[ccp4bb] Difference map
Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] Difference map
Hi Intekhab Alam, an Fobs-Fobs' map usually works only if the two crystals are isomorphous, which means that there are neither large cell constant changes nor any other larger structural changes (like overall rotations, domain movements, other rearrangements) than the bound compound (ligand, heavy atom, ...). Scaleit produces a plot of Riso against resolution which ideally should resemble the scattering curve of the bound compound (~ like an atomic scattering curve for instance), plus a mild increase at high resolution due to the increasing noise component at higher resolution. If the curve starts at high Riso values and increases steeply, this would indicate anisomorphism, and there is probably no chance to detect your compound signal. All this is qualitative, but you could estimate the expected change with the Crick-Magdoff equation by replacing the heavy atoms with a sum over your compound atoms. The Crick-Magdoff equations has been recently discussed on this board: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg10039.html Good luck, Dirk. Am 29.09.10 09:09, schrieb intekhab alam: Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
[ccp4bb] The color of GFP crystals?
Dear CCP4 community, I wonder if anybody has experience about the color of GFP crystals. Is there any chance that the GFP crystal will become colorless? We are working on one membrane protein fused with GFP. Since we got some crystals which didn't show any color (Condition 01), I am not sure if I should go for optimization. Please check the link below for the image details: http://www.docstoc.com/docs/55950590/GFP-crystals Because of the limited amount of sample, we used Topaz system (screening chip) to do the screening and there is no way to confirm these crystals are actually protein crystals. I noticed that the crystals are bigger day by day. Is it a good sign to confirm the protein crystals? Thank you very much for your helpful information! Best wishes, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
Re: [ccp4bb] Difference map
That seems the right procedure. I presume the two crystals have similar cell, etc? What is the Riso plot from scaleit look like - if it is 55% + then there is no isomorphism, but if it is 30% then the map should be reasonable. which phase did you use for the map calculation? Eleanor intekhab alam wrote: Hi Folks I have a query regarding the difference map between the two structures ligand bound data (2.5A) and native (2.8A). I tried to calculate the fourier difference map between two data sets ligand bound- native. The protocol in CCP4 that i used is as: 1.merge the mtz file of native nad ligand bound using cad 2. scaling this combine file with scaleit program followed by map generation uisng fft. I got the map but i did not find the fu;ll map of the ligand,i can only see small density nera the ligand binding site at 5 sigma level. I have calculated omit map that cleraly showed the ligand. why is such discrepency in the two cases, is there is something missing from the calculation. kindly help me out. Thanks and regards Intekhab alam
Re: [ccp4bb] The color of GFP crystals?
Hi Tri, To me, the first 3 pictures suggest that there is a faint greenish tinge in the little shapes (hopefully crystalline) there. I guess the idea of the Topaz chip is that you can take it as is to the x-ray beam or am I wrong? Cheers, Boaz - Original Message - From: Tri Ngo ngoduc...@gmail.com Date: Wednesday, September 29, 2010 10:50 Subject: [ccp4bb] The color of GFP crystals? To: CCP4BB@JISCMAIL.AC.UK Dear CCP4 community, I wonder if anybody has experience about the color of GFP crystals. Is there any chance that the GFP crystal will become colorless? We are working on one membrane protein fused with GFP. Since we got some crystals which didn't show any color (Condition 01), I am not sure if I should go for optimization. Please check the link below for the image details: http://www.docstoc.com/docs/55950590/GFP-crystals Because of the limited amount of sample, we used Topaz system (screeningchip) to do the screening and there is no way to confirm these crystals are actually protein crystals. I noticed that the crystals are bigger day by day. Is it a good sign to confirm the protein crystals? Thank you very much for your helpful information! Best wishes, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150 Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan
[ccp4bb] Expressions of Interest in Diamond’s Phase III Beamline proposals
Dear Everyone, As an MX representative of the Diamond User Committee we are writing to remind you that the deadline is fast approaching for Expressions of Interest in Diamond’s Phase III Beamline proposals, three of which are aimed at MX. The three relevant beamlines and their representatives are 1) Coherent and sub micron focus x-rays for macromolecular crystallography – Colin Nave and Ian Robinson 2) Beamline for Advanced Structure – Function Studies in Life and Physical Sciences – Arwen Pearson and Samar Hasnain 3) i-plate: in-situ diffraction screening beamline – Jonathan Grimes and Frank von Delft You can read more about the Phase III Prioritization process at http://www.diamond.ac.uk/Home/Science/phase-III.html where you’ll find a list of all the preliminary concepts for beamlines. The deadline for receipt of letters of interest is 30th September so please write or email diamond.scie...@diamond.ac.uk showing your support for any or all of these ideas explaining why they are important for driving your science forward. This a chance to push the boundaries of what we will be able do at Diamond. The letters do not have to be long. Best wishes, Peter Johan Peter Moody 1/56 Henry Wellcome Laboratories University of Leicester Lancaster Road Leicester LE1 9HN UK tel. (0)116 229 7097 fax. (0)116 229 7084 http://www2.le.ac.uk/departments/biochemistry/staff/moody Dr. Johan P. Turkenburg York Structural Biology Laboratory University of York Phone (+) 44 1904 328251 York YO10 5DD UK Fax (+) 44 1904 328266
Re: [ccp4bb] The color of GFP crystals?
