues of 1) how these force generating machines are recruited at cortical sites, and 2) how they are coordinated with the polarity proteins Par3/Par6/aPKC. The general goal of the project is the structural characterization of protein complexes involved in mitotic spindle placement. The strategy to achieve this aim will combine X-ray crystallography, biochemistry, and cell biology. I am seeking a talented and enthusiastic postdoctoral fellow who enjoys working as part of a collaborative and multidisciplinary team. The ideal candidate should have a PhD in protein structure, biochemistry or equivalent qualification, and extensive experience in expression and characterization of multi-subunit protein complexes. Knowledge of protein crystallography will be advantageous. The Structural Biology Department of the IFOM-IEO Campus is equipped with the state-of-the-art apparatus for protein purification, characterization and crystallization, including a nanodrop crystallization robot and an automated imaging system, and has good access to synchrotron beamlines. Successful candidates will benefit from a stimulating and collaborative environment within the Campus. Candidates will be encouraged to apply to the Marie Curie co-founded SIPOD postdoc program (http://www.semm.it/PostDoc.php). The position is available from December 2013, with an initial salary compared to the candidate experience. Postdoc applicants should send their enquiries by e-mail to Marina Mapelli (marina.mape...@ieo.eu). They should also ask two referees to send letters of recommendation at the same electronic address. With all my kind regards, Marina --- Marina Mapelli, PhD Department of Experimental Oncology European Institute of Oncology Via Adamello 16, 20139 Milan, Italy tel: +39-02-94375018 email: marina.mape...@ieo.eu web: http://www.ieo.it/Italiano/Ricerca/Experimental_Oncology/research/Basic/Pages/MarinaMapelli.aspx ---
[ccp4bb] Staining Crystals with comassie
Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] Staining Crystals with comassie
Izit is an aqueous solution of methylene blue, which you can prepare simply and cheaply. Look here: http://www.ysbl.york.ac.uk/ccp4bb/2000/msg00387.html One word of warning: not every crystal stains. I found poking crystal with hair or glass fibre to see if it cracks or breaks is more reliable. Salt crystals react to attacks from glass fibres like rocks (as indeed what they are). From: Swastik Phulera Sent: Tuesday, October 15, 2013 3:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Staining Crystals with comassie Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] High Rsyms between 5-7 A
Hi Ursula, if you look at the Wilson plot, you see a minimum near 6A resolution. This means that in the range 7-5A, intensities are low, and Rsyms are therefore elevated. best, Kay On Mon, 14 Oct 2013 11:52:03 -0700, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: Here is some more info on the data: lowest res. shell (50-8.0A): Rsym=4.4%, I/sig=100. detector ADSC315, 1 degree oscillations, mosaicity around 0.3 The Rsym stays high whether I merge just the first 30 frames or 200 frames. Wouldn't that speak against radiation damage? The crystals were grown from 2.5 M sodium formate and cryoprotected in 2.8 M sodium formate, 30% glycerol. Ursula On Mon, Oct 14, 2013 at 11:39 AM, Dominika Borek domin...