Re: [ccp4bb] Enhancing Crystal Quality
Hi Lucas, The funky diffraction pattern is most likely due to a cracked crystal, resulting in a mixture of slightly differently aligned diffraction patterns. Were the cracks there before you added the cryprotectant? If not, the cryoprotectant is definitively to blame. As has mentioned before, you have to take a shot at room temperature without any cryoprotectant added, to make sure the bad quality is not due to the cryoprotectant. Mitegen sells plastic capillaries, which you can slide over your loop to prevent the crystal from drying out. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yi-Liang Liu Sent: Thursday, August 02, 2012 4:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Enhancing Crystal Quality Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some patterns (cracks (?)) on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote: Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
Re: [ccp4bb] Process multiple data sets
An earlier post said the point-group is P2, and these reported cells do not quote the beta angles: what are these angles?. In the monoclinic system it is possible to have two closely-similar alternative cells in certain special cases, and if the crystals have been indexed differently this could prevent their merging. The program Pointless would sort that out for you. Phil On 2 Aug 2012, at 00:45, Edwin Pozharski wrote: The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. This is very substantial difference and with unit cell expanding by ~20% one would expect scaling problems. Try using the same unit cell/orientation on all datasets, it's easy to do if you abandon gui and feed input file directly to denzo. -- Edwin Pozharski, PhD University of Maryland, Baltimore
[ccp4bb] space group and multiplicity
Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina
Re: [ccp4bb] space group and multiplicity
Hi Careina The obvious answer is to look it up in International Tables vol. A. If you don't have access to that you can look up the text file $CLIBD/syminfo.lib in the CCP4 distribution and work it out from there. Fpr a given space group you need to count the number of 'symop' lines. That's the number of asymmetric units in the primitive cell. If you want the number in a centred cell, also count the number of 'cenop' lines: that's the number of centring translations, so to get the total no of a.u.'s in the centred cell you would multiply these two numbers. I'm afraid there isn't an easier way, for example you can't get it straightforwardly from the space group symbol. HTH -- Ian On 2 August 2012 09:37, Careina Edgooms careinaedgo...@yahoo.com wrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina
Re: [ccp4bb] space group and multiplicity
I don't want to confuse things further, but as a PS to Ian's answer that clearly tells you how to get Z.. You should be aware that a crystal might also have non-crystallographic symmetry. Ian's answer is right for Z, but as you also mentioned monomers I thought I should mention that if the a.u. comprises a multimer then you would have to multiply. i.e. symops will give the number of copies of the asymmetric unit in the unit cell, this might not be the number of monomers. Peter On 2 August 2012 10:12, Ian Tickle ianj...@gmail.com wrote: Hi Careina The obvious answer is to look it up in International Tables vol. A. If you don't have access to that you can look up the text file $CLIBD/syminfo.lib in the CCP4 distribution and work it out from there. Fpr a given space group you need to count the number of 'symop' lines. That's the number of asymmetric units in the primitive cell. If you want the number in a centred cell, also count the number of 'cenop' lines: that's the number of centring translations, so to get the total no of a.u.'s in the centred cell you would multiply these two numbers. I'm afraid there isn't an easier way, for example you can't get it straightforwardly from the space group symbol. HTH -- Ian On 2 August 2012 09:37, Careina Edgooms careinaedgo...@yahoo.com wrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina
[ccp4bb] REGISTER NOW: Murnau Conference 2012 on Structural Biology of Molecular Transport
