[ccp4bb] Amazing B-factor
Dear all, Recently we have collected one set of data which is processed to 2.9A and 3.0A and the Wilson-B values are 50.1 and 59.1, respectively. As for the completeness of the highest shell is only 66% in 2.9A (80% in 3.0A), we use the dataset with 3.0A for phasing and refinement. Everything is OK during phasing. Routinely, we use CNS (rigid, minimize and bgroup) for primary refinement. -- Jian Wu Ph.D. Student Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS) Tel: 0086-21-54921117 Email: prote...@gmail.com
Re: [ccp4bb] 3D LCD and glasses
Robert Esnouf wrote: Dear All, I see that the UK is catching up and the NVIDIA Geforce 3D Vision Bundle (Samsung 2233RZ 22 LCD + NVIDIA GeForce 3D Vision Glasses) is now available for ~£350. Can I reveal my ignorance and ask whether (with a suitable graphics card) this kit gives decent 3D with Coot and PyMol? And are there recommendations for appropriate graphics cards? Dear Robert, I can hopefully speak for Bernhard who has implemented the necessaries for Coot: we are testing and so Coot should be able to support this. the next release of ccp4mg will support this kit - I have it working. I believe Pymol has supported this for some time. Best Wishes, Stuart McNicholas
Re: [ccp4bb] coupling between occupancy and b-values in refinement
Dear Patrick Long ago we (Cheetham et a (1992) J Molec Biol 224:613-628) did some refinements on hen lysozyme + substrate complexes at 1.75A and 2A resolution and showed that B and occupancy are negatively correlated, especially at 2.0A. In simplistic terms this is because at medium resolution low density at the atomic position could be the result of either low occupancy or high B factor. We ran parallel refinements using assumed occupancies at 0.1 intervals. We looked at R factors but they werent much help - this was pre-Rfree so the latter might be a valuable criterion now. I think a good strategy is to refine your substrate complex at intervals of o=0.1, 0.2 ... (with a non-complex model at 1-o, ass positions of side chains and waters may well be different) and look at the B factors. At the correct(ish) occupancy substrate Bs will be expected to be similar to those of the ligating residues from the protein. But it does get to be quite subjective until you have higher resolution. best wishes Pete On 31 May 2009, at 16:58, Patrick Loll wrote: Hi all, I'm looking for a reference to bolster my response to a referee, in which I defend my decision not to refine the occupancy of a ligand in structure refined at around 2 A resolution (note the ligand binding slte lies on a two-fold crystallographic axis, so the maximum occupancy is 0.5) I recall reading a paper a LONG time ago (decades) in which someone described some careful refinement experiments, and concluded that the correlation between occupancy and B-value is so strong that it simply makes no sense to independently refine both parameters (at least for light atoms, and in the absence of super high resolution data). Alas, all that I recall is this take-home message. I have no idea of where the paper appeared, or the names of the authors (or indeed, if I'm even remembering the paper's message correctly). I've tried trolling through Acta, without success. Does anyone have a better idea of where I might find this paper, or one espousing a similar message? Thanks, Pat - Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexel.edu
[ccp4bb] Postdoc ad: PX at SGC (U of Oxford)
Hi I am recruiting for a postdoc position in my group, available immediately. This is the Protein Crystallography group of the Structural Genomics Consortium, Oxford. In particular, hard-core crystallographers strong on theory and (optionally) with a bent for programming, are invited to apply. For details, please see: http://www.sgc.ox.ac.uk/jobs/H909009.html, or contact me for further info. (If you miss the deadline, don't despair, send your application anyway, directly to me.) The remit of the Structural Genomics Consortium (SGC) is to solve human proteins of medical relevance and place them in the public domain without restrictions; it is funded by a consortium of public and industrial funders, and consists of independently operating departments in the Universities of Oxford and