Re: [ccp4bb] how to ignore spot overlap in imosflm?
Hi This is all good advice, but there is more that you could do if you're desperate to use these images. Having made sure that your spots on the images are not actually overlapping (i.e. by looking closely at the images), but just flagged as overlapping in iMosflm, you may be able (i.e. no guarantees) to persuade Mosflm to use them by - (i) go to Processing Options-Advanced Integration and increase the values for the Profiles-Tolerance. There's a tool-tip that becomes visible if you hover the mouse cursor over the entry boxes which gives more advice on values. (ii) if you're *really* desperate, and you have noticed that the overall box width and box height has increased to values much bigger than your spots (e.g. your spots are really only ~5-10 pixels across, but the box size has increased to ~30) you could try setting the overall box size to ~the separation distance in pixels and UNchecking the Optimise overall box size check box - this will fix the overall dimensions but allow the spot size within it to optimise. This might get you out of a hole... As Zhije says, the proper solution is to collect the data without overlaps, though. Either of the above steps will reduce the measurement quality, though (i) is better than (ii). A rule of thumb is to set the crystal to detector distance (in mm) to at least [maximum cell edge(Å)/wavelength(Å)] (pedants might want to multiply the RHS of that by 1mm. There are better methods than rules of thumb, though, e.g. the strategy options that are now widely available. On 14 May 2012, at 04:48, Zhijie Li wrote: Hi Xinghua, The total intensity of each reflection needs to be accurately quantitated in order to calculate the structure factors. Not only the dots need to be well separated in the 3D reciprocal space, but also a small area around the dots are often needed to calculate the background for subtraction. That is why when two dots are getting too close, the programs will reject both dots. The first thing you need to do is to inspect the images reported with large number of overlaps to see if the dots are really overlapping or just close to each other. If the dots are barely touching or just too close to each other, you can manipulate the SEPERATION parameter to force the program to take the closely spaced spots. But keep in mind that you may get less accurate integration by doing so. If many spots are really touching each other, normally we won't force the programs to use them. Then the proper remedy is to move the detector farther and collect the dataset again (also, try to optimize your freezing to get the mosaicity as low as possible). For how to play with the mosflm parameters, please read here: http://www.mrc-lmb.cam.ac.uk/harry/cgi-bin/keyword2.cgi?SEPARATION. What you need is probably CLOSE. The hazard of high percentage of overlaps: If the overlaps are only scattered in a whole dataset, it is OK, even if they make up 5-10% or even 20% of the whole dataset. It will only give you a lower completeness, which is not too detrimental to the structure solution. However, if large, continuous regions in the dataset are missing, that will cause you to have poorly defined regions in the calculated map, often seen as featureless stripes or layers in the map. Unfortunately, when you have closely spaced reflections, the latter is often the case. The proper solution is to collect the data at a greater detector distance to resolve the spots (after taking the test images, both imosflm and HKL2000 can simulate the collection run to help you to decide what distance you need). In cases that you have a long unit cell (200A), the first thing you need to do is to align the long edge of the Unix cell with the rotational axis of the pin. In the difficult cases, you probably even need to shoot multiple crystals and combine the datasets to get enough completeness. Zhijie From: Xinghua Qin Sent: Sunday, May 13, 2012 10:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to ignore spot overlap in imosflm? Dear CCP4ers, We collected a diffraction dataset with high percentage of spot overlaps, It would be so kind to tell me how to ignore spot overlap in imosflm and explain the hazard of high percentage of spot overlaps. Thanks in advance. Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] Reminder: Deadline 20th of May for application to the PEPC8 EMBO Practical course