Dear colleagues, this is to remind you that the Murnau Conference 2012 on Structural Biology of Molecular Transport will be taking place from 17-20 October. Online registration is OPEN (http://www.murnauconference.de/2012/registration.html). We would be delighted to meet you at the conference. Please also spread the information amongst your colleagues to help us attract a broad audience and an interesting cross-section of the community. If you want the conference poster (PDF file) for your notice boards, please let us know. Murnau Conference 2012 on Structural Biology of Molecular Transport October 17-20, 2012 - Murnau/Germany SESSIONS I Channels and Transporters I: Transport through Membrane II RNA, Nuclear and ER Transport / Nucleocytoplasmic Transport III Endosomal / Synaptic Transport IV Cytoskeleton and Cellular Motility V Channels and Transporters II: Molecular Machines PLENARY SPEAKERS Marc Baldus (Utrecht) Tamir Gonen (Seattle) Reinhard Jahn (Göttingen) Hartmut Michel (Frankfurt) You Min Chook (Dallas) Poul Nissen (Arhus) Tom Pollard (New Haven) Jim Rothman (Yale) Mike Rosen (Dallas) Helen Saibil (London) Irmi Sinning (Heidelberg) Daniela Stock (Darlinghurst) Gerhard Wagner (Harvard) BACKGROUND INFO The Murnau Conference is an international meeting (~180 participants) covering current issues in the wide field of modern structural biology. A clear goal of the conference, which will take place this year for the 4th time, is to bring together the most eminent scientists in the field with young researchers in a casual atmosphere in the heart of Europe. The first three meetings in the series took place in 2005 (Structural Biology of Molecular Recognition), 2007 (Structural Biology of Disease Mechanisms) and 2010 (Structural Biology of the Modern RNA World). Murnau is a picturesque small town located directly at lake Staffelsee in the Bavarian alpine upland between Munich and Garmisch-Partenkirchen. There will be an exciting social program including a typical Bavarian evening in a fashionable microbrewery and ample time for stimulating discussions with participants from all over the world. Please contact us in case of further questions. With best regards, Prof. Dr. Dirk Heinz in the name of the organization committee www.murnauconference.dehttp://www.murnauconference.de murnauconference2...@gmail.commailto:murnauconference2...@gmail.com Murnau Conference 2012 -Office- Christine Bentz GF/W (Scientific Director's Office) Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig, Germany christine.be...@helmholtz-hzi.demailto:christine.be...@helmholtz-hzi.de +49 (0)531-6181-1003 Protect the environment - please don't print this e-mail unless you really need to Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 Braunschweig | www.helmholtz-hzi.de Vorsitzende des Aufsichtsrates: MinDir'in Bärbel Brumme-Bothe, Bundesministerium für Bildung und Forschung Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches Ministerium für Wissenschaft und Kultur Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA Gesellschaft mit beschränkter Haftung (GmbH) Sitz der Gesellschaft: Braunschweig Handelsregister: Amtsgericht Braunschweig, HRB 477
Re: [ccp4bb] Process multiple data sets
Dear All: Thank you very for your comments and advices. ' I am getting to know why are the problems. And will try again. I appreciate you all for your inputs regards Uma On Thu, Aug 2, 2012 at 4:11 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: An earlier post said the point-group is P2, and these reported cells do not quote the beta angles: what are these angles?. In the monoclinic system it is possible to have two closely-similar alternative cells in certain special cases, and if the crystals have been indexed differently this could prevent their merging. The program Pointless would sort that out for you. Phil On 2 Aug 2012, at 00:45, Edwin Pozharski wrote: The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. This is very substantial difference and with unit cell expanding by ~20% one would expect scaling problems. Try using the same unit cell/orientation on all datasets, it's easy to do if you abandon gui and feed input file directly to denzo. -- Edwin Pozharski, PhD University of Maryland, Baltimore
Re: [ccp4bb] Process multiple data sets
Uma, Before this discussion goes much further, you need to provide more details: 1) putative space group? 2) observed resolution and diffraction anisotropy? 3) how big was the crystal and what was its shape? Was the crystal split? 4) were the data sets taken at different points on the crystal? Is radiation damage a factor? 5) did you just rotate around phi (or omega) to collect the different data sets or did you change the other angular settings? 