Re: [ccp4bb] I/sigma continued
I agree absolutely with James - be as succinct as you like in a table but include the verbose definition for each entry in the log file - or at the very least in the manual. It should be easy to search for with the table tag. People will not go and read a reference.. Eleanor James Holton wrote: I think the best way to deal with issues like this can be found in Strunk White The Elements of Style (1918). Among other things, these authors put forward a rather simple yet often overlooked rule to writing in general, which I think applies equally well to computer programs: Be clear. The sentence itself is an example of how brevity need not sacrifice clarity. Yes, you need labels in the table itself to be short, but there is space immediately below (and above) every table that (IMHO) ought to contain the definitions of each and every variable/abbreviation used in the table, spelled out no matter how obvious it may seem to the author. I can tell you many long and painful stories about me trying to figure out what some variable in some equation in some paper actually meant! Context is everything. If you are tight for space, cite a reference (such as the manual). That, and scientists talking about such quantities in email, papers, etc. (such as myself) should also heed Strunk and White and also not just assume that everyone knows exactly what structure factor means as opposed to a structure amplitude, let alone I/sigma. Indeed, the word intensity is an incredibly ill-defined unit all by itself, to the point of being useless. It can have units of photons, photons/s, photons/area/s, photons/area, energy/volume, and many many more. Often even in the same equation! I would strongly advise against changing the variable names printed out in log files by SCALA and other programs, especially when a given name has persisted for a decade or more. Adding an inline definition is fine, but changing names not only breaks programs that were written to read these logs (and sometimes even humans reading the log), but it also confines the meaning of I/SIGMA from SCALA to a particular period in history. So, what statistic do we want to look at? That depends on what you are trying to do with the data. There is no way for Phil to know this, so it is good that he prints out lots of different statistics. That said, when talking about the data quality requirements for structure solution by MAD/SAD, I suggest looking at I/sigma(I) where: I - merged intensity (proportional to photons) assigned to a reciprocal lattice point (hkl index) sigma(I) - the error assigned to I Exactly what I/sigma(I) is required to solve a structure, or make some conclusion about a solved structure is a topic for another day. -James Holton MAD Scientist Phil Evans wrote: “I/sigma” statistics seem to be contentious confusing (see recent discussions on CCP4BB), particularly in what the various measures should be called (and how they should be labelled in a table, where there is only room for a very short name). I thought it worth commenting on this issue at a little more length. There are several interacting issues: 1) Statistics can be calculated either for individual observations Ihl or for intensities averaged over multiple (symmetry-related or replicate) measurements Ih(avg): both are useful, but they need to be distinguished 2) The statistic can be (a) the ratio of means I/sigma or (b) the mean of ratios I/sigma . These are not the same. 3)The “sigma” used in 2(a) can be either (a) the estimated corrected SD or (b) the RMS scatter of observations ie the RMS deviation (which is itself generally used to estimate a “correction” to the SD). The RMS scatter cannot be used for 2(b) of course, since that needs individual sigmas for each reflection. 4) Values will depend on how many outliers have been rejected. For what it’s worth, Scala outputs two such statistics:- (i) “I/sigma”: this is calculated for individual observations Ihl and is the (mean intensity Ihl)/(RMS scatter of Ihl). RMS scatter = RMS [Ihl – Ih(avg)]. This is some measure of the average significance of individual observations, but does not take into account multiplicity. In my new program under development (a Scala replacement) I have relabelled this column “I/RMS” but I don’t really know what best to call it. This value is a ratio of means (see 2(a) above). (ii) “Mn(I/sd)”: this is the mean value of (Ih(avg)/sd(Ih(avg))), where Ih(avg) is the (weighted) average over all observations for reflection h, and sd(Ih(avg)) is the estimated SD of this average, after any “corrections” have been applied. This is, I think, the best estimate of “signal-to-noise ratio”, but does depend on realistic estimates of sd(Ih(avg)), which is not entirely straightforward (and certainly doesn’t allow for systematic errors!). This value is a mean of ratios (see 2(b) above). The “corrected” sd(Ihl) is calculated
[ccp4bb] failed compilation for Pointless