@work.swmed.eduwrote: There are several possibilities: (1) Radiation damage causes such bump in R-merge at medium resolution (2) Crystallization in very high salt that absorbs causes such behaviour, e.g. if your crystallized from 2M sulfate. (3) Very short exposure time -- very short means shorter than 1 second per image -- that results in large errors due to goniostat instability. (4) Very short exposure may also result in errors due to shutter problems. (5) Very weak diffraction. All errors will be better visible with weak diffraction. Any combination of the above factors is also possible. What are R-merges in the lowest resolution shell i.e. 50-8 A? If they are high too -- this is probably goniostat, shutter or beam instability. If they are low in the lowest resolution shell -- this probably radiation damage. What is the mosaicity? Low means lower than 1 degree for some people, for others less than 0.1 degree? What detector was used to collect data? Oscillation step? What is I/s(I) in the lowest resolution shell? Dominika 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 Dominika Borek, Ph.D. *** UT Southwestern Medical Center 5323 Harry Hines Blvd. *** Dallas, TX 75390-8816 214-645-6378 (phone) *** 214-645-6353 (fax) -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220
[ccp4bb] Molrep and Refmac5 errors
Dear all, I recently started using CCP4i (Windows 8) software for data processing and model building but I get the following error messages whenever I use molrep and refmac5: MOLREP #CCP4I TERMINATION STATUS 0 MOLREP(ccp4): Error in label assignments in LABIN #CCP4I TERMINATION TIME 08 Oct 2013 16:27:56 #CCP4I MESSAGE Task failed REFMAC5 #CCP4I TERMINATION STATUS 0 Program received signal 11 (SIGSEGV): Segmentation fault. #CCP4I TERMINATION TIME 08 Oct 2013 16:26:50 #CCP4I MESSAGE Task failed I get those error with every data set I tried, therefore, I would be really appreciative if you could help me with it. Thanks, Rajiv
[ccp4bb] POSTDOC POSITION at UC Irvine, SoCal
Immediate opening for a postdoc in structure-based drug discovery on any of these projects: 1) inhibitor discovery against the Helicobacter pylori acid-gated urea channel (ulcers, stomach cancer) 2) reactivation of p53 cancer mutants 3) inhibitors inosine monophosphate dehydrogenase (anti-P. falciparum and anti-T. foetus compounds) 4) trypanosomal uridylyl transferase (anti-trypanosomal compounds) 4) annexin A2 and S100A4 (cancer, anti-angiogenesis) US citizenship or US green card preferred. Please contact Hudel Luecke via email at hu...@uci.edu with a c.v. and name and emails of three references Cheers, Hudel -- Prof. Hartmut Luecke Director, Center for Biomembrane Systems Depts. of Biochemistry, Biophysics Computer Science 3205 McGaugh Hall University of California Irvine, CA 92697-3900 +1 (949) 394-7574 (mobile, email much preferred) +1 (949) 824-8551 (fax, email much preferred) hu...@uci.edu http://bass.bio.uci.edu/~hudel/
[ccp4bb] CCP4 Study weekend 2014
CCP4 Study weekend 2014 on Complementary Techniques - programme now live at: http://www.cse.scitech.ac.uk/events/CCP4_2014/programme.html What techniques can be combined with crystallographic analysis? Of all deposited structures in the PDB, more than half are oligomeric, and three quarters have a ligand bound. In-solution methods like SAXS or biophysical techniques allow us to validate the information on the molecular assembly. Biophysical techniques are also essential to probe ligands and co-factors. We address how to make ligand complexes with novel methods to mount and manipulate crystals. A session on drug design gives the perspective and discusses additional techniques such as NMR. Simulations and NMR also address the dynamic nature of macromolecules. EM techniques can be used to address macromolecular complexes of changing composition and go full circle with the X-ray analysis. You will learn how complementary methods help in structure determination and analysis, leading to a better understanding of the bio-macromolecules we study. Interested? Then click here to registerhttps://eventbooking.stfc.ac.uk/login?EVENT=147. The organisers. Ivo Tews, University of Southampton Jon Cooper, UCL.
[ccp4bb] A case of perfect pseudomerehedral twinning?
