[ccp4bb] merge weak anomalous signal from multiple datasets
Dear CCP4 users, Recently, I got some datasets with weak anomalous Br signal. I tried to merge them according to Q. Liu et al Science 336, p1033 (2012). I am using the script multiscale@SSRL. The merged dataset has WEAKER anomalous signals. Liu et al used SCALA for scaling and merging while multiscale@SSRL using AIMLESS. Should this cause such a difference? The SCALA@SSRL has a limitation on the number of frames it can process. So I cannot directly check if this caused the difference. Any suggestions? Thanks! Charles -- *** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology **
Re: [ccp4bb] CCP4BB Digest - 28 Feb 2015 to 1 Mar 2015 (#2015-61)
Instead of Br which has a weak signal at its K edge, why not use Iodide at some wavelength (or energy) where it gives a stronger signal? The longer wavelength used will help with spot spatial resolution as well. One may be fighting radiation damage, too. One may be fighting a moving (vibrating?) crystal, too. Sure, you already have some diffraction images, but a better experiment may make one's life easier. My advice would be to not use higher than a 200 mM (4% saturated) KI quick soak, but you didn't tell us how Br ended up in your crystals. Jim Recently, I got some datasets with weak anomalous Br signal. I tried to merge them according to Q. Liu et al Science 336, p1033 (2012). I am using the script multiscale@SSRL. The merged dataset has WEAKER anomalous signals. Liu et al used SCALA for scaling and merging while multiscale@SSRL using AIMLESS. Should this cause such a difference? The SCALA@SSRL has a limitation on the number of frames it can process. So I cannot directly check if this caused the difference. Any suggestions?
Re: [ccp4bb] Skin on drops
In my practice, I never worried about the skin. You need extra care while you mount the crystal. In my case I have used the skin to mount my crystals without any problem. In fact it was much helpful. While mounting, you should not take the crystal covered with the skin. Best Syed On Wed, 2/25/15, Gyanendra Kumar gyanendr...@gmail.com wrote: Subject: Re: [ccp4bb] Skin on drops To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, February 25, 2015, 10:45 PM Adding DTT in your protein buffer or crystallization solution may also help.You could try increasing amounts of DTT/BME/TCEP in your crystallization solution and find a balance between reduction of skin formation vs getting crystals. Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on the drop in which the crystals were tightly embedded. -Gyan On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut han_c...@yahoo.co.uk wrote: Dear Ulrike, you could try avoid the drop-air interface by overlying sitting drops with silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will alter the kinetics with which your drops reach equilibrium, and hence may alter your ability to get crystals of the protein. Batch crystallization under oil is another option of course. Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO for example) in your crystallization conditions may also be something to consider. If none of these work, I'd concentrate on harvesting the crystals from the skin. I tend to cut these open from the side, flip over the skin so that one has better access to the crystals that generally are associated with the inner face of the skin. You can try peel the crystals off the skin, or cut out a piece of skin surrounding a crystal. That will not hurt diffraction quality of the crystals. Hope that helps. Han On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: Dear crystallographers, I am trying to crystallize a soluble protein which tends to form aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the crstallization process a thick skin is formed on top of the sitting-drops. As well the crystals are buried in precipitate. Before I start harvesting I try to remove the skin but still it is hardly possible to get any crystals out of these drops. Any suggestions how to avoid the formation of skin on crystallization drops ? Cheers, Ulrike Han Remaut, PhD Laboratory of Structural Molecular Microbiology VIB / Vrije Universiteit Brussel Building E4, Pleinlaan 2 1050 Brussel han.rem...@vib-vub.be tel. +32-2-629 1923 / +32-499 708050 http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital,Department of Structural Biology,262, Danny Thomas Place, MS-311Memphis, TN 38105Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Higher Rmerge at lower resolution