Dear all, this is a reminder that the deadline for application to the EMBO Practical Course Protein expression, purification and characterization (PEPC8) is on Sunday the 20th of May. The course will be held at EMBL Hamburg from the 3rd of September until the 11th of September 2012. This is a hands-on course with practicals in cloning, expression (E. coli Baculo virus), purification (with and without tags) and characterization (thermofluor, circular dichroism, SAXS, crystallization and in-situ dynamic light scattering, ITC and thermophoresis). Speakers include: Imre Berger, EMBL Grenoble Huseyin Besir, EMBL Heidelberg Christian Betzel, University of Hamburg Louise Bird, Oxford University Uwe Bierfreund, GE Healthcare Richard Burgess, University of Wisconsin-Madison Stefan Duhr, NanoTemper David Hacker, EPFL Lausanne Josan Marquez, EMBL Grenoble Alex McPherson, University of California Preben Morth, University of Oslo Jochen Mueller-Dieckmann, EMBL Hamburg Joanne Nettleship, University of Oxford Ilme Schlichting, Max Planck Institute, Munich Dmitri Svergun, EMBL Hamburg Participants are encouraged to bring their own sample to the course for cloning and expression in the pOPIN vectors, crystallization, SAXS and biophysical characterization. A more extensive characterization of samples is sponsored in context of the course by the co-sponsor Pcube. For more information, please visit our website: http://events.embo.org/12-pepc/index.html or contact us by email: pep...@googlemail.com Best regards, Rob Meijers, Stephane Boivin, Annabel Parret Dmitri Svergun EMBL Hamburg Notkestrasse 85 D-22603, Hamburg, Germany
[ccp4bb]
Dear All, I would like to draw your attention to the DIALS-I workshop on X-ray Diffraction Data Analysis to be held at Diamond Light Source on June 12th/13th 2012. For information about the programme and how to register for the workshop please go to the home page http://diamond.ac.uk/Home/Events/DIALS-1.html. For any further information about the scope and purpose of the meeting please contact Gwyndaf Evans (gwyndaf.ev...@diamond.ac.ukmailto:gwyndaf.ev...@diamond.ac.uk). The number of places on the workshop is limited so please register as soon as possible. Regards, The Organisers Gwyndaf Evans, Graeme Winter, Alun Ashton and David Waterman
[ccp4bb] DIALS-I workshop on X-ray Diffraction Data Analysis
[Just to correct a few issue with my earlier announcement here it is again with subject line!] Dear All, I would like to draw your attention to the DIALS-I workshop on X-ray Diffraction Data Analysis to be held at Diamond Light Source on June 12th/13th 2012. For information about the programme and how to register for the workshop please go to http://www.diamond.ac.uk/Home/Events/DIALS-1.html. A more detailed programme will appear soon. For any further information about the scope and purpose of the meeting please contact Gwyndaf Evans (gwyndaf.ev...@diamond.ac.ukmailto:gwyndaf.ev...@diamond.ac.uk). The number of places on the workshop is limited so please register as soon as possible. Regards, The Organisers Gwyndaf Evans, Graeme Winter, Alun Ashton and David Waterman
[ccp4bb] position in HTP crystallization
A position in crystallization is now available at the New York Structural Genomics Research Consortium (NYSGRC; www.nysgrc.org). The NYSGRC is one of four NIH-funded high throughout structure determination centers for PSI:Biology. Extensive macromolecular cystallization experience is an absolute requirement and familiarity with automation and high throughput efforts is highly desirable. The successful candidate will also possess experience with cryoprotection, mounting, and freezing protein crystals. Interested candidates should send a CV, including the names of three references, to Dr. Jeffrey Bonanno at jeffrey.bonanno AT einstein.yu.edu. Jeff
Re: [ccp4bb] question on metal refinement in a protein structure
On Sat, 2012-05-12 at 08:48 -0400, Dave Roberts wrote: However, I just want to make sure the metal environment is not due to the fact that I did something wrong in my refinement script - thus making it tetrahedral because it was refined as tetrahedral. ... I don't use CCP4 for refinement, I use CNS, but I'm hoping somebody will be able to guide me here. AFAIK, CNS does not generate any geometry restraints for metal ions *automatically*. You may want to make sure (if you didn't yet) that you use a correct residue name, e.g. NI2 (and atom name NI+2) for Ni++. CNS libraries would use different values for the radii and scattering factors of these. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Deposition of riding H
On Sat, 2012-05-12 at 19:28 +0100, Yuri Pompeu wrote: Dear community, I am probably disturbing a sleeping bear definitely so Reading the thread on hydrogen deposition with the model, I came accross several arguments that make sense on their own, but when put together are puzzling and dont seem to converge to an answer. this is true for most other recurring threads -Some argued that depositing riding hydrogens with the model may imply that your data had enough information for you to include hydrogen atoms in the final model. Unless there is a remark that explicitly states that these are riding hydrogens, but nobody reads those This is definetely a problem especially when dealing with non-experienced users that may think the model is more accurate than it really is. they always do and they have no idea how accurate the model actually is -It seemed to be consensus, that when softwares use hydrogen restraints it can be beneficial geometrically and also can make your model a better description of your x-ray data. I think nobody disputes that, although the benefit may vary from structure to structure Based on these two main arguments, many would agree