Hello crystallography experts, I think I may have a case of perfect pseudomerehedral twinning where P222 Laue symmetry is actually P2 with twinning and aniostropic data masks intensity based twinning statistics. I would like some input to determine if my reasoning makes sense. I would like to perform a twin test using local intensity differences (J E Padilla and T O Yeates, 2003) but I am not sure how to do this. I am a little worried that using a twin law in a lower symmetry group than appropriate will give the drop in Rfree/Rwork that I am seeing and the crystal is not really twinned. 1. I have processed in P222 and P21. In each case I get good scaling statistics using XDS (seebelow) 2. Systematic absences clearly point to a single 2(1)-fold screw axis 3. There appears to almost be two more 2(1) screw axis except that there are some significant violations in these absences leading me to believe that they are not true srew axis. 4. Patterson map shows no peaks relating molecules by translation in P222 (1 molec/asu) 5. I have difficulty interpreting the patterson map in P2 (2molec/asu) (I am new to this so could be my own fault) 7. Intensity statistics show no twinning but the data is very aniosotropic suggesting that twinning can be masked. The cumulative distribuiton of z= (I/I) deviates for centric reflections my a max of 0.104 and the curve falls well below for all values of (Phenix triage). 8. Molecular replacement (Phaser) in P222 gives a single solution in P212121. Every other choice of space group fails to give a solution. 9. Refinement in P212121 space group stalls around 34/31% Rfree/Rwork. Althrough there are geometry problems to correct and water has not been added. 10. Molecular replacement in P2 give single solution in P21 11. Refinement against the same model used above gives 36.5 33.8 Rfree/Rwork without twin law and 32/30 Rfree/Rwork with twin law = h, -k, -l I have shown scaling for P212121. Statistics are nearly the same for P222 scaling except XDS throws up a warning that I have outliers in my systematic absences. P212121 20-1.7A CELL 56.27967.257 106.264 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13696 1911 1989 96.1% 2.6% 2.7% 13681 61.96 2.8% 99.9* 23* 1.044 1400 3.56 24387 3163 3167 99.9% 2.5% 2.8% 24386 64.65 2.7% 99.9*9 0.888 2672 2.93 22483 3969 3995 99.3% 3.2% 3.1% 22463 46.05 3.5% 99.9*1 0.880 3292 2.54 20108 4685 4712 99.4% 4.2% 4.1% 20076 30.45 4.8% 99.8*1 0.839 3710 2.28 20966 5221 5268 99.1% 5.5% 5.6% 20923 22.69 6.3% 99.7*-1 0.779 3932 2.08 21599 5702 5779 98.7% 7.3% 7.7% 21521 16.73 8.5% 99.4* 0 0.756 3932 1.93 21589 6216 6289 98.8% 11.1% 12.1% 21478 10.73 13.1% 98.5* 0 0.735 3755 1.80 19392 579 6709 98.1% 17.5% 19.7% 19089 6.32 21.3% 96.1* -3 0.695 2421 1.70 10889 5093 7126 71.5% 24.8% 27.6% 9561 3.63 31.7% 89.2*2 0.750 350 total 175109 42539 45034 94.5% 4.0% 4.2% 173178 23.25 4.6% 99.9*1 0.808 25464 0 210 3.203 0.6806E+03 0.3320E+0220.50 3* 0 220 3.057 0.9423E+04 0.3288E+0328.66 1 0 230 2.924 0.3205E+03 0.4586E+02 6.99 1* 0 240 2.802 0.1231E+03 0.4612E+02 2.67 1 0 250 2.690 0.2306E+02 0.4566E+02 0.51 1* 0 260 2.587 0.1540E+04 0.7494E+0220.56 1 0 270 2.491 0.3212E+03 0.5579E+02 5.76 1* 0 280 2.402 0.2706E+04 0.1156E+0323.42 1 0 290 2.319 0.3761E+02 0.4931E+02 0.76 1* 0 300 2.242 0.4053E+03 0.6758E+02 6.00 1 0 310 2.170 0.4495E+03 0.6956E+02 6.46 1* 0 320 2.102 0.3573E+04 0.1442E+0324.78 1 0 330 2.038 0.5625E+02 0.7052E+02 0.80 1* 0 340 1.978 0.2167E+04 0.1090E+0319.88 1 0 350 1.922 0.3802E+02 0.6082E+02 0.63 1* 0 360 1.868 0.1628E+04 0.9449E+0217.23 1 30018.760 0.6193E+01 0.5268E+01 1.18 2* 40014.070 0.4882E+04 0.1672E+0329.19 1 50011.256 0.3065E+02 0.8641E+01 3.55 2* 700 8.040 0.1511E+03 0.1280E+0211.81 2* 800 7.035 0.5131E+04 0.1255E+0340.87 2 900 6.253 -0.2260E-01 0.1419E+02 0.00 2* 1000 5.628 0.7113E+03 0.2502E+0228.43 2 1100 5.116 0.3705E+03 0.2093E+0217.70 2* 1300 4.329 0.4986E+03 0.2448E+0220.37
[ccp4bb] A case of perfect pseudomerehedral twinning?