Dear Gerard, Eleanor et al., In the graphs of Rmeas vs. batch number (and I or I/sigma vs. batch number), everything looks roughly constant, which made me think that significant radiation damage was not the issue--but are you saying that radiation damage might also (or instead) produce a smiley in the graph of Rmeas vs. resolution? Looking back at the diffraction images, they do seem to degrade as each sweep progresses, though there are always some clear diffraction spots past my current resolution cutoff (1.6 A). The beam was 100 um and the crystal was about 700x500x300 um; I translated the beam 110 um along the long axis between shots, so the spots are probably mostly but not entirely fresh. Another thing I noticed is that in the sweeps with the smiley-face Rmeas vs. resolution trace (lower in the middle, roughly equal at high and low resolution) the mean I/sigma vs resolution trace also looks different: instead of being highest at 4-5 A and dropping steadily as resolution increases, it's lower than I would expect at high resolution and higher at low resolution. Perhaps the weakest high-res spots are disappearing altogether and not being counted? Tables from an early sweep and a late sweep from this crystal are below, in case that's interesting. Thanks, Veronica Good sweep: Overall InnerShell OuterShell Low resolution limit 21.77 21.77 1.63 High resolution limit 1.60 8.76 1.60 Rmerge (within I+/I-) 0.025 0.028 0.113 Rmerge (all I+ and I-) 0.027 0.031 0.122 Rmeas (within I+/I-) 0.030 0.033 0.133 Rmeas (all I+ I-) 0.030 0.034 0.132 Rpim (within I+/I-) 0.016 0.018 0.069 Rpim (all I+ I-) 0.012 0.015 0.050 Rmerge in top intensity bin 0.024 - - Total number of observations 108218 524 5456 Total number unique 16662 121 804 Mean((I)/sd(I)) 43.0 62.8 15.0 Mn(I) half-set correlation CC(1/2) 0.999 0.998 0.993 Completeness 99.9 92.3 100.0 Multiplicity 6.5 4.3 6.8 Questionable sweep: Overall InnerShell OuterShell Low resolution limit 21.77 21.77 1.63 High resolution limit 1.60 8.76 1.60 Rmerge (within I+/I-) 0.027 0.036 0.041 Rmerge (all I+ and I-) 0.029 0.039 0.045 Rmeas (within I+/I-) 0.032 0.044 0.049 Rmeas (all I+ I-) 0.032 0.044 0.049 Rpim (within I+/I-) 0.017 0.025 0.027 Rpim (all I+ I-) 0.013 0.020 0.020 Rmerge in top intensity bin 0.034 - - Total number of observations 102687 511 4744 Total number unique 16644 121 799 Mean((I)/sd(I)) 53.8 53.5 34.1 Mn(I) half-set correlation CC(1/2) 0.999 0.997 0.998 Completeness 99.8 92.1 99.9 Multiplicity 6.2 4.2 5.9 On Sat, Feb 28, 2015 at 6:41 AM, Gerard Bricogne g...@globalphasing.com wrote: Dear Veronica, At first glance, it looks as if you have a textbook example of progressive radiation damage, i.e. the structure is changing between the start and the end of your experiment, and the scaling gets skewed towards finding a best compromise between all the measurements at medium (rather than low) resolution. The smiley shape in the Rmeas is something that Zbyszek Dauter identified many years ago as a tell-tale sign of progressive radiation damage. You say that each of your 6 datasets is taken from a fresh spot on the crystal, but how big is your beam relative to the crystal? How far apart are these spots? Most of all: at room temperature, free radicals will diffuse much more readily than in a frozen crystal, so that your later datasets may not in fact come from truly fresh spots. I hope this is helpful. With best wishes, Gerard. -- On Fri, Feb 27, 2015 at 03:40:38PM -0500, Veronica Pillar wrote: Hi all, I have a data set from a large room-temperature lysozyme crystal consisting of 6 90-degree sweeps, each taken from a fresh spot on the crystal. When I scale merge the first sweep by itself, the R[meas/merge] vs. resolution trace as reported by aimless looks fairly normal (lowest values in the 6-2.5 A range and steadily increasing at higher resolution). However, as I look at the subsequent sweeps individually, the traces get progressively stranger until the final sweep's R vs. resolution trace looks like a smiley face, lowest around 2.5 A and about the same in the lowest and highest resolution bins. The overall Rmeas is about the same for all sweeps (3.0-3.2%). Do you have any ideas on what has happened (either during data collection or data processing) to cause this? Thanks, Veronica