What GFP did you use, your pH is 4.6 or something around there according to your picture. If the pH is true GFP will be colorless. You certainly have some more GFP lying around (without your protein fused to it), just titrate it and see what pH dependency you have. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 29, 2010, at 4:41 AM, Tri Ngo wrote: Dear CCP4 community, I wonder if anybody has experience about the color of GFP crystals. Is there any chance that the GFP crystal will become colorless? We are working on one membrane protein fused with GFP. Since we got some crystals which didn't show any color (Condition 01), I am not sure if I should go for optimization. Please check the link below for the image details: http://www.docstoc.com/docs/55950590/GFP-crystals Because of the limited amount of sample, we used Topaz system (screening chip) to do the screening and there is no way to confirm these crystals are actually protein crystals. I noticed that the crystals are bigger day by day. Is it a good sign to confirm the protein crystals? Thank you very much for your helpful information! Best wishes, TriNgo Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
Re: [ccp4bb] Micromatrix seeding using the mosquito
Not with membrane proteins, however we use our Mosquito with 20 nl seeding standard. Strip 1 Protein, Strip 2 Seed stock solution We first copy the plate by either picking up protein, then reservoir and dispensing together 200-400 nl or we multidispense protein and add the reservoir later (larger drops, in particular if we can't concentrate the protein well then we add 600 nl and hope that enough evaporates before we come with the reservoir solution). As last step we add the 20 nl seed solution. We've also done seed titrations then we use three strips 1)Protein 2) seed buffer solution 3) seed. In that case the combination of 2+3 is always 100nl and we start out with 5 nl Seed stock. In the IntelliPlates (3well) we play the protein/reservoir ratio game + seed Reproducibility is pretty good. We keep our seed stock where we grew the crystals e.g. 20˚C or 4˚C. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 29, 2010, at 5:23 AM, Patrick Shaw Stewart wrote: Hi Jeroen I think that part of the point of the method is that it’s incredibly quick and easy to do – you can make seeds and run an experiment in 20 mins. But I’m sure it’s important to use the seed stock quickly and freeze it. Maria I think most people use 100 nl seed stock, 200 nl reservoir, 300 nl protein as D’Arcy and co originally suggested. Maybe more seed stock would be better as Roberto says, to increase the additive effect. The main reasons not to put seed stock into the reservoir are (1) it’s more work – compared to your robot picking up the seed stock from a PCR tube - and (2) sometimes your seed stock is very valuable, for example if you only have one crystal and you make up say 5 ul of seed stock. Has anyone had success with MMS microseeding with membrane proteins? I wonder if membrane protein crystals dissolve more easily. Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of mesters Sent: 28 September 2010 07:26 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito Hello Maria, the success of this method depends on a few things. First, your seed stock-solution. Most people prepare it by smashing up seeds in mother liquor (i.e. reservoir solution). Looking at the phase diagram, you will quickly understand that this might not be the best way to go as you will dissolve your seeds fairly quickly over time. That is why people prepare really fresh stocks, keep them on ice and freeze them as soon as possible in order to stabilize the seeds. Keeping on ice will only help if your protein is less soluble at lower temperatures, which true for roughly 45% of proteins (45% will react retrograde, 10% do not show a clear preference). Looking at the phase diagram again, you will also understand that seeding only works for near equilibrated drops that are metastable-supersaturated. If undersaturated, seeding will not work as the seeds will dissolve over time. Keeping the above considerations in mind, best is to seed after the drops have equilibrated. Next best option is to add the seeds or the protein as the last component to the drop... Jeroen. On 20.09.10 09:53, Maria Håkansson wrote: Dear all, Anyone who have had success using the mosquito for micromatrix seeding? Which way is the optimal way of adding seed solution to sitting drops on an MRC plate? Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir solution or to add concentrated seed solution (1 microliter) directly to the reservoir solution and then pipett 100 nl protein + 100 nl reservoir solution. Best regards and thanks in advance, Maria Håkansson __ Maria Håkansson, Ph.D. Senior Scientist, Max-lab, Lund University Phone: +46 (0) 76 8585 706 Fax: +46 (0) 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) SE-221 00 Lund, Sweden Web address: www.maxlab.lu.se Email: maria.hakans...@maxlab.lu.se __ -- image002.jpg-- Dr. Jeroen R. Mesters Gruppenleiter Strukturelle Neurobiologie und Kristallogenese Institut für
[ccp4bb] Postdoctoral Position in Structural Biology lab
Postdoctoral candidates: Postdoctoral Position in Membrane Transport The Pinkett laboratory at Northwestern University is seeking a highly motivated individual for a postdoctoral fellow position in the field of protein structural biology. The individual will join a research team studying the structure and function of ABC transporters. Our laboratory is equipped with state-of-the art facilities for structural biology and protein biochemistry research. We also have access to nearby Argonne National labs (APS). An ideal applicant would be highly motivated with strong analytical skills and enjoy working independently as well as in a collaborative environment. A strong background in X-ray crystallography, protein expression and/or biochemistry is a plus. The position requires a Ph.D. in a related field and less than five years of postdoctoral experience. Applicants should email their CV and a summary of their research experience to Dr. Heather Pinkett (h-pink...@northwestern.edu). -- Heather W Pinkett, PhD Assistant Professor Department of Molecular Biosciences Northwestern University 2205 Tech Drive Evanston, IL 60208-3500 Office: Cook Hall 4133 Lab: Cook Hall 4161 Tel. (847) 467-4048 h-pink...@northwestern.edu http://groups.molbiosci.northwestern.edu/pinkett/Pinkettlab/Welcome.html
Re: [ccp4bb] The color of GFP crystals?
Dear experts, Thank you very much for your input. Here are my answers for your questions: To me, the first 3 pictures suggest that there is a faint greenish tinge in the little shapes (hopefully crystalline) there. I guess the idea of the Topaz chip is that you can take it as is to the x-ray beam or am I wrong? - I didn't see the greenish tinge in the original images. I think the color of the images was modified when I convert to pdf format. - The screening Topaz chip cannot apply for diffraction. They have a different chip for in-situ diffraction but we didn't use that chip in this case. What GFP did you use, your pH is 4.6 or something around there according to your picture. If the pH is true GFP will be colorless. You certainly have some more GFP lying around (without your protein fused to it), just titrate it and see what pH dependency you have. - The protein was fused with GFPuv. It also shows the beautiful green color at the concentration 30mg/ml in the low pH buffer (20mM sodium acetate 4.6, 100mM NaCl, 0.2% Cymal5). Do you think that the crystal getting bigger is a good sign of protein crystal? My best regards, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
[ccp4bb] refmac 5.6 and the ccp4i task interface
Is it possible to use refmac 5.6 with the ccp4i Refmac5 task? Can I just copy the 5.6 binary over the 5.5 binary distributed with CCP4? It looks like the new refmac has a number of new keywords which wouldn't be accessible doing this, but would it otherwise be compatible? If so, where does the dictionary go? Is there some elegant way of letting users choose between the two? Thanks. -ben -- | Ben Eisenbraun | Software Sysadmin | | Structural Biology Grid | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] ? steps after detwinning
@R.Brown I'm using DENZO I'm trying POINTLESS but I don't understand unmerged data -what's that? is it the spot images which are used for peak search during data processing? Both SFCHECK phenix.xtriage showed the information about pst twinning. However I've tried a thorough process of reindexing,phaser,refmac ...etc. now in P31 is showing R-factor R-free as 0.37 0.40 respectively with a poly-ala model.I think I'm going towards correct solution. Many many thanks for your help.