that hydrogens should be included throughout refinement but not deposited. I do agree and I won't deposit them myself, but then what others choose to deposit is really their choice. So this brings me to last point that was also mentioned in the old thread. If you used riding hydrogens throughout refinement and arrived at a final model that you believe best describes your x-ray data to a certain level of accuracy (Rvalues, geometry, map CC, etc...) would you not be invalidating the whole refinement process by going in and removing the hydrogen atoms right before deposition? Not really. You report that you used riding hydrogens and you report the program you used to generate them. In theory, anyone can dig up the appropriate version and reproduce your results. So how would one avoid this Catch-22? I don't think it's strictly a catch-22 situation. The issue is that depending on what the structural model is used for, different forms of the pdb file may become most useful. The only situation I can imagine when having riding hydrogens is beneficial is for algorithm development and perhaps verification of how much differences in riding hydrogen treatment contribute to differences in things like R-values. Both are quite esoteric tasks and you already provide sufficient information (vide supra). Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Off topic: thermal melt
Hi Anita, You don't state the WL of the melting and back folding CDs, I'm guessing it's around 210 nm? In any case, the refolding that you see might be just the formation of aggregates. For saying if it's really refolding you'd need a new spectrum WL vs AU in the end of the reverse thermal melt and superimpose it with the first one. Having the protein more concentrated than that, or using a longer path length could reduce your noise. Carlos Em 14/05/2012, às 09:10, anita p escreveu: Hi All, Sorry for this off topic. I am stuck with a plant protein which is not crystallizing. I am expressing this in E coli. I have done the CD and thermal melt of the deletion constructs of this protein @ 0.2 mg/ml. Please refer to the doc file attached to this email. The CD looks okay but the thermal melt looks kind of wierd. I would expect a sigmoid curve for the thermal melts and then when we do a reverse temperature scan, the protein should denature and may not fold back (with some exceptions) Please let me know if any one had the same experience before and could explain me what this meant. Kind regards Anita Cd.doc
[ccp4bb] 2 postdoctoral positions available@EMBL-Hamburg
Dear All, I would like to inform you that 2 postdoctoral positions are available at the EMBL Hamburg Unit in the research group of Matthias Wilmanns. I attach brief descriptions of the Vacancy Notices below. Deadline for application is 15th June 2012. Further Detailed Information can be found under : http://www.embl-hamburg.de/aboutus/jobs/searchjobs/index.php?newlang=1newms=srsearchregion=668 Regards Margret Fischer --- *Postdoctoral Positon, Reference HH_00025* Applications are invited for research projects of the Wilmanns group in the EC-funded consortium SystemTB (www.systemtb.org), which aims to understand the pathology of /Mycobacterium tuberculosis/ during infection. While the core expertise of our group is in structural biology, we have established methods in mycobacterial genetics for the creation of full deletion mutants and specific point mutants using non-pathogenic model systems (M. smegmatis, BCG) (for examples, see: Noens et al. (2011) PMID: 2143903, Poulsen et al. (2010) Mol. Syst. Biol. PMID: 21439037). We have initialized several new collaborations using different --omics techniques, namely in interactomics (using high throughput mass spectrometry) (IBB Warsaw), metabolomics (ETH Zurich), proteomics (ETH Zurich), and lipidomics (CNRS Toulouse). The fellow is expected to carry out and to coordinate experiments in mycobacterial genetics in the context of ongoing and future projects. Most of them will be in cooperation with partners from the SystemTB consortium * Postdoctoral Positon, Reference HH_00026* Applications are invited for research projects of the Wilmanns group (www.embl-hamburg.de/research/unit/wilmanns) in the new European consortium GoMoA with partners from Spain, France and Germany. The key aim of this network is to identify and to characterize protein targets from /Mycobacterium tuberculosis/ for lead compounds against tuberculosis that already have been validated by an industrial partner. The objective of our group is to contribute to the identification of protein targets, in collaboration with the group of Dr. John Overington from the EMBL-EBI Unit in Cambridge (UK), and on their biochemical and structural characterization. This novel research direction will complement our record in M. tuberculosis structural biology and functional characterization. The fellow is expected to carry out and to coordinate experiments in functional and subsequent structural characterization of protein targets from /M. tuberculos/ / /
Re: [ccp4bb] how to ignore spot overlap in imosflm?