I am trying compile Pointless from source as this is the only CCP4 bit which hasn't compiled on my x86_64 machine (linux Ubuntu 8). I cd into src/pointless and then try to run configure.sh. This stops at a point where it complains that it can't find ccp4_errno.h. That seems odd, as I have declared all CCP4 environment variables, and could check that the file is actually where it should be. Anyone could help me with this? J Dr James Foadi PhD Membrane Protein Laboratory Diamond Light Source Ltd. Diamond House Harwell Science and Innovation Campus Didcot Oxfordshire OX11 0DE United Kingdom office email: james.fo...@diamond.ac.uk alternative email: j.fo...@imperial.ac.uk
Re: [ccp4bb] RESUME: long term data backup
Dear All, I wonder how people currently do their long term backups. I see DATs/DLTs being slowly dropped off at the beamlines and most people brings their data home in external HDs. Anyone using blue-ray or double layer DVDs for long term backups? If so what kind of hardware? Do you use HDs for long term storage? If so, do you do a second copy and how do you store them? I will try to compile the answers and relay back to the list a resume. -- David Aragao (our own setup): We currently have online NAS server (QNAP 209 Pro - http://www.qnap.com/pro_detail_feature.asp?p_id=93) with 2x hotswappable 1 Tb disks. The system allows ftp, nfs, samba over the network and also to directly connect our USB2 transport HD. The drawback is that the system is very very slow (4h-6h to transfer 150 Gb) and has crashed a few times needing reboot. We are not using any of the RAID options on the QNAP since we use 1 Tb for x-ray diffraction data (latest trips) and 1 Tb for automatic office/windoze backups over the network. We keep an extra 1 Tb HD for failures. Then we have been using an extra external USB2 750 Gb HDs for second offline copy of the data. One of the reasons that triggered my question was that we cannot rely on single HD type for backup. Unfortunately our QNAP has exactly this hardisk: http://www.theinquirer.net/inquirer/news/374/1050374/seagate-barracudas-7200-11-failing -- Graeme Winter: I have quite a lot of data, as you know, and I have a three-phase way of handling data... I keep the data on hard drive on computers as far as possible, primary backup of everything to firewire with secondary backup to DVD as bzip2'd images. This way if I lose something I can fetch the data from firewire (or process from there if I run out of space) then if one of those fails (and they do) I can recover the data from DVD. Overall I have a few TB of data kept in this way. Redundancy is good, as is orthogonality i.e. DVD and Firewire, not 2x firewire disk or 2x DVD. Follow up: Ok, so I don't accumulate data at this kind of rate, so I write the DVD's manually. I know at Brookhaven they have a DVD robot, which would probably do what you want Otherwise probably the two HD backups is probably actually the best you are going to get. -- Roger Rowlett: We keep our backups on hard drives of two servers (master and backup) in separate locations on campus. Some data is kept on CD-ROM, but we're doing that less now. -- Stephen Graham: If at all possible you should consider outsourcing it. You might have access to some kind of large university of national facility for archiving scientific/academic 'data'. Otherwise there are companies who specialise in archiving data - for a fee they will take the problem out of your hands (and you don't need to worry about what format to use, what to do once the media you currently use become scarce, etc). Either way, we should all lobby the PDB or someone to archive all the images for us pronto! -- Kay Diederichs: Burning DVDs must be a nightmare, and recovering from DVD failure even more so. Whenever I burn a DVD with important data I also create a CD with the ECC data (see http://www.dvdisaster.de). I have my synchrotron data since 1999 online on harddisk (_all_ our data, not only those datasets that gave structures). Disks are cheap and convenient. Whenever we start to be short on disk space, I go shopping for bigger disks. The hardware currently is an eSATA 4 TB RAID5 in a €340,- RaidSonic Stardom ST6600-5S-S2 5-disk case (http://www.raidsonic.de/de/pages/search/search_list.php?we_objectID=4239pid=0). A terabyte disk now is less than €100, so the whole thing costs €800,- . RAID5 guards against single-disk failures, and I keep a spare terabyte disk in case I have to exchange one of the five internal ones. The unit is hooked up to a Linux machine with a recent kernel (which supports the SATA port multipier feature) and a eSATA adapter (e.g. Adaptec 1225SA). We have two of those in different buildings, and I do a daily (rsync) copy of the master to the backup. I'm running this for over a year, and am happy with it. -- Patrick Loll: We're currently using normal DVD-Rs. I don't know how robust these will prove to be in the long term, but for right now it's cheap and easy and requires no fancy hardware. -- Wladek Minor: Hard drive. 1TB cost around $120. 1.5TB are not as reliable yet. Plus Thermaltake BlacX ST0005U - storage enclosure