Hello crystallography experts, I think I may have a case of perfect pseudomerehedral twinning where P222 Laue symmetry is actually P2 with twinning and aniostropic data masks intensity based twinning statistics. I would like some input to determine if my reasoning makes sense. I would like to perform a twin test using local intensity differences (J E Padilla and T O Yeates, 2003) but I am not sure how to do this. I am a little worried that using a twin law in a lower symmetry group than appropriate will give the drop in Rfree/Rwork that I am seeing and the crystal is not really twinned. 1. I have processed in P222 and P21. In each case I get good scaling statistics using XDS (seebelow) 2. Systematic absences clearly point to a single 2(1)-fold screw axis 3. There appears to almost be two more 2(1) screw axis except that there are some significant violations in these absences leading me to believe that they are not true srew axis. 4. Patterson map shows no peaks relating molecules by translation in P222 (1 molec/asu) 5. I have difficulty interpreting the patterson map in P2 (2molec/asu) (I am new to this so could be my own fault) 7. Intensity statistics show no twinning but the data is very aniosotropic suggesting that twinning can be masked. The cumulative distribuiton of z= (I/I) deviates for centric reflections my a max of 0.104 and the curve falls well below for all values of (Phenix triage). 8. Molecular replacement (Phaser) in P222 gives a single solution in P212121. Every other choice of space group fails to give a solution. 9. Refinement in P212121 space group stalls around 34/31% Rfree/Rwork. Althrough there are geometry problems to correct and water has not been added. 10. Molecular replacement in P2 give single solution in P21 11. Refinement against the same model used above gives 36.5 33.8 Rfree/Rwork without twin law and 32/30 Rfree/Rwork with twin law = h, -k, -l I have shown scaling for P212121. Statistics are nearly the same for P222 scaling except XDS throws up a warning that I have outliers in my systematic absences. P212121 20-1.7A CELL 56.27967.257 106.264 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13696 1911 1989 96.1% 2.6% 2.7% 13681 61.96 2.8% 99.9* 23* 1.044 1400 3.56 24387 3163 3167 99.9% 2.5% 2.8% 24386 64.65 2.7% 99.9*9 0.888 2672 2.93 22483 3969 3995 99.3% 3.2% 3.1% 22463 46.05 3.5% 99.9*1 0.880 3292 2.54 20108 4685 4712 99.4% 4.2% 4.1% 20076 30.45 4.8% 99.8*1 0.839 3710 2.28 20966 5221 5268 99.1% 5.5% 5.6% 20923 22.69 6.3% 99.7*-1 0.779 3932 2.08 21599 5702 5779 98.7% 7.3% 7.7% 21521 16.73 8.5% 99.4* 0 0.756 3932 1.93 21589 6216 6289 98.8% 11.1% 12.1% 21478 10.73 13.1% 98.5* 0 0.735 3755 1.80 19392 579 6709 98.1% 17.5% 19.7% 19089 6.32 21.3% 96.1* -3 0.695 2421 1.70 10889 5093 7126 71.5% 24.8% 27.6% 9561 3.63 31.7% 89.2*2 0.750 350 total 175109 42539 45034 94.5% 4.0% 4.2% 173178 23.25 4.6% 99.9*1 0.808 25464 0 210 3.203 0.6806E+03 0.3320E+0220.50 3* 0 220 3.057 0.9423E+04 0.3288E+0328.66 1 0 230 2.924 0.3205E+03 0.4586E+02 6.99 1* 0 240 2.802 0.1231E+03 0.4612E+02 2.67 1 0 250 2.690 0.2306E+02 0.4566E+02 0.51 1* 0 260 2.587 0.1540E+04 0.7494E+0220.56 1 0 270 2.491 0.3212E+03 0.5579E+02 5.76 1* 0 280 2.402 0.2706E+04 0.1156E+03 23.42 1 0 290 2.319 0.3761E+02 0.4931E+02 0.76 1* 0 300 2.242 0.4053E+03 0.6758E+02 6.00 1 0 310 2.170 0.4495E+03 0.6956E+02 6.46 1* 0 320 2.102 0.3573E+04 0.1442E+03 24.78 1 0 330 2.038 0.5625E+02 0.7052E+02 0.80 1* 0 340 1.978 0.2167E+04 0.1090E+0319.88 1 0 350 1.922 0.3802E+02 0.6082E+02 0.63 1* 0 360 1.868 0.1628E+04 0.9449E+0217.23 1 30018.760 0.6193E+01 0.5268E+01 1.18 2* 400 14.070 0.4882E+04 0.1672E+0329.19 1 500 11.256 0.3065E+02 0.8641E+01 3.55 2* 700 8.040 0.1511E+03 0.1280E+0211.81 2* 80 0 7.035 0.5131E+04 0.1255E+0340.87 2 90 0 6.253 -0.2260E-01 0.1419E+02 0.00 2* 1000 5.628 0.7113E+03 0.2502E+0228.43 2 1100 5.116 0.3705E+03 0.2093E+0217.70 2* 1300 4.329 0.4986E+03 0.2448E+0220.37 2* 1500 3.752 0.8513E+03 0.3547E+0224.00 2* 1600
[ccp4bb] A case of perfect pseudomerehedral twinning?