[ccp4bb] Python Developer, 15 months
Dear All, We are looking for a Python programmer to improve our robot at the MX beamlines, Australian Synchrotron. Details below. Position Title: Senior Controls Engineer (Python Developer), 15 months, fixed term contract position Job Description Reporting directly to a Principal Controls Engineer, the role will be part of a robotics development group. The role will be expected to work closely with engineers and scientists to support the development of a new sample mounting system for macromolecular crystallography. Qualifications A degree or higher level in an engineering discipline or suitable qualification in a technical field. Required Skills This role is hands-on technical and experience in the following areas would be essential. 3-5 years of experience with Python including: -Applications experience -Design experience -Requirement gathering -Object orientated development -Logging -Threading -Documentation Experience in the following areas would be beneficial. -Experience with C/C++ -GUI Development experience such as Qt / wx Accountabilities also include, -Managing projects that cover a broad range of control system activities to ensure timely delivery that meets expectations -Developing conceptual / detailed design, development and implementation of control systems across the facility and within the area of responsibility to provide functional and reliable control systems that support, the needs of the facility. -Programing, building or configuring software that is used in control systems, using defined engineering processes and procedures. -Performing an appropriate level of test and integration activities for the software and hardware systems deployed in the facility. -Providing operational support by trouble shooting, diagnosing and solving problems with the installed control systems across the facility. -Developing and communicating procedures and work instructions related to controls systems to ensure appropriate level of knowledge and skill is available to support key aspects of the control system. -Developing and maintaining collaborative relationships with subject matter experts at other comparable facilities and within industry to ensure the AS remains competitive internationally and fosters collaboration Applications close : Friday, 6th March 2015 Job Location Clayton, Victoria, Australia Applications via http://www.synchrotron.org.au/about-us/synchrotron-careers/working-at-the-synchrotron/current-openings -- David Aragão, PhD | BL Scientist - MX | Australian Synchrotron p: (03) 8540 4121 | f: (03) 8540 4200 | m: 0467 775 203 david.ara...@synchrotron.org.au | www.synchrotron.org.au 800 Blackburn Road, Clayton, Victoria 3168, Australia -- orcid.org/-0002-6551-4657
Re: [ccp4bb] merge weak anomalous signal from multiple datasets
Hi Charles, I don't know what multiscale does. Probably the right thing. If the anomalous signal is weaker after scaling of multiple datasets, non-isomorphism might be at fault. Try Blend to scale your datasets. Blend is part of ccp4 and gives good graphical diagnostic feedback. Andreas On 01/03/2015 4:20, CPMAS Chen wrote: Dear CCP4 users, Recently, I got some datasets with weak anomalous Br signal. I tried to merge them according to Q. Liu et al Science 336, p1033 (2012). I am using the script multiscale@SSRL. The merged dataset has WEAKER anomalous signals. Liu et al used SCALA for scaling and merging while multiscale@SSRL using AIMLESS. Should this cause such a difference? The SCALA@SSRL has a limitation on the number of frames it can process. So I cannot directly check if this caused the difference. Any suggestions? Thanks! Charles -- *** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology **
Re: [ccp4bb] Higher Rmerge at lower resolution
Hi Veronica, as others have said, at RT the radicals travel much further than at cryo temperature, so your lateral shifts are probably not sufficient. But it is not clear to me that radiation damage is to blame at all. To me it sounds like you might have to mask your beamstop better, or you are having overloads, or something else going on in the lowest resolution range. If you put your first dataset (if that one already displays the problem), after compression, into a Dropbox folder, I'll have a look. best, Kay