6) are all your data sets indexed in exactly the same way (a tricky and non-obvious factor for a novice to appreciate). Using pointless on unmerged data sets helps with this. You have a number of unknowns here, and your problem in merging the data sets may be due to radiation damage, non-uniformity in a large crystal, index refinement problems due to diffraction anisotropy, etc. We routinely merge different data sets from a single crystal, which has been translated and rotated about one axis only. We try to index and process the data sets using a common setting matrix (which is easy with XDS). However, sometimes it just does work, but merging pairs of data sets often allowed us to discard the worst offender(s). Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Aug 1, 2012, at 4:37 PM, Uma Ratu wrote: I notice one thing with my data sets. The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. Is this the reason that I can't index both with same parameter in HKL? And subsequently, can't integrate and scala together. If so, is there a way that I can fix it? Thank you for your advice Uma On Wed, Aug 1, 2012 at 8:50 AM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
[ccp4bb] Protein-Protein Interactions
Dear Colleagues, I have a question for all of you bioinformatics oriented structural biologists: How do I predict the sites of protein-protein interactions between two receptors that have been proven to interact biochemically but lack specific details regarding proximity. This is not a straightforward question for me, and I believe it is somewhat complicated. The complicated scenario involves a multitude of different subunits and isoforms. Also, there is not structural data to support all components involved, and thus I presume I should use the sequence based software. I am aware that there are different types of prediction software, either sequence or structure based predictions using different algorithms:http://rosettadesigngroup.com/blog/58/10-protein-protein-interface-prediction-servers/Receptor 1:-Has 5 predicted subunits (Alpha)2-(Beta)2-(Gamma)11. Alpha (6 isoforms)2. Beta (3 Isoforms)3. Gamma (3 Isoforms)Receptor 2:-Is believed to be composed of (Alpha)3-(Beta)21. Alpha (4 isoforms)2. Beta(1 isoform)Any advice or recommendation will be well appreciated! Sincerely, lorenzo Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China
[ccp4bb] Crystallization Robots
Hello ccp4BBers, Our lab is thinking about trying to purchase a crystallization robot. We need to be able to do LCP in addition to sitting drop experiments. Does anyone have experience with the NT8 robot from formulatrix? The other two robots I am aware of are the LCP Mosquito from TTP and the LCP Gryphon from ARI. Does anyone have any experiences they could share with me about these instruments? Feel free to email off list if you want. Thanks! -Sam
Re: [ccp4bb] Crystallization Robots
Dear Sam, I spent quite some time trying to get up and running a HT LCP setup in the past years, and all I can say is that TTP has successfully automated in its Mosquito LCP all the homemade workarounds I employed back then. For instance, they tackle the dehydration problem by covering the drop with precipitant subsequently to LCP dispensing. They also use a chamber to control RH (I believe the NT8 uses a similar device too). They create a droplet of precipitant prior to dispensing in order to avoid touching the viscous LCP with a dry tip, which might cause it to stick. They even calibrate the position of the syringe before starting the procedure; this is really critical, since you have to manually fix the syringe in position after making the LCP. I've only watched brief demonstrations of the NT8 and Gryphon, and specially the Gryphon experienced severe calibration problems which prevented the LCP drops to be hydrated. NT8 seemed to be a fine piece of hardware though, and it even spares you of the pain of changing the tip roll every once in a while. Jon 2012/8/2 Sam Jimmeson samjimme...@gmail.com Hello ccp4BBers, Our lab is thinking about trying to purchase a crystallization robot. We need to be able to do LCP in addition to sitting drop experiments. Does anyone have experience with the NT8 robot from formulatrix? The other two robots I am aware of are the LCP Mosquito from TTP and the LCP Gryphon from ARI. Does anyone have any experiences they could share with me about these instruments? Feel free to email off list if you want. Thanks! -Sam -- Jon Agirre, PhD Biophysics Unit (CSIC-UPV/EHU) http://www.ehu.es/jon.agirre +34656756888
[ccp4bb] CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC
*CALL FOR PROPOSALS FOR ESRF BEAM TIME WITH ONLINE MICROSPEC*Proposal Deadline *4th September 2012* There will be beam time available at the ESRF for MX data collection with a setup that allows online monitoring of UV/VIS absorbance or fluorescence spectral changes of the crystal during the X-ray diffraction experiment. These complementary techniques will aid in the interpretation of X-ray data in the context of reaction intermediate state trapping and/or radiation damage monitoring, most particularly in the case of redox or photoactive proteins. Users who are interested in using this beam time (including those who are members of BAG Groups) should use the following mechanism:_http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal _and *it must be clearly indicated in the title of the proposal form that the online monitoring of spectral changes is necessary for the project*.A brief description of the device is given below however users are encouraged to consult the web pages for detailed information:_http://www.esrf.fr/UsersAndScience/Experiments/MX/How_to_use_our_beamlines/Run_Your_Experiment/Microspectrophotometer_User_Guide_The device is also described in: McGeehan, J., Ravelli, R.B., Murray, J.W., Owen, R.L., Cipriani, F., McSweeney, S., Weik, M. and Garman, E.F. (2009) Colouring cryo-cooled crystals: online microspectrophotometry. /J.Synchrotron Radiat./ *16*, 163-172. As this is not a standard set-up, it might take a significant amount of time to train users, align the device, and analyze the data in order to derive relevant data collection schemes. *We will therefore schedule 24 hours for each project*. The *deadline *for this specific application is *Tuesday 4th September 2012*. It is strongly recommended to, beforehand, record an absorption (fluorescence) spectrum of the crystal on a home microspectrophotometer such as the 4dx one, or at an off-line facility such as the ESRF Cryobench, and to provide it in the application form. Such a spectrum would greatly help to determine the feasibility of the experiment. For optimal experimental conditions, crystals should be flash-cooled in minimal amounts of cryosolution, especially when the crystals are small. Finally, please note that *the ESRF sample changer cannot be operated at the same time as the on-line microspec*. The use of a specific laser is possible if the device is compliant with the beam line safety system (interlock on device power). Recent examples of research using the ESRF online microspectrophotometer include: de la Mora /et al./ /Acta Cryst. D/ (2012), Ferraroni et al., /J. Inorg. Biochem./ (2012), Gumiero /et al./ /J. Biol. Chem./ (2011), Hersleth /et al./ /Biochim. Biophys. Acta/(2011), Kiontke /et al./ /EMBO J./ (2011), Orru et al., /J. Biol. Chem./(2011), Regis-Faro et al., /J. Am. Chem. Soc./(2011) Dates of beam-time: *4th October - 7th October 2012* Storage Ring: 7/8 +1 (200mA) Beamline: ID14-1 Energy: 13.27 keV (not tunable) Specifications: UV/VIS-range: 250-1100 nm Light source: Mikropack DH-2000-BAL (Deuterium/Halogen) Fluorescence/Actinic excitation wavelength: 405, 440, 473, 532, 561, 671 nm ODmax for UV-vis absorbance spectra: 2-2.5 Monitoring light size: 0.03 (min) - 0.15mm(max) Sampling freq (to disk): 10Hz or lower -- Dr David FLOT Beam-Line Operation Manager Tel : (+33) 4 76 88 17 63 Structural Biology GroupFax : (+33) 4 76 88 26 24 ESRF B.P. 220, 6 rue Jules Horowitz e-mail : david.f...@esrf.fr F-38043 GRENOBLE CEDEX http://www.esrf.eu
Re: [ccp4bb] space group and multiplicity
On 08/02/2012 04:37 AM, Careina Edgooms wrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina If you want to derive this number yourself (instead of looking it up in ITC), do this: 1. Write down all the symmetry operators for the spacegroup. To save time , I'll use P2 for an example: (x,y,z) (-x,y,-z) 2. Keep applying them until you get a closed list of symmetry mates: (x,y,z) - primitive (-x,y,-z) - second copy Now apply second operator to the second copy and you get (-(-x),y,-(-z)) = (x,y,z) - but that is the same as the primitive operator, so further application of symmetry will not lead to new copies. 3. Count the unique symmetry copies you found - in this case 2 of them and you are done. Other space groups are not really more complicated, the same steps apply, you just have more operators. Notice that symmetry mates should always be shifted back into the origin unit cell, e.g. in P21 the second operator is (-x, y+1/2, -z) which after two applications results in (-(-x), (y+1/2)+1/2, -(-z)) = (x, y+1, z) but this is the same as (x,y,z) after you translate it back by (0,-1,0). Where do you find symmetry operators? You can derive them yourself from the space group symbol or look them up in ITC. Once you master this, you will be able to explain to others why there is no P22 space group :) Cheers, Ed.