[ccp4bb] Protein Expression Group Leader Position
Associate Director Protein Expression Group, Novartis Institutes for Biomedical Research, Basel, Switzerland Job description: As Group Leader in the Structural Biology Platform (SBP) within the Center for Proteomic Chemistry you will lead a group dedicated to all aspects of protein expression including molecular biology and expression in insect cells and bacteria. You and the members of your group will work in a multidisciplinary environment to contribute to the discovery of new drugs. Your responsibility is to manage group resources in accordance with Platform priorities, to create and maintain an innovative, efficient, competitive and result-oriented research environment with high scientific standards, to develop and coach associates, and to recruit new talent. As a member of the scientific leadership team you will contribute to the overall strategy and priorities of the Platform. In addition, you will represent the Platform in protein production related matters. Minimum requirements: We are looking for a highly talented and motivated scientific leader with a PhD in biology or biochemistry, significant postdoctoral experience in an academic setting and more than 5 years of professional experience in the pharmaceutical or biotech industry. The ideal candidate will have a strong background in molecular biology and protein expression and should have a sound knowledge in drug discovery and biochemistry. Experience of working in the field of structural biology is an advantage. He/she should have excellent leadership skills and a passion for working in a professional, multi-disciplinary, team-oriented and science-driven environment. Outstanding oral and written communication skills are required as well as a high level of creativity and commitment. Applications must be submitted on the Novartis web site ( http://www.novartis.com/ under careers, Job ID 50681BR).
Re: [ccp4bb] failed compilation for Pointless
Ii is commented out in mine as well. But even deleting the # does not help In fact this is the full log from configuration: . . . checking for a BSD-compatible install... /usr/bin/install -c checking whether build environment is sane... yes checking for gawk... gawk checking whether make sets $(MAKE)... yes checking whether to enable maintainer-specific portions of Makefiles... no checking for gfortran... gfortran checking for Fortran compiler default output file name... a.out checking whether the Fortran compiler works... yes checking whether we are cross compiling... no checking for suffix of executables... checking for suffix of object files... o checking whether we are using the GNU Fortran compiler... yes checking whether gfortran accepts -g... yes checking how to get verbose linking output from gfortran... -v checking for Fortran libraries of gfortran... -L/usr/lib/gcc/x86_64-linux-gnu/4.2.4 -L/usr/lib/gcc/x86_64-linux-gnu/4.2.4/../../../../lib -L/lib/../lib -L/usr/lib/../lib -L/usr/lib/gcc/x86_64-linux-gnu/4.2.4/../../.. -lgfortranbegin -lgfortran -lm checking for xlc++... no checking for CC... no checking for cxx... no checking for c++... c++ checking whether we are using the GNU C++ compiler yes checking whether c++ accepts -g... yes checking for style of include used by make... GNU checking dependency style of c++... gcc3 checking how to run the C++ preprocessor... c++ -E checking for grep that handles long lines and -e... /bin/grep checking for egrep... /bin/grep -E checking for ANSI C header files... yes checking for a BSD-compatible install... /usr/bin/install -c checking for ccp4_errno in CCP4... no configure: error: Failed to find ccp4 libs . . . J Dr James Foadi PhD Membrane Protein Laboratory Diamond Light Source Ltd. Diamond House Harwell Science and Innovation Campus Didcot Oxfordshire OX11 0DE United Kingdom office email: james.fo...@diamond.ac.uk alternative email: j.fo...@imperial.ac.uk web page: - Original Message From: James M. Vergis jver...@virginia.edu To: James Foadi james_fo...@yahoo.co.uk Sent: Tuesday, 31 March, 2009 14:50:08 Subject: RE: [ccp4bb] failed compilation for Pointless James, I checked my pointless configure file from my install (CentOS 5 64-bit machine) and the include ccp4_errno.h line is commented out in my configure file. I did not modify the file, that’s how it came. Maybe that will solve your problem. Good luck! James M. Vergis, Ph.D. University of Virginia Molecular Physiology and Biological Physics KVWEINR 360A Snyder Building 480 Ray C. Hunt Drive Charlottesville, VA 22908-0886 phone: 434-243-2730 FAX: 434-243-8271 jver...@virginia.edu -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James Foadi Sent: Tuesday, March 31, 2009 6:50 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] failed compilation for Pointless I am trying compile Pointless from source as this is the only CCP4 bit which hasn't compiled on my x86_64 machine (linux Ubuntu 8). I cd into src/pointless and then try to run configure.sh. This stops at a point where it complains that it can't find ccp4_errno.h. That seems odd, as I have declared all CCP4 environment variables, and could check that the file is actually where it should be. Anyone could help me with this? J Dr James Foadi PhD Membrane Protein Laboratory Diamond Light Source Ltd. Diamond House Harwell Science and Innovation Campus Didcot Oxfordshire OX11 0DE United Kingdom office email: james.fo...@diamond.ac.uk alternative email: j.fo...@imperial.ac.uk