Hello crystallography experts, I think I may have a case of perfect pseudomerehedral twinning where P222 Laue symmetry is actually P2 with twinning and aniostropic data masks intensity based twinning statistics. I would like some input to determine if my reasoning makes sense. I would like to perform a twin test using local intensity differences (J E Padilla and T O Yeates, 2003) but I am not sure how to do this. I am a little worried that using a twin law in a lower symmetry group than appropriate will give the drop in Rfree/Rwork that I am seeing and the crystal is not really twinned. 1. I have processed in P222 and P21. In each case I get good scaling statistics using XDS (seebelow) 2. Systematic absences clearly point to a single 2(1)-fold screw axis 3. There appears to almost be two more 2(1) screw axis except that there are some significant violations in these absences leading me to believe that they are not true srew axis. 4. Patterson map shows no peaks relating molecules by translation in P222 (1 molec/asu) 5. I have difficulty interpreting the patterson map in P2 (2molec/asu) (I am new to this so could be my own fault) 7. Intensity statistics show no twinning but the data is very aniosotropic suggesting that twinning can be masked. The cumulative distribuiton of z= (I/I) deviates for centric reflections my a max of 0.104 and the curve falls well below for all values of (Phenix triage). 8. Molecular replacement (Phaser) in P222 gives a single solution in P212121. Every other choice of space group fails to give a solution. 9. Refinement in P212121 space group stalls around 34/31% Rfree/Rwork. Althrough there are geometry problems to correct and water has not been added. 10. Molecular replacement in P2 give single solution in P21 11. Refinement against the same model used above gives 36.5 33.8 Rfree/Rwork without twin law and 32/30 Rfree/Rwork with twin law = h, -k, -l I have shown scaling for P212121. Statistics are nearly the same for P222 scaling except XDS throws up a warning that I have outliers in my systematic absences. P212121 20-1.7A CELL 56.27967.257 106.264 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13696 1911 1989 96.1% 2.6% 2.7% 13681 61.96 2.8% 99.9* 23* 1.044 1400 3.56 24387 3163 3167 99.9% 2.5% 2.8% 24386 64.65 2.7% 99.9*9 0.888 2672 2.93 22483 3969 3995 99.3% 3.2% 3.1% 22463 46.05 3.5% 99.9*1 0.880 3292 2.54 20108 4685 4712 99.4% 4.2% 4.1% 20076 30.45 4.8% 99.8*1 0.839 3710 2.28 20966 5221 5268 99.1% 5.5% 5.6% 20923 22.69 6.3% 99.7*-1 0.779 3932 2.08 21599 5702 5779 98.7% 7.3% 7.7% 21521 16.73 8.5% 99.4* 0 0.756 3932 1.93 21589 6216 6289 98.8% 11.1% 12.1% 21478 10.73 13.1% 98.5* 0 0.735 3755 1.80 19392 579 6709 98.1% 17.5% 19.7% 19089 6.32 21.3% 96.1* -3 0.695 2421 1.70 10889 5093 7126 71.5% 24.8% 27.6% 9561 3.63 31.7% 89.2*2 0.750 350 total 175109 42539 45034 94.5% 4.0% 4.2% 173178 23.25 4.6% 99.9*1 0.808 25464 0 210 3.203 0.6806E+03 0.3320E+0220.50 3* 0 220 3.057 0.9423E+04 0.3288E+0328.66 1 0 230 2.924 0.3205E+03 0.4586E+02 6.99 1* 0 240 2.802 0.1231E+03 0.4612E+02 2.67 1 0 250 2.690 0.2306E+02 0.4566E+02 0.51 1* 0 260 2.587 0.1540E+04 0.7494E+0220.56 1 0 270 2.491 0.3212E+03 0.5579E+02 5.76 1* 0 280 2.402 0.2706E+04 0.1156E+03 23.42 1 0 290 2.319 0.3761E+02 0.4931E+02 0.76 1* 0 300 2.242 0.4053E+03 0.6758E+02 6.00 1 0 310 2.170 0.4495E+03 0.6956E+02 6.46 1* 0 320 2.102 0.3573E+04 0.1442E+03 24.78 1 0 330 2.038 0.5625E+02 0.7052E+02 0.80 1* 0 340 1.978 0.2167E+04 0.1090E+0319.88 1 0 350 1.922 0.3802E+02 0.6082E+02 0.63 1* 0 360 1.868 0.1628E+04 0.9449E+0217.23 1 30018.760 0.6193E+01 0.5268E+01 1.18 2* 400 14.070 0.4882E+04 0.1672E+0329.19 1 500 11.256 0.3065E+02 0.8641E+01 3.55 2* 700 8.040 0.1511E+03 0.1280E+0211.81 2* 80 0 7.035 0.5131E+04 0.1255E+0340.87 2 90 0 6.253 -0.2260E-01 0.1419E+02 0.00 2* 1000 5.628 0.7113E+03 0.2502E+0228.43 2 1100 5.116 0.3705E+03 0.2093E+0217.70 2* 1300 4.329 0.4986E+03 0.2448E+0220.37 2* 1500 3.752 0.8513E+03 0.3547E+0224.00 2* 1600 3.517 0.7354E+03 0.3602E+0220.42 2 1700 3.311 0.2188E+03 0.3146E+02
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton
Re: [ccp4bb] High Rsyms between 5-7 A