Re: [ccp4bb] space group and multiplicity
The space group decoder does exactly these steps and lists additional useful information. http://www.ruppweb.org/new_comp/spacegroup_decoder.htm Other examples including unit cell packing in C2 are in BMC chapter 5. Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edwin Pozharski Sent: Thursday, August 02, 2012 8:08 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] space group and multiplicity On 08/02/2012 04:37 AM, Careina Edgooms wrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina If you want to derive this number yourself (instead of looking it up in ITC), do this: 1. Write down all the symmetry operators for the spacegroup. To save time , I'll use P2 for an example: (x,y,z) (-x,y,-z) 2. Keep applying them until you get a closed list of symmetry mates: (x,y,z) - primitive (-x,y,-z) - second copy Now apply second operator to the second copy and you get (-(-x),y,-(-z)) = (x,y,z) - but that is the same as the primitive operator, so further application of symmetry will not lead to new copies. 3. Count the unique symmetry copies you found - in this case 2 of them and you are done. Other space groups are not really more complicated, the same steps apply, you just have more operators. Notice that symmetry mates should always be shifted back into the origin unit cell, e.g. in P21 the second operator is (-x, y+1/2, -z) which after two applications results in (-(-x), (y+1/2)+1/2, -(-z)) = (x, y+1, z) but this is the same as (x,y,z) after you translate it back by (0,-1,0). Where do you find symmetry operators? You can derive them yourself from the space group symbol or look them up in ITC. Once you master this, you will be able to explain to others why there is no P22 space group :) Cheers, Ed.
Re: [ccp4bb] space group and multiplicity
Hi, cctbx Explore symmetry will do this and lot more: http://cci.lbl.gov/cctbx/explore_symmetry.html Pavel On Thu, Aug 2, 2012 at 1:37 AM, Careina Edgooms careinaedgo...@yahoo.comwrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina
Re: [ccp4bb] space group and multiplicity
Also I see it works on all settings, not just the limited set of standard symbols, doesn't need spaces in the names (which are redundant anyway), and uses the correct xHM symbols (such as R32:r). It also accepts the PDB-only symbols H3 H32. Well done Pavel - Like + :). Cheers -- Ian On 2 August 2012 18:06, Pavel Afonine pafon...@gmail.com wrote: Hi, cctbx Explore symmetry will do this and lot more: http://cci.lbl.gov/cctbx/explore_symmetry.html Pavel On Thu, Aug 2, 2012 at 1:37 AM, Careina Edgooms careinaedgo...@yahoo.com wrote: Dear ccp4 I ask a very fundamental question because I have not had formal training in this and I would like to understand. How can I obtain the multiplicity (z) from the space group? So for example if the space group is P222 how do I know that there are 4 monomers in the unit cell? Or if it is P422 then there is 8? I am only concerning myself with a primitive lattice for now because I am sure the others are more complicated. thanks Careina
Re: [ccp4bb] Protein-Protein Interactions
Hi Lorenzo, If the structure for your receptor is unknown, then you can use Homology Modeling methods to get a rough idea of the structure, MODELLER is a well know tool for this (http://salilab.org/modeller/). Of course depending on your % similarity to the template, the higher the % similarity, the more reliable your structure may be (of course assuming there are no major conformational changes, etc.) Now, to figure out the sites of interaction, you could use a shape based complementarity approach like the one used in the ZDOCK algorithm (http://zdock.umassmed.edu/software/). This gets to be a little bit trickier if your % similarity to your template is low, because the dissimilarity is often due to surface residue differences, which are obviously the ones you're interested on. On the other hand, if the source of interaction is driven mainly by hydrophobic forces, then an analysis using the spatial aggregation propensity method (http://pubs.acs.org/doi/abs/10.1021/jp911706q?journalCode=jpcbfk) may reveal interesting sites of aggregation. This method is a little bit more forgiving that the shape complementarity one because of the intrinsic averaging that goes on to determine the site of