Toronto, and Karolinska Institutet (Stockholm). The Oxford site has solved almost 300 such structures in the last five years, and is now halfway through Phase II which is funded till June 2011; this phase has an increased emphasis on chemical biology and membrane proteins. (http://www.sgc.ox.ac.uk) The Protein Crystallography group collaborates tightly with the 5 other groups to get their purified proteins crystallized and solved (five per month). Additionally, our research revolves around methods development, for which we're ideally positioned thanks to accumulated historic data, extensive automation equipment, close links to vendors, and especially, access to the many proteins of high biological relevance. The current emphasis is on methodology for rapidly and systematically generating co-crystal structures, by expanding on the high-throughput approaches that underpin our success with novel targets; this is of high relevance to the the SGC's exploration of open source chemical biology. Frank -- Dr Frank von Delft Principal Investigator: Protein Crystallography Structural Genomics Consortium Oxford University +44 1865 617583
Re: [ccp4bb] coupling between occupancy and b-values in refinement
Hi Pat I concur with George, we routinely refine together the group B factor and occupancy of our ligands and frequently see significant deviations of the group occupancy from the starting value even if it is highly correlated with the group B factor (the significance test takes account of the correlation), sometimes at 2.8 Ang, but rather easily detectable at 2 Ang. This often eliminates negative difference density, particularly at the heavier atoms such as S, Cl, Br (of course this could also be explicable by inducement of disorder by radiation damage as opposed to the ligand binding with partial occupancy on soaking or co-crystallisation). Whenever I see a significant difference (say 10 Ang.^2) between the average B factor of the ligand and the average B factor of the protein atoms in the binding site I suspect that partial occupancy of the ligand is the true explanation. There is in fact no reason why the occupancy and B factor should be completely correlated, unless of course the data errors are large and/or the resolution is very low and/or the group only makes a small contribution to the total scattering, in which the errors will dominate and the results will not be significant. This is because increasing the B factor of an atom or group of course makes the density broader and lower but does not change the integral of the density (i.e. the number of electrons scattering), whereas changing the occupancy clearly does change the number of electrons (in fact proportionally). Hence the effects of changing the B factor and occupancy are quite different. Cheers -- Ian PS I wasn't your referee either! -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of George M. Sheldrick Sent: 31 May 2009 17:20 To: Patrick Loll Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] coupling between occupancy and b-values in refinement Dear Pat, You don't say how large your ligand is, but if the occupancy is refined as a single parameter so that all the atoms in the ligand are constrained to have the same occupancy, it should be rather well-defined and not highly correlated with the B-values. By the way, I was not your referee! Best wishes, George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Sun, 31 May 2009, Patrick Loll wrote: Hi all, I'm looking for a reference to bolster my response to a referee, in which I defend my decision not to refine the occupancy of a ligand in structure refined at around 2 A resolution (note the ligand binding slte lies on a two-fold crystallographic axis, so the maximum occupancy is 0.5) I recall reading a paper a LONG time ago (decades) in which someone described some careful refinement experiments, and concluded that the correlation between occupancy and B-value is so strong that it simply makes no sense to independently refine both parameters (at least for light atoms, and in the absence of super high resolution data). Alas, all that I recall is this take-home message. I have no idea of where the paper appeared, or the names of the authors (or indeed, if I'm even remembering the paper's message correctly). I've tried trolling through Acta, without success. Does anyone have a better idea of where I might find this paper, or one espousing a similar message? Thanks, Pat - Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexel.edu Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of
Re: [ccp4bb] 3D LCD and glasses
Stuart, Robert, Others, I believe Stuart is confusing the Samsumg 2233rz frame-sequential stereo 3D display with the Zalman M220W interlaced stereo 3D solution. Due to lack of driver support, as far as I know, the Samsung isn't yet supported by any OpenGL-based software, although nVidia has indicated on its web site that 3D Vision driver support is to be expected soon (at least for Windows). PyMOL and other products (VMD, Chimera, ...) do already support the Zalman display without need of any special graphics card. Cheers, Warren -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Stuart McNicholas Sent: Monday, June 01, 2009 5:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] 3D LCD and glasses Robert Esnouf wrote: Dear All, I see that the UK is catching up and the NVIDIA Geforce 3D Vision Bundle (Samsung 2233RZ 22 LCD + NVIDIA GeForce 3D Vision Glasses) is now available for ~£350. Can I reveal my ignorance and ask whether (with a suitable graphics card) this kit gives decent 3D with Coot and PyMol? And are there recommendations for appropriate graphics cards? Dear Robert, I can hopefully speak for Bernhard who has implemented the necessaries for Coot: we are testing and so Coot should be able to support this. the next release of ccp4mg will support this kit - I have it working. I believe Pymol has supported this for some time. Best Wishes, Stuart McNicholas
[ccp4bb] Amazing B-factor
Dear all, Recently we have collected one set of data which is processed to 2.9A and 3.0A and the Wilson-B values are 50.1 and 59.1, respectively. As for the completeness of the highest shell is only 66% in 2.9A (80% in 3.0A), we use the dataset with 3.0A for phasing and refinement. Everything is OK during phasing. Routinely, we use CNS (rigid, minimize and bgroup) for primary refinement and the R and freeR values go to 31.1 and 34.1. Surprisingly, the B-factor is 137.2. I have modified the B-factor to a fixed value (59.1 A^2) in the searching model coordinate file, however it changed to 127.6 after 'rigid' and subsequently to 131.9 after 'minimize'. In all the scripts above I used the default option anisotrpic at overall B-factor correction. I don't know whether it is right to use this option at low resolution, so I have tried the other two options no and isotropic. In the 1st choice, the B-factor is seemingly reasonable (55~59) but the R and freeR are very high (45.9 and 51.9). In the 2nd choice, the B-factor, R and freeR values are between those of the other two options (111.3, 40.6, and 45.3). I want to know what cause these surprising changes especially for B-factor value. Any suggestion is appreciated. Jian Wu -- Jian Wu Ph.D. Student Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS) Tel: 0086-21-54921117 Email: prote...@gmail.com
Re: [ccp4bb] 3D LCD and glasses
Warren DeLano wrote: Stuart, Robert, Others, I believe Stuart is confusing the Samsumg 2233rz frame-sequential stereo 3D display with the Zalman M220W interlaced stereo 3D solution. Due to lack of driver support, as far as I know, the Samsung isn't yet supported by any OpenGL-based software, although nVidia has indicated on its web site that 3D Vision driver support is to be expected soon (at least for Windows). PyMOL and other products (VMD, Chimera, ...) do already support the Zalman display without need of any special graphics card. Cheers, Warren My apologies. Warren is correct. I had just answered another email on the Zalman display and had my wires well and truly crossed. Sorry.
[ccp4bb] Request recommendation for the peptide synthesis company
Dear All, I would appreciate if anyone could recommend any company for long peptide synthesis? It will be better if it is reliable and reasonable priced. The peptide we want to synthesis is around 40-50aa long, should be HPLC grade and above 98% purity. Thank you! Ping Sun National Cancer Institute at Frederick Frederick, MD 21702
Re: [ccp4bb] BioRad CFX96 for thermofluor/DSF?
Use the FRET channel - it's ideal for SYPRO orange. Artem Sorry Charlie for the late reply. I'm using the CFX96 5 channel with Sypro and other dyes. I tested the machine against the Eppendorf product nd decided to go for the Biorad version as running identical samples gave better results on the Biorad. This could be a machine specific problem but on a 96 well plate the detection level was ~1.5x better, you can use the HEX channel for detection. Jürgen On 29 Apr 2009, at 03:06, Charlie Bond wrote: Hi Folks, Has anyone here had any success running a thermofluor (differential scanning fluorimetry) experiment on a BioRad CFX96 with SYPRO orange as the fluorophore? Our local 'technical expert' tells us it should work and gave us some settings to test, but was very sketchy about any detail, and in a test we ran we got some strange results (every well that contains SYPRO orange - even those with no protein - have a peak in fluorescence at 58oC). Before we tinker, or go and persuade a friendly molbiol lab to let us play with the filters in their iq5, I thought I'd see if anyone here had any advice. I'll summarise any responses I get. Cheers, Charlie -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia charles.b...@uwa.edu.au +61 8 6488 4406 - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.me.com/bosch_lab/
Re: [ccp4bb] Request recommendation for the peptide synthesis company
ping, we had great success as far as price and level of synthesis with a company called Synbiosci, Livermore, CA. I am no way connected to this company but just a suggestion. hope it helps psp On Mon, Jun 1, 2009 at 10:31 AM, ping sun ccp4@gmail.com wrote: Dear All, I would appreciate if anyone could recommend any company for long peptide synthesis? It will be better if it is reliable and reasonable priced. The peptide we want to synthesis is around 40-50aa long, should be HPLC grade and above 98% purity. Thank you! Ping Sun National Cancer Institute at Frederick Frederick, MD 21702 -- Pius S Padayatti Scientist, Polgenix, Inc. 11000 Cedar Ave, Suite 260 Cleveland, OH 44106 Phone: 216-658-4528 Fax: 216-658-4529
Re: [ccp4bb] coupling between occupancy and b-values in refinement
Hi, I have to take issue with one of Ian's points. His statement that the integral of the electron density does not change with the change in a B factor but does change with a change in occupancy is the equivalent to stating that the integral of an occupancy difference map feature is nonzero while the integral of a B factor difference map feature is zero. These conditions only occur when all of the Fourier coefficients are used to calculate the difference map, which only truly occurs with a very high resolution map, and one that includes the F000 reflection. Refinements that occur in practice only approximate these conditions and this approximation results in the oft observed difficulty in identifying a difference map feature as either due to an error in occupancy or B factor. The problem gets worst as the resolution of the map decreases and the series termination effects become larger. To overcome this difficulty of interpreting the difference map feature one needs some other source of information, as others in this thread have suggested. The classics are assuming that the B factors of the mystery group are similar to the surrounding atoms that are presumed to be at full occupancy, or assuming that the occupancy of a significantly large group of atoms are equal to each other. Each of these assumptions have been used in real refinements to help solve this problem. Each are made more difficult if atoms in question are sitting atop atoms of the alternative structure. When your compound is not present something else probably is (water most likely) and if you don't have a model for the alternative structure you will end up overestimating the occupancy of your compound. In your particular case, your molecule superimposes upon its symmetry image. This situation introduces additional correlations due to the apparently superimposed atoms of the two images, including correlations with the positional parameters. You will have to be very careful to enforce strict stereochemical restraints, more strict than you would for the rest of the model. In addition you will likely have to run more cycles of refinement to achieve convergence than you normally expect. With your resolution, an assumption of a group occupancy, strict geometry restraints, and selecting the occupancy that gives the B factors you want, you will come up with a number for the occupancy of your compound. Verifying the accuracy of that number will be difficult. I predict that the R value and R free will not change by a significant amount. (That is the definition of a correlation between two parameters, that you can change them in compensating ways and not affect the fit to your data.) Your best hope would be that the new difference map will show you the water molecules that occupy this site when your compound does not. Failing that you will have an occupancy for your group but no error bars for that value. Dale Tronrud Ian Tickle wrote: Hi Pat I concur with George, we routinely refine together the group B factor and occupancy of our ligands and frequently see significant deviations of the group occupancy from the starting value even if it is highly correlated with the group B factor (the significance test takes account of the correlation), sometimes at 2.8 Ang, but rather easily detectable at 2 Ang. This often eliminates negative difference density, particularly at the heavier atoms such as S, Cl, Br (of course this could also be explicable by inducement of disorder by radiation damage as opposed to the ligand binding with partial occupancy on soaking or co-crystallisation). Whenever I see a significant difference (say 10 Ang.^2) between the average B factor of the ligand and the average B factor of the protein atoms in the binding site I suspect that partial occupancy of the ligand is the true explanation. There is in fact no reason why the occupancy and B factor should be completely correlated, unless of course the data errors are large and/or the resolution is very low and/or the group only makes a small contribution to the total scattering, in which the errors will dominate and the results will not be significant. This is because increasing the B factor of an atom or group of course makes the density broader and lower but does not change the integral of the density (i.e. the number of electrons scattering), whereas changing the occupancy clearly does change the number of electrons (in fact proportionally). Hence the effects of changing the B factor and occupancy are quite different. Cheers -- Ian PS I wasn't your referee either! -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of George M. Sheldrick Sent: 31 May 2009 17:20 To: Patrick Loll Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] coupling between occupancy and b-values in refinement Dear Pat, You don't say how large your ligand is, but if the
Re: [ccp4bb] Amazing B-factor
Dear Jain, The Wilson plot is based on the assumption that you can model your structure with a collection of atoms randomly distributed in the unit cell. This model is very poor at low resolution, say, 4.5A and lower. Since the Wilson B depends on the change in scattering as a function of resolution, a Wilson B calculated for 3A data will be very unreliable, being based only on the data from 4.5A to 3A. Once you begin refinement you have a proper model of your crystal and all the low resolution data can be used to estimate the average B. At this point the Wilson B should be ignored, it was only a stepping stone to get you here. A Wilson B of 50 to 60A^2 for a 3A data set collected on frozen crystals at a synchrotron (I'm making an assumption here.) is quite surprising. 137A^2 seems much more likely to me. I'd trust CNS's results. The overall anisotropic correction should be fine at this resolution too. Dale Tronrud Jian Wu wrote: Dear all, Recently we have collected one set of data which is processed to 2.9A and 3.0A and the Wilson-B values are 50.1 and 59.1, respectively. As for the completeness of the highest shell is only 66% in 2.9A (80% in 3.0A), we use the dataset with 3.0A for phasing and refinement. Everything is OK during phasing. Routinely, we use CNS (rigid, minimize and bgroup) for primary refinement and the R and freeR values go to 31.1 and 34.1. Surprisingly, the B-factor is 137.2. I have modified the B-factor to a fixed value (59.1 A^2) in the searching model coordinate file, however it changed to 127.6 after 'rigid' and subsequently to 131.9 after 'minimize'. In all the scripts above I used the default option anisotrpic at overall B-factor correction. I don't know whether it is right to use this option at low resolution, so I have tried the other two options no and isotropic. In the 1st choice, the B-factor is seemingly reasonable (55~59) but the R and freeR are very high (45.9 and 51.9). In the 2nd choice, the B-factor, R and freeR values are between those of the other two options (111.3, 40.6, and 45.3). I want to know what cause these surprising changes especially for B-factor value. Any suggestion is appreciated. Jian Wu -- Jian Wu Ph.D. Student Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences (CAS) Tel: 0086-21-54921117 Email: prote...@gmail.com mailto:prote...@gmail.com
Re: [ccp4bb] BioRad CFX96 for thermofluor/DSF?
Hi, I've used the CFX96 with the FRET channel for DSF and found it to work well. Gary -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of ar...@xtals.org Sent: Monday, June 01, 2009 11:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] BioRad CFX96 for thermofluor/DSF? Use the FRET channel - it's ideal for SYPRO orange. Artem Sorry Charlie for the late reply. I'm using the CFX96 5 channel with Sypro and other dyes. I tested the machine against the Eppendorf product nd decided to go for the Biorad version as running identical samples gave better results on the Biorad. This could be a machine specific problem but on a 96 well plate the detection level was ~1.5x better, you can use the HEX channel for detection. Jürgen On 29 Apr 2009, at 03:06, Charlie Bond wrote: Hi Folks, Has anyone here had any success running a thermofluor (differential scanning fluorimetry) experiment on a BioRad CFX96 with SYPRO orange as the fluorophore? Our local 'technical expert' tells us it should work and gave us some settings to test, but was very sketchy about any detail, and in a test we ran we got some strange results (every well that contains SYPRO orange - even those with no protein - have a peak in fluorescence at 58oC). Before we tinker, or go and persuade a friendly molbiol lab to let us play with the filters in their iq5, I thought I'd see if anyone here had any advice. I'll summarise any responses I get. Cheers, Charlie -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310 35 Stirling Highway Crawley WA 6009 Australia charles.b...@uwa.edu.au +61 8 6488 4406 - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.me.com/bosch_lab/