Thanks for all the great suggestions. They included beamline problems, detector-specific problems, translational NCS, narrow dynamic range problem for strong low resolution reflections,radiation damage, and more. The agreement seems to be that no matter what the reason for the high Rsym is, it is probably save to publish the structure. I am curious about one specific suggestion about interference observed in mostly helical proteins: Helices, due to their regular structure, cause an interference effect that reduces the intensities of reflections in that resolution range. It should be visible in the diffraction images. It appears as if there is a broad region with weak or even missing reflections. The reflections are stronger again at higher resolution. The effect on diffraction is exactly what I am seeing and my proteins are probably about 80% helical. Does anybody have an opinion about this? Ursula On Tue, Oct 15, 2013 at 2:13 AM, Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Hi Ursula, if you look at the Wilson plot, you see a minimum near 6A resolution. This means that in the range 7-5A, intensities are low, and Rsyms are therefore elevated. best, Kay On Mon, 14 Oct 2013 11:52:03 -0700, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: Here is some more info on the data: lowest res. shell (50-8.0A): Rsym=4.4%, I/sig=100. detector ADSC315, 1 degree oscillations, mosaicity around 0.3 The Rsym stays high whether I merge just the first 30 frames or 200 frames. Wouldn't that speak against radiation damage? The crystals were grown from 2.5 M sodium formate and cryoprotected in 2.8 M sodium formate, 30% glycerol. Ursula On Mon, Oct 14, 2013 at 11:39 AM, Dominika Borek domin...@work.swmed.edu wrote: There are several possibilities: (1) Radiation damage causes such bump in R-merge at medium resolution (2) Crystallization in very high salt that absorbs causes such behaviour, e.g. if your crystallized from 2M sulfate. (3) Very short exposure time -- very short means shorter than 1 second per image -- that results in large errors due to goniostat instability. (4) Very short exposure may also result in errors due to shutter problems. (5) Very weak diffraction. All errors will be better visible with weak diffraction. Any combination of the above factors is also possible. What are R-merges in the lowest resolution shell i.e. 50-8 A? If they are high too -- this is probably goniostat, shutter or beam instability. If they are low in the lowest resolution shell -- this probably radiation damage. What is the mosaicity? Low means lower than 1 degree for some people, for others less than 0.1 degree? What detector was used to collect data? Oscillation step? What is I/s(I) in the lowest resolution shell? Dominika 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 Dominika Borek, Ph.D. *** UT Southwestern Medical Center 5323 Harry Hines Blvd. *** Dallas, TX 75390-8816 214-645-6378 (phone) *** 214-645-6353 (fax) -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220 -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
R factors cannot be used to detect twining. The traditional R is calculated using structure factors (roughly the square root of intensity) but you can't do that calculation in the presence of twining because each structure factor contributes to two intensities. The formula for the R in the presence of twining is very different than that of the formula used in its absence. It would have been better to have used a different name and prevent the confusion. If you are worried about your systematic absences you need to figure out which images they were recorded on and judge the spot for yourself. Everything you have said points to your crystal being P212121 (or very nearly P212121). Dale Tronrud On 10/15/2013 02:31 PM, Yarrow Madrona wrote: Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton
[ccp4bb] Methods to detect/distinguish carbohydrates, lipids, proteins and nucleic acids
Dear CCP4 members: Sorry for the off-topic subject, but I'd appreciate it if you could share your experience with methods that could easily detect or distinguish carbohydrates, proteins, lipids and nucleic acids. The story began with a 2D gel of a sample from IP. Instead of seeing different focused spots, we saw a line of bands across the gel. We then speculate that it might not be protein. Carbohydrate? Basically, we are looking for a simple assay that can tell what we have in the sample. Thanks in advance for sharing your experience! Hua Yuan