aggregation. All of these methods and other simulations tools are available in the Discovery Studio suite from Accelrys. Disclaimer: I work for Accelrys as their Product Manager for the Life Science Modeling and Simulations suite of products. So, if you're interested in evaluating and gain access to these tools please contact me directly. Kind regards, Francisco Sr. Product Manager http://accelrys.com From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dr. Lorenzo Finci Sent: Thursday, August 02, 2012 6:07 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein-Protein Interactions Dear Colleagues, I have a question for all of you bioinformatics oriented structural biologists: How do I predict the sites of protein-protein interactions between two receptors that have been proven to interact biochemically but lack specific details regarding proximity. This is not a straightforward question for me, and I believe it is somewhat complicated. The complicated scenario involves a multitude of different subunits and isoforms. Also, there is not structural data to support all components involved, and thus I presume I should use the sequence based software. I am aware that there are different types of prediction software, either sequence or structure based predictions using different algorithms: http://rosettadesigngroup.com/blog/58/10-protein-protein-interface-prediction-servers/ Receptor 1: -Has 5 predicted subunits (Alpha)2-(Beta)2-(Gamma)1 1. Alpha (6 isoforms) 2. Beta (3 Isoforms) 3. Gamma (3 Isoforms) Receptor 2: -Is believed to be composed of (Alpha)3-(Beta)2 1. Alpha (4 isoforms) 2. Beta(1 isoform) Any advice or recommendation will be well appreciated! Sincerely, lorenzo Lorenzo Ihsan FInci, Ph.D. Postdoctoral Scientist, Wang Laboratory Harvard Medical School Dana-Farber Cancer Institute Boston, MA Peking University The College of Life Sciences Beijing, China
Re: [ccp4bb] Enhancing Crystal Quality
Hi Herman and other CCP3BBers, Thanks for your suggestions. I didn't see any cracks in the crystal drops initially. I will certainly try to shot crystals under room temperature and see what happens. Does the plastic loops fit into the cryo stands Molecular Dimension sells? LUcas On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote: Hi Lucas, The funky diffraction pattern is most likely due to a cracked crystal, resulting in a mixture of slightly differently aligned diffraction patterns. Were the cracks there before you added the cryprotectant? If not, the cryoprotectant is definitively to blame. As has mentioned before, you have to take a shot at room temperature without any cryoprotectant added, to make sure the bad quality is not due to the cryoprotectant. Mitegen sells plastic capillaries, which you can slide over your loop to prevent the crystal from drying out. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yi-Liang Liu Sent: Thursday, August 02, 2012 4:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Enhancing Crystal Quality Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some patterns (cracks (?)) on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote: Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
Re: [ccp4bb] Enhancing Crystal Quality
Mitegen makes a nice little product that is a plastic tube that will slide over one of their magnetic cap/loops. If you put some well solution in the tube and seal the base with apiezon, you can collect quite a bit of data on the loop mounted crystal before it dries out. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu yiliang...@gmail.com wrote: Hi Herman and other CCP3BBers, Thanks for your suggestions. I didn't see any cracks in the crystal drops initially. I will certainly try to shot crystals under room temperature and see what happens. Does the plastic loops fit into the cryo stands Molecular Dimension sells? LUcas On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote: Hi Lucas, The funky diffraction pattern is most likely due to a cracked crystal, resulting in a mixture of slightly differently aligned diffraction patterns. Were the cracks there before you added the cryprotectant? If not, the cryoprotectant is definitively to blame. As has mentioned before, you have to take a shot at room temperature without any cryoprotectant added, to make sure the bad quality is not due to the cryoprotectant. Mitegen sells plastic capillaries, which you can slide over your loop to prevent the crystal from drying out. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yi-Liang Liu Sent: Thursday, August 02, 2012 4:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Enhancing Crystal Quality Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some patterns (cracks (?)) on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote: Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