[ccp4bb] Position available in Frankfurt
A PhD and a Postdoctoral position are immediately available in the Ribosome Crystallography Group led by Prof. Dr. Paola Fucini, in the Cluster of Excellence for Macromolecular Complexes and Johann Wolfgang Goethe University. The candidates will participate in the ongoing research of the group, which aims to elucidate the role played by the ribosome and ancillary factors in regulating the translational apparatus. Working in a multidisciplinary and collaborative research environment, the candidates will have the opportunity to use state-of-the-art research facilities and techniques in Molecular Biology, Protein Chemistry, X- ray Crystallography, Cryo electron microscopy and Nuclear Magnetic Resonance. The main aim of the research projects will be the investigation of the structure and biological function of macromolecular complexes composed by the ribosome, nascent chain, translational factors, chaperones or antibiotics. The final goal of the research is to further our understanding in the vital process of the translation of the genetic code into functional proteins and its regulation by natural factors or de-novo designed translational inhibitors. The successful candidates (in particular for the postdoctoral position) should have a strong interest – and possibly experience - in the field of Structural Biology and have demonstrated the ability to conduct high quality research. Prospective PhD students should have an undergraduate degree in related field and possess good laboratory skills. Applicants should send a brief letter of motivation, curriculum vitae, statement of research accomplishments and interests, and the names of three referees, preferably by e-mail to fuc...@chemie.uni-frankfurt.de or by post to: Prof. Dr. Paola Fucini Cluster of Excellence for Macromolecular Complexes J.W. Goethe University Frankfurt am Main Max-von-Laue-Strasse 7 D-60438 Frankfurt am Main, Germany. Applications will be accepted until the positions are filled. Further information about the Group and the Research Centre can be found at: http://www.cef-mc.de/, http://www.uni-frankfurt.de/english/, http://user.uni-frankfurt.de/~joharms/akfucini/ The J.W. Goethe University is an equal opportunity employer, and particularly encourages female applicants. It also tries to increase the proportion of scientists with physical disabilities. Respective applications are welcome. Tatsuya KAMINISHI, Ph.D. Fucini Laboratory Institute for Organic Chemistry and Chemical Biology Johann Wolfgang Goethe University Max-von-Laue-Strasse 7, N160/B413 D-60438 Frankfurt am Main, Germany Tel: +49 69 798-29165 Fax: +49 69 798-29268 Web: http://user.uni-frankfurt.de/~joharms/akfucini/
Re: [ccp4bb] Seeding
Dear HengChiat Tai (a) According to me Seed Solutions should be stored depending on the stability of the crystals in the drop. If your crystal goes bad quite early then you have to make fresh seed frequently. Its always a good habit to make fresh seed, everytime you do streaking. (b) The number of drops which you can streak after dipping the whisker in seed solution once ! Depends on your swiftness. There are robots which can streak 12 drops continuously without the rquirement of washing in between. I can streak upto 6 drops continuously with a gap of less than a second. But it's always good to wash the whisker once in a while with water. If your crystallization condition has salts like Ammonium sulfate in it, then, the salt crystallizes quite fast on the whisker, as it is exposed to air for some time and you may land up seeding salt crystals in to your drop. (c)The inconsistency, which, you have been seeing in the crystal growth is quite common with streaking. Since it depends on how many micronuclei or seeds get in to the drop. Some times only one seed goes into the drop and you get a better quality single crystal in the entire drop. If two or three seeds go into the drop during streaking the effective protein concentation gets distribued between the seeds and as a result you have small and numerous crystals. The only solution to this can beStreak as many drops as possible . Your protocol for seed preparation looks fine. I think you can minimize the vortex steps. If possible vortex or mix it well with pipette. Choose those ends of the crystal for making seed solution which seem to be growing. It is like inoculating the Fresh media with bacteria which are in their early log phase of growth. Work with higher dilutions like, 100X and so on. Set up and streak as many drops as possible. All the best Regards Vikas Navratna -- Hotmail® goes with you. Get it on your BlackBerry or iPhone.http://windowslive.com/Tutorial/Hotmail/Mobile?ocid=TXT_TAGLM_WL_HM_Tutorial_Mobile1_052009