[ccp4bb] how to present the electron density map
Hi,everyone, I want to present the ommit density map on my structure, It will be great if someone can suggest the software and a brief instruction on how to use it. _ More than messages–check out the rest of the Windows Live™. http://www.microsoft.com/windows/windowslive/
Re: [ccp4bb] how to present the electron density map
You can transfer the omit map from cns format to ccp4 or o map using mapman . Then you can load and show it in pymol. Good luck! liu xu zhen wrote: Hi,everyone, I want to present the ommit density map on my structure, It will be great if someone can suggest the software and a brief instruction on how to use it. check out the rest of the Windows Live™. More than mail–Windows Live™ goes way beyond your inbox. More than messages http://www.microsoft.com/windows/windowslive/
[ccp4bb] Halide soaking
Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http://hk.promo.yahoo.com/security/ 了解更多!
Re: [ccp4bb] Halide soaking
Hi! Normally the cell parameters, etc change very very little. You'll only know if the bromides got in at the synchrotron by looking at the fluorescence spectrum and at the anomalous signal. Normally some will make it in and some will be in ordered sites, then it becomes mostly a question of data quality to detect it. You could also try the equivalent iodide soak. Iodine has a decent anomalous signal at the copper wavenght and thus the anomalous signal can be detected at your home source and many times the structure can be solved by SAD or SIRAS. I would also thing that conditions that give ordered iodide sites are likely to result in ordered bromide sites, although the ions are not identical. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca ** On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http:// hk.promo.yahoo.com/security/ 了解更多!
Re: [ccp4bb] Halide soaking
On Tuesday 31 March 2009 09:57:13 Jose Antonio Cuesta-Seijo wrote: Hi! Normally the cell parameters, etc change very very little. You'll only know if the bromides got in at the synchrotron by looking at the fluorescence spectrum That won't help, normally. It only tells you that there is bromide in the solution, not that it found ordered positions in your crystal. and at the anomalous signal. Normally some will make it in and some will be in ordered sites, then it becomes mostly a question of data quality to detect it. Right. You have to process the data and look for a real signal. The mere presence of Br is not enough. In my experience, the frustrating thing is that even if the Br soak works in the sense that it introduces Br into your lattice, the signal is often/usually not sufficient to phase the structure de novo. Nevertheless, when you eventually do solve and refine the structure, you can go back to your anomalous difference data and calculate a Bijvoet Difference Fourier that clearly shows the Br sites. Ethan You could also try the equivalent iodide soak. Iodine has a decent anomalous signal at the copper wavenght and thus the anomalous signal can be detected at your home source and many times the structure can be solved by SAD or SIRAS. I would also thing that conditions that give ordered iodide sites are likely to result in ordered bromide sites, although the ions are not identical. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca ** On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http:// hk.promo.yahoo.com/security/ 了解更多! -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Halide soaking
We were on a Br-soak flurry for awhile, and when I also talked to others, it would work about 20-30% of the time. A few years I compared a structure with Se-Met and native with Br-soak. There were four Br's at about 30% occupancy, and it phased fine. It wasn't as good as the Se-Met phasing, but we could trace and build the structure independently. It's so easy to try that my suggestion has been to try it out first, and if it works, then great. Also, if I had redone it, I would have soaked for longer than 20-30 seconds to try and increase the occupancy. Bernie Santarsiero On Tue, March 31, 2009 12:17 pm, Ethan Merritt wrote: On Tuesday 31 March 2009 09:57:13 Jose Antonio Cuesta-Seijo wrote: Hi! Normally the cell parameters, etc change very very little. You'll only know if the bromides got in at the synchrotron by looking at the fluorescence spectrum That won't help, normally. It only tells you that there is bromide in the solution, not that it found ordered positions in your crystal. and at the anomalous signal. Normally some will make it in and some will be in ordered sites, then it becomes mostly a question of data quality to detect it. Right. You have to process the data and look for a real signal. The mere presence of Br is not enough. In my experience, the frustrating thing is that even if the Br soak works in the sense that it introduces Br into your lattice, the signal is often/usually not sufficient to phase the structure de novo. Nevertheless, when you eventually do solve and refine the structure, you can go back to your anomalous difference data and calculate a Bijvoet Difference Fourier that clearly shows the Br sites. Ethan You could also try the equivalent iodide soak. Iodine has a decent anomalous signal at the copper wavenght and thus the anomalous signal can be detected at your home source and many times the structure can be solved by SAD or SIRAS. I would also thing that conditions that give ordered iodide sites are likely to result in ordered bromide sites, although the ions are not identical. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca ** On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!é¦æ¸¯æä¾ç¶²ä¸å®å ¨æ»ç¥ï¼æä½ å¦ä½é²ç¯é»å®¢! è«åå¾ http:// hk.promo.yahoo.com/security/ äºè§£æ´å¤! -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Halide soaking
You always get the entry of Bromide into crystal by quick soaking, because it does not require the incorporation of Bromide into the protein. But whether the signal is good enough for phasing is another story. You have to collect the full data set to know the answer. Nian 2009/3/31 tat cheung cheng theif...@yahoo.com.hk: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http://hk.promo.yahoo.com/security/ 了解更多!
Re: [ccp4bb] Halide soaking
I'd like to remind folks here that if one's crystals are sturdy enough to survive halide soaking, they're *probably* also sturdy enough to live through covalent iodination. Iodination is easy to set up and if it does work out - one gets awesome quality derivatives with multiple sites (as long as there are enough Tyrosines in the protein). The drawback is that iodination can and often does kill the crystal - which is the reason for my earlier comment regarding crystal machismo/resilience. If you want to try it, I would be happy to supply an easy protocol that worked for me several times :) Artem You always get the entry of Bromide into crystal by quick soaking, because it does not require the incorporation of Bromide into the protein. But whether the signal is good enough for phasing is another story. You have to collect the full data set to know the answer. Nian 2009/3/31 tat cheung cheng theif...@yahoo.com.hk: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!»´ä´£¨Ñºô¤W¦w¥þ§ð²¤¡A±Ð§A¦p¦ó¨¾½d¶Â«È! ½Ð«e©¹ http://hk.promo.yahoo.com/security/ ¤F¸Ñ§ó¦h!
[ccp4bb] IPTG induced protein expression
Dear CCP4bb users, I was wondering if anyone has used regular IPTG (not IPTG-dioxane free, special grade) for protein expression. Are there any problems using such regular samples (5mg dioxane per Kg of IPTG) for protein expression? It would be great to have an idea for real problems if using this. Cheers Ronaldo.
[ccp4bb] Error with molrep when using locked srf.
Why do i keep getting an error when running molrep with locked rotation functions? Open failed: Unit: 8, File: /tmp/francisreyes/ qv_9_molrep_crossrot_alo.dat (logical: /tmp/francisreyes/ qv_9_molrep_crossrot_alo.dat) MOLREP(ccp4): Open failed: File: /tmp/francisreyes/ qv_9_molrep_crossrot_alo.dat Times: User: 58.3s System:2.7s Elapsed: 1:06 /pre /html *** * Information from CCP4Interface script *** The program run with command: /sw/share/xtal/ccp4-6.1.1/bin/molrep PATH_SCR /tmp/francisreyes/qv_9_molrep_ PATH_OUT /tmp/francisreyes/ qv_9_molrep_ HKLIN /Users/francisreyes/Public/QV/Pm12_1.1.mtz MODEL /Users/francisreyes/Public/QV/Pm12_1.1.pdb has failed with error message Last system error message: No such file or directory MOLREP(ccp4): Open failed: File: /tmp/francisreyes/ qv_9_molrep_crossrot_alo.dat *** It doesn't seem to want to calculate the spherical coefficients for F_obs? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Halide soaking
Hi, it worked very nice for me in 1 out of 1 case where I tried it :-). Very well diffracting crystals (1.8 Ang), rather small protein 20 kDa, 50 %solvent content, 1 mol/ASU. 20-30 s soak in 0.5 M NaBr resulted in 6 nice ordered sites. It was crucial for us to collect a 3 wavelength MAD data set. A SAD data set (using just the peak, even if with high redundancy ) was not enough to obtain traceable electron density map, even-though one could distinguish clearly protein boundaries and solvent channels. Good luck Ale On 31 Mar 2009, at 18:19, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑客! 請前往 http://hk.pro mo.yahoo.com/security/ 了解更多!
Re: [ccp4bb] IPTG induced protein expression
I was wondering if anyone has used regular IPTG (not IPTG-dioxane free, special grade) for protein expression. Are there any problems using such regular samples (5mg dioxane per Kg of IPTG) for protein expression? I have. No problem whatsoever. I belive the whole dioxane-free thing is about dioxane being carcinogen and has nothing to do with minute amounts of it affecting E.coli in any way. The amounts of dioxane in regular grade IPTG is nothing in comparison to the carcinogenicity of the heavy metals we use for phasing (or even cigarette smoking). Dima
Re: [ccp4bb] Halide soaking
Somewhat to our surprise, we have found that, even when the halide signal is too weak to solve the substructure from scratch, when you find the sites (in our case, by using the protein model as a substructure in Phaser), they can still add significant phase information. So you can get more out of it than just seeing which sites are halides, especially since the model can be a rough and incomplete one. But if all you're after is seeing which sites are halides, the procedure in Phaser (which is iterative, in that sites found in the first round help to find further sites) is more sensitive and convincingly finds more sites than an anomalous difference Fourier (which is not iterative). There's a tutorial on our web page with real (albeit lysozyme) data illustrating how this works. Regards, Randy Read On 31 Mar 2009, at 18:17, Ethan Merritt wrote: On Tuesday 31 March 2009 09:57:13 Jose Antonio Cuesta-Seijo wrote: Hi! Normally the cell parameters, etc change very very little. You'll only know if the bromides got in at the synchrotron by looking at the fluorescence spectrum That won't help, normally. It only tells you that there is bromide in the solution, not that it found ordered positions in your crystal. and at the anomalous signal. Normally some will make it in and some will be in ordered sites, then it becomes mostly a question of data quality to detect it. Right. You have to process the data and look for a real signal. The mere presence of Br is not enough. In my experience, the frustrating thing is that even if the Br soak works in the sense that it introduces Br into your lattice, the signal is often/usually not sufficient to phase the structure de novo. Nevertheless, when you eventually do solve and refine the structure, you can go back to your anomalous difference data and calculate a Bijvoet Difference Fourier that clearly shows the Br sites. Ethan You could also try the equivalent iodide soak. Iodine has a decent anomalous signal at the copper wavenght and thus the anomalous signal can be detected at your home source and many times the structure can be solved by SAD or SIRAS. I would also thing that conditions that give ordered iodide sites are likely to result in ordered bromide sites, although the ions are not identical. Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: jcue...@uhnres.utoronto.ca ** On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供網上安全攻略，教你如何防範黑 客! 請前往 http:// hk.promo.yahoo.com/security/ 了解更多! -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www- structmed.cimr.cam.ac.uk
Re: [ccp4bb] Halide soaking
Hi everyone, the in situ iodination reaction described in the following classic paper by the late Paul Sigler works quite well. Iodination of a single tyrosine in crystals of alpha-chymotrypsin. Sigler PB. Biochemistry. 1970 Sep 1;9(18):3609-17. The primary purpose of my experiment (which took place 11 years ago according to my notebook) was indeed to iodinate tyrosines, but difference fourier analysis using calculated phases from the final refined MIR structure to reveal the complete iodination model (out of curiosity), showed that in addition to iodination of two tyrosines, two histidines had also been iodinated! In retrospect, I had actually run across those peaks in my cross-difference fourier maps but thought that they were too 'secondary' to be included in the heavy atom model. Best wishes Savas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Alessandro Vannini Sent: Tuesday, March 31, 2009 9:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Halide soaking Hi, it worked very nice for me in 1 out of 1 case where I tried it :-). Very well diffracting crystals (1.8 Ang), rather small protein 20 kDa, 50 %solvent content, 1 mol/ASU. 20-30 s soak in 0.5 M NaBr resulted in 6 nice ordered sites. It was crucial for us to collect a 3 wavelength MAD data set. A SAD data set (using just the peak, even if with high redundancy ) was not enough to obtain traceable electron density map, even-though one could distinguish clearly protein boundaries and solvent channels. Good luck Ale On 31 Mar 2009, at 18:19, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!香港提供�W上安全攻略，教你如何防��黑客! ��前往 http://hk.pro mo.yahoo.com/security/ 了解更多! E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.12080 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] Halide soaking