Although the question was asked for Mosflm I would like to briefly p[oint out that you might be able to also rescue your data by using a program that does 3D profile fitting e.g. d*trek and XDS. However as Andrew pointed out the shoot first try to fix it later mentality might have ruined the possibility of solving your crystal structure. Carefully deciding how to collect your data is worth the time. Jürgen On May 14, 2012, at 4:29 AM, A Leslie wrote: On 14 May 2012, at 03:22, Xinghua Qin wrote: Dear CCP4ers, We collected a diffraction dataset with high percentage of spot overlaps, It would be so kind to tell me how to ignore spot overlap in imosflm and explain the hazard of high percentage of spot overlaps. Thanks in advance. Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.commailto:xhqin1...@gmail.com mailto:xhqin1...@gmail.com .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] how to ignore spot overlap in imosflm?
On Mon, 2012-05-14 at 13:01 -0400, Bosch, Juergen wrote: Although the question was asked for Mosflm I would like to briefly p[oint out that you might be able to also rescue your data by using a program that does 3D profile fitting e.g. d*trek and XDS. For the sake of completeness (and nothing more), denzo may be used to get around the overlap issue by using a smaller spot radius while keeping background radius large enough to prevent the spots from contaminating the background measurements. I recall this was also a useful trcik for really large unit cells (i.e. when you still have severe overlaps even after pushing detector all the way back). -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
[ccp4bb] Postdoc position at UC-Davis (Al-Bassam lab)
Dear Colleagues, A postdoctoral position is available in the Al-Bassam Laboratory at UC-Davis, studying the structural basis of tubulin assembly and disassembly regulators into dynamic microtubules. Our laboratory focuses on combining Structural biology and Single molecule biophysics approaches to study mechanisms of tubulin and microtubule regulators, including: single particle cryo-EM, x-ray crystallography and single molecule total internal reflection fluorescence (TIRF) microscopy. For information regarding ongoing projects, please visit our lab website: http://microtubule.mcb.ucdavis.edu Our laboratory is equipped with a state of the art protein expression and purification setups, crystallization and crystal imaging robots. Our laboratory has full access to a state of the art, newly renovated, electron microscopy facility, that includes an aberration corrected JEOL 2100F (200 kV), a cryo-capable JEOL 2100F (200 kV), and a JEOL 1230 (120kV) as well as ancillary equipment for specimen preparation and transfer. For a detailed description of the facility, see website: http://www.mcb.ucdavis.edu/cryoem/ Candidates should hold a PhD in biophysics, biochemistry or a related field. Candidates with experience in biochemistry, three-dimensional electron microscopy or x-ray crystallography are preferred, but outstanding candidates with expertise in other disciplines will also be considered. Interested candidates should send a CV and names of references to Jawdat Al-Bassam: jaw...@mcb.ucdavis.edu The University of California is an Equal Opportunity/Affirmative Action Employer.
[ccp4bb] dm: Error in opening input map file.