Re: [ccp4bb] refmac 5.6 and the ccp4i task interface
Yes. There are two ways: 1) replace $CBIN/refmac5 with refmac5.6 2) on ccp4i click System administration, select configure interface and jus below Give full path name for CCP4 programs to overcome name conflicts click Add a program There will appear two fields. On the right field type refmac5 and on the left field actual address of the program. I hope it helps regards Garib On 29 Sep 2010, at 16:39, Ben Eisenbraun wrote: Is it possible to use refmac 5.6 with the ccp4i Refmac5 task? Can I just copy the 5.6 binary over the 5.5 binary distributed with CCP4? It looks like the new refmac has a number of new keywords which wouldn't be accessible doing this, but would it otherwise be compatible? If so, where does the dictionary go? Is there some elegant way of letting users choose between the two? Thanks. -ben -- | Ben Eisenbraun | Software Sysadmin | | Structural Biology Grid | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] The color of GFP crystals?
In my hands GFP fusions can produce colorless crystals (but still have GFP!) depending on the condition. Sometimes the crystals gradually become colorless with time - bleaching or perhaps some chemical effect - no idea. I've not tested the colorless crystals with UV to see if they still glow. Artem On Wed, Sep 29, 2010 at 3:41 AM, Tri Ngo ngoduc...@gmail.com wrote: Dear CCP4 community, I wonder if anybody has experience about the color of GFP crystals. Is there any chance that the GFP crystal will become colorless? We are working on one membrane protein fused with GFP. Since we got some crystals which didn't show any color (Condition 01), I am not sure if I should go for optimization. Please check the link below for the image details: http://www.docstoc.com/docs/55950590/GFP-crystals Because of the limited amount of sample, we used Topaz system (screening chip) to do the screening and there is no way to confirm these crystals are actually protein crystals. I noticed that the crystals are bigger day by day. Is it a good sign to confirm the protein crystals? Thank you very much for your helpful information! Best wishes, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
[ccp4bb] Seeking Postdoctoral Researcher
Laboratory: Zhang Initiative Research Unit, Advanced Science Institute, RIKEN, Japan. (Unit Leader: Dr. Kam Zhang) Job title and Job description: Postdoctoral Researchers Job description: We are seeking a highly motivated and experienced computational structural biologist to join the Zhang Initiative Research Unit at RIKEN. The successful candidate will use computational tools to tackle problems in the area of protein folding, structure prediction, and structure-based drug design. Our ability to predict the structure of proteins from their primary sequences will greatly facilitate our understanding of the important biological functions that proteins play in their host systems. The 3D structure information will also facilitate the rationale design of drugs for the treatment of various diseases with unmet medical needs. Qualifications: This position requires a PhD in areas related to biophysics, biochemistry, computational chemistry, bioinformatics, or structural biology with experience in employing computational tools to solve biological or chemical problems. Experiences in protein folding, protein structure prediction, protein structure analysis, protein crystallography, or structure-based drugs are highly desired. Proficiency in object-oriented programming such as C++, Java, scripting languages such as Python, Perl are also desired. Experiences in high performance computing, network computing, or grid computing would be a plus. The ideal candidate should possess a track record of accomplishments demonstrating technical proficiency, independent thinking, and scientific creativity. The research environment will be international and hence communication in English is encouraged. The complete job ad, as well as how to apply can be viewed here: http://www.riken.jp/engn/r-world/info/recruit/k100817_s_asi.html Best regards, Francois.