The conditions in the drop play a huge role in the success of iodination. If you see iodinated histidines, this means that you had high pH - higher than 8 at least, as histidines are much harder to iodinate than tyrosines (which will work even at pH 5, and definitely at 6). Paul's classic experiment is very nice :) If you're into even more classic experiments, check out: INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN ROGER M. HERRIOTT, 1936 http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2141501blobtype=pdf The heavily modified method that I will be posting shortly does not involve adding iodine directly to the crystals, which is its sole (but I think significant) benefit :) Artem Hi everyone, the in situ iodination reaction described in the following classic paper by the late Paul Sigler works quite well. Iodination of a single tyrosine in crystals of alpha-chymotrypsin. Sigler PB. Biochemistry. 1970 Sep 1;9(18):3609-17. The primary purpose of my experiment (which took place 11 years ago according to my notebook) was indeed to iodinate tyrosines, but difference fourier analysis using calculated phases from the final refined MIR structure to reveal the complete iodination model (out of curiosity), showed that in addition to iodination of two tyrosines, two histidines had also been iodinated! In retrospect, I had actually run across those peaks in my cross-difference fourier maps but thought that they were too 'secondary' to be included in the heavy atom model. Best wishes Savas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Alessandro Vannini Sent: Tuesday, March 31, 2009 9:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Halide soaking Hi, it worked very nice for me in 1 out of 1 case where I tried it :-). Very well diffracting crystals (1.8 Ang), rather small protein 20 kDa, 50 %solvent content, 1 mol/ASU. 20-30 s soak in 0.5 M NaBr resulted in 6 nice ordered sites. It was crucial for us to collect a 3 wavelength MAD data set. A SAD data set (using just the peak, even if with high redundancy ) was not enough to obtain traceable electron density map, even-though one could distinguish clearly protein boundaries and solvent channels. Good luck Ale On 31 Mar 2009, at 18:19, tat cheung cheng wrote: Hi all I am now trying to do bromide soaking, but i am not really sure does the bromide atom enter my crystal. So is there any signs that indicate the entry of bromide atom? e.g. does the space group, cell dimension change? or just nothing change, and the bromide atom just get in? Thanks very much. T.C. Cheng Yahoo!Ïã¸ÛÌṩ¾WÉÏ°²È«¹¥ÂÔ£¬½ÌÄãÈçºÎ·À¹ ºÚ¿Í! ÕÇ°Íù http://hk.pro mo.yahoo.com/security/ Á˽â¸ü¶à! E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.12080 http://www.pctools.com/en/spyware-doctor-antivirus/
[ccp4bb] fermentation position available
Greetings All -- I am posting this on behalf of the HR department here at Exelixis. Our Fermentation department provides all of our protein resources for high-throughput assay, cell biology and structural biology. Our structural biology protein consumption rates are comparable to the larger structural genomics initiatives, so someone with that sort of background and experience would be an especially ideal candidate. Please mention this to anyone you think is appropriate and might be interested in relocation to the San Francisco Bay Area of sunny* California! Cheers, Tom *Full disclosure: yes, we have some fog too... But snow is generally properly kept to where it belongs: about a 3 hour drive into the Sierra Nevada mountains... :) Here are the details: Assistant Research Scientist II Job ID #: 667 Location: South San Francisco, CA Position Type: Full-Time Regular Department: Genome Biochemistry Education Required: Bachelors Degree Experience Required: 1 - 3 Years Apply: Online at www.exelixis.com We are seeking a professional to join our fermentation team in a hands-on laboratory role. Duties include cell culture maintenance, scale-up and optimization of protein expression, and biomass production for multiple drug discovery projects. The successful candidate will assist in planning and development of new methodologies and laboratory protocols to improve the efficiency and productivity of the fermentation process. You will prepare and organize data for presentation and project status reports at individual, group, and departmental research meetings. You will be expected to maintain familiarity with current scientific literature relevant to the research experiments and programs. BS/BA degree in Biology, Biochemistry, or Chemical Engineering and one year of related experience; or MS/MA degree in related discipline or combination of experience and education/training. Familiarity with protein expression analysis tools such as Western blotting, SDS-PAGE, and activity analysis are desired, as would knowledge of expression construct design and protein purification techniques. The qualified candidate should have experience in the production of recombinant proteins from systems such as E. coli and/or insect cells using fermentors and bioreactors from 5 to 80 L scale. Must be able to lift at least 40 lbs. This email (including any attachments) may contain material that is confidential and privileged and is for the sole use of the intended recipient. Any review, reliance or distribution by others or forwarding without express permission is strictly prohibited. If you are not the intended recipient, please contact the sender and delete all copies. Exelixis, Inc. reserves the right, to the extent and under circumstances permitted by applicable law, to retain, monitor and intercept e-mail messages to and from its systems.
[ccp4bb] Iodination - posted
Hi, Since a surprisingly large number of people asked for the protocol, I compiled a quickie PDF document and posted it here: http://www.xtals.org/pdfs/iodination.pdf Please excuse the inevitable consequences of haste - I am sure that the file is riddled with spelling errors and poor grammar. On the flip side, it also contains two sample images of electron density maps for mono- and bis- iodinated tyrosine (from the same crystal structure). Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind.
Re: [ccp4bb] Halide soaking
Around here, I would guesstimate the success rate to be in the 5-10% range. Still, I always try it as it's so easy. I don't think longer soak times will usually do much to increase occupancy. Instead, try higher halide concentrations. I also like to try monovalent cations (Rb, Cs) but haven't had any success with those yet. ho UC Berkeley
[ccp4bb] Building structure in COOT
Hi, Good day I am currently building my structure by using COOT. My protein is a tetramer protein and I have fit my protein sequence into one of the monomer of the homologous model. May I know how can I replace other monomer with the amended monomer?? Thanks Chong-wai
[ccp4bb] Choosing weighing term
Dear All: Hi. I have a question about selecting weighing term during restrained refinement using Refmac5 of CCP4 packages. For a 300kDa homodimer protein structure at 2.5A, 91% complete. I obtain optimal R and Rfree by using NCS tight restraints of the peptides of the two monomers. Weighing term 0.15 gives (R=0.22, Rfree=0.28) and weighing term 0.1 gives(R=0.23, Rfree=0.28). Higher weighing term gives larger difference between R and Rfree. Is there a criteria or special range of choosing weighing term? Is weighing term 0.1 too small? I read the references by Ian Tickle (Acta Cryst D, 1998 and 2000)on R and Rfree ratio, those helped a lot but I still do not know the key of weighing term. Thanks so much. Best, Hongnan Cao Biochemistry Department UC Riverside _ 梦幻K图，百变造型，让你的照片与众不同，快来MClub试试吧！ http://club.msn.cn/?form=3
Re: [ccp4bb] Building structure in COOT
Liew Chong Wai wrote: Hi, Good day I am currently building my structure by using COOT. My protein is a tetramer protein and I have fit my protein sequence into one of the monomer of the homologous model. May I know how can I replace other monomer with the amended monomer?? Thanks *Chong-wai* Hi Chong-wai, Several possibilities to generate the tetramer from the monomer, let say A. You can calculate the RT matrices ( in CCP4 or using LSQMAN and MOLEMAN2 or using O for example) to superpose the monomer A onto B, C and D and then apply these matrices to the amended monomer (A) in order to generate the tetramer. You can also superpose the monomer A onto B in COOT by using SSM or LSQ and save the new position as monomer B in COOT etc... and then you can generate 4 monomers and the tetramer. Do not forget to change the chain ID before refinement. Best regards, David -- David Cobessi Institut de Biologie Structurale 41, Rue Jules Horowitz 38027 Grenoble Cedex-1, France Tel:33(0)438789613 33(0)608164340 Fax:33(0)438785122