Dear CCP4ers, I have a problem when I use DM to do NCS averaging. If I input 9 NCS averaging masks, DM works OK. However, if I input 10 NCS averaging masks, DM can not open input map file. The masks should be OK because they are generated by the same method. Do you have any idea how to solve the problem? Thank you in advance! Yu DM script is as below: /usr/share/CCP4-6.0.2/ccp4-6.2.0/bin/dm \ hklin /home/crystal/Documents/Ecoli/datasets/F111062011/130_10/reprocess/DM/x_refine33_phase.mtz \ ncsin1 E.msk \ ncsin2 F1.msk \ ncsin3 F2.msk \ ncsin4 D4.msk \ ncsin5 D5.msk \ ncsin6 D3.msk \ ncsin7 D2.msk \ ncsin8 D1.msk \ ncsin9 C5.msk \ ncsin10 C6.msk \ hklout x_dm.mtz EOF-dm SOLC 0.62 #RESOL 50 4.0 #NCSMASK SIZE 1 #NCSMASK UPDATE 3 #GRID 144 144 144 MODE SOLV hist AVER MULTI combine weight 0.15 combine pert scheme res from 4.5 NCYCLE 10 ncsmask overlap AVER DOMAIN 1 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 1 REFINE ROTA MATRIX -0.26665 0.52763 0.80654 0.60893 -0.55642 0.56533 0.74706 0.64187 -0.17293 TRAN -31.41600 172.23766 -96.59856 AVER DOMAIN 1 REFINE ROTA MATRIX 0.82271 0.34200 -0.45406 -0.55866 0.63407 -0.53465 0.10506 0.69353 0.71272 TRAN -141.48692 39.40455 -17.81890 AVER DOMAIN 1 REFINE ROTA MATRIX -0.35001 0.06658 0.93438 0.03939 -0.99554 0.08569 0.93592 0.06679 0.34582 TRAN -75.12174 220.50938 35.16329 AVER DOMAIN 2 REFIN ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 2 REFINE ROTA MATRIX -0.42731 0.24363 0.87066 0.39149 -0.81818 0.42109 0.81494 0.52080 0.25424 TRAN -7.42336 185.84557 -68.83428 AVER DOMAIN 2 REFINE ROTA MATRIX -0.42918 0.04126 0.90228 0.01801 -0.99837 0.05422 0.90304 0.03952 0.42774 TRAN -74.51554 219.69878 39.63858 AVER DOMAIN 2 REFINE ROTA MATRIX 0.94716 0.32039 -0.01558 -0.30765 0.89358 -0.32691 -0.09082 0.31442 0.94493 TRAN -121.99072 28.41423 20.60830 AVER DOMAIN 3 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 3 REFINE ROTA MATRIX -0.42590 0.08845 0.90044 -0.00887 -0.99557 0.09361 0.90473 0.03188 0.42480 TRAN -77.38764 220.87816 40.52480 AVER DOMAIN 4 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 4 REFINE ROTA MATRIX -0.34404 0.07038 0.93631 -0.03070 -0.99750 0.06370 0.93845 -0.00683 0.34534 TRAN -79.74300 222.43433 43.52758 AVER DOMAIN 5 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 5 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 AVER DOMAIN 6 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 6 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 AVER DOMAIN 7 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 7 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 AVER DOMAIN 8 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 8 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 AVER DOMAIN 9 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 9 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 AVER DOMAIN 10 REFINE ROTATE EULER 0.0000.0000.000 TRANSLATION0.0000.0000.000 AVER DOMAIN 10 REFINE ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309 -0.01068 0.35948 TRAN -70.14525 224.81303 43.77335 LABIN FP=FOBS SIGFP=SIGFOBS PHIO=PHIFMODEL FOMO=FOM LABOUT PHIDM=PHIDM FOMDM=FOMDM EOF-dm -- Part of the log file is as below: -- Grid dimensions 96 108 162 must contain the following prime factors for agreement with symmetry restrictions-2 2 2 CCP4 library signal library_file:Cannot open file (Error) raised in ccp4_file_open2 CCP4 library signal ccp4_map:Cannot open file (Error) raised in ccp4_cmap_open CCP4 library signal ccp4_map:Cannot open file (Error) raised in MRDHDS dm: Error in