Re: [ccp4bb] Crystallization Robots
We have a Crystal Gryphon and it works fine for sitting drop plates. We usually set 200+200 nL drops for screens, but it can of course dispense much larger volumes if required. It's affordable and easy to maintain, not much in the way of consumables required. The software is very easy to use, and pretty adaptable to non-standard protocols. I don't have the LCP attachment, but have seen it demonstrated twice. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On Thu, Aug 2, 2012 at 10:04 AM, Sam Jimmeson samjimme...@gmail.com wrote: Hello ccp4BBers, Our lab is thinking about trying to purchase a crystallization robot. We need to be able to do LCP in addition to sitting drop experiments. Does anyone have experience with the NT8 robot from formulatrix? The other two robots I am aware of are the LCP Mosquito from TTP and the LCP Gryphon from ARI. Does anyone have any experiences they could share with me about these instruments? Feel free to email off list if you want. Thanks! -Sam
Re: [ccp4bb] asking for a reference for cacodylate decomposition in protein crystals upon X-ray exposure
I do not have the reference you are seeking, but I have seen cacodylate-containing xtals diffract to better than 1.2 and hold up very well. Also, arsenic has an anomalous signal which may be exploited for phasing, peak ~ 1.04 A. On 07/29/12 18:53, Tatyana Sysoeva wrote: Hi! I heard a couple of times that use of cacodylate buffers in crystallization is bad, and not only because of the compound toxicity. As I understood, presence of the cacodylate in a protein crystal will cause a particular crystal degradation pattern upon X-ray exposure - darkening of the crystals, gas formation I tried to find some references on that and failed in doing so. I found some earlier discussions like this one: http://www.proteincrystallography.org/ccp4bb/message23691.html but don't have anything to reference in literature. I would appreciate if someone can point me to a right direction. I am sorry if this question is out of the groups topic range. Thank you in advance! Sincerely, Tanya -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] MR with Phaser
A few thoughts: 1. Search for all possible space groups (e.g., P2 and P21 in this case). Be happy it isn't C222, which means 8 possible combinations of screw axes to search! As mentioned already, P21 is far more common than P2. I think P21 is one of the most common space groups in protein crystallography. 2. How are you determining how many copies of the search model go in the ASU? It is not necessarily one biological unit, or an integral number of biological units. Run a cell content analysis in Phaser (e.g. Matthews probability calculator) and start there, but consider the results a suggestion only. For larger ASUs, the predicted number is not very accurate. Six might actually be 4 or 8 chains. 3. Look at the crystal packing in Pymol, Coot, or in you favorite tool. You can do this by enabling a large symmetry molecule radius. If you see a regular lattice of proteins with nice solvent channels and protein-protein contacts, things are looking up. (But you can be fooled into a premature victory at times.) 4. Partial Phaser solutions may provide a big hint about how many molecules are in the ASU when packing is examined. Often the placement of the missing molecules is quite obvious, as it completes a solvent channel or fills in symmetrical protein-protein contacts. 5. Finally, look at the maps. Crappy maps probably mean the wrong space group, especially if chains don't pack well. Good maps with good packing usually mean you are on the right track. Cheers and good luck, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/1/2012 2:27 PM, Uma Ratu wrote: Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma
