Re: [ccp4bb] moelcular replacement with large cell
Self rotation is not very clear - and the top peaks are almost certainly generated partly by the high crystallographic symmmetry. Things to check: 1) native Patterson - is there a non-crystallographic translation? ctruncate checks this for you. 2) twinning - if there is a non-crystallographic translation many of the twinning tests are somewhat invalidated. again I have found the Ltest which is now incorporated into ctruncate and only applied to reliable data is quite informative. Probably phenix.xtriage has looked for this but you should check the loggraphs too. PG 622 can be twinned in several ways ( see $CHTML/winned info) 3) Only the old self rotation programs show you all symmetry equivalents of your peaks - I cant work out in my head whether any of the ones you list indicate NCS 3-folds.. But I would expect to find a solution with the larger protein with 100% sequence ID. Maybe try searching at low resolution with the whole molecule instead of splitting it into domains? Eleanor Wei Zhang wrote: Dears, I am doing molecular replacement of a protein complex with a P622 data set with large cell parameters (a=b=135, c=480). The data set seems well. R merge is 0.17 for all and 0.70 for the last shell of 2.9 angstrom. I am not sure it is a complex in the crystal. Phenix analysis reveal there is no twin. The proposed protein complex is about 70 kDa with a larger subunit of 50 kDa and a small subunit of 20 kDa. The matthews analysis indicates that there might be 3 complexes in the ASU. The structure of the 50 kDa subunit is known while the 20 kDa one is unknown. But molecular replacement failed with either Phaser or Molrep. Self-rotation with CNS reported the result as below: ! index, psi, phi, kappa, RF-function ( 0.25) 1 0.000 0.000 180.000 29.7217 Self-ratation with Molrep reported the result as below: Number of RF peaks : 30 thetaphi chialphabeta gamma Rf Rf/sigma Sol_RF 1 0.000.000.000.000.000.000.3444E+05 17.13 Sol_RF 290.00 -80.07 179.990.00 180.00 -19.86 5061. 2.52 Sol_RF 390.00 -65.53 179.990.00 180.00 -48.94 4890. 2.43 Sol_RF 490.00 -76.12 179.990.00 180.00 -27.76 4722. 2.35 Sol_RF 511.42 90.00 61.00 30.00 11.53 30.00 2438. 1.21 Sol_RF 6 164.38 60.00 179.99 60.00 -31.24 120.00 1850. 0.92 Sol_RF 785.13 -141.54 179.97 38.32 -170.26 141.40 1805. 0.90 Sol_RF 890.00 -60.00 90.00 30.00 -90.00 -30.00 1764. 0.88 Sol_RF 984.07 -144.87 179.96 34.98 -168.15 144.73 1743. 0.87 Sol_RF 1072.25 -60.00 89.30 46.76 -84.03 -13.24 1665. 0.83 Sol_RF 11 138.24 -130.77 180.00 49.23 83.52 130.77 1608. 0.80 Sol_RF 12 170.36 30.00 179.99 30.00 -19.28 150.00 1590. 0.79 Sol_RF 1382.04 60.00 179.99 60.00 -164.07 120.00 1571. 0.78 Sol_RF 14 141.01 30.00 179.99 30.00 -77.99 150.00 1554. 0.77 Sol_RF 15 123.63 -155.38 179.98 24.62 112.74 155.38 1517. 0.75 Sol_RF 16 148.80 30.00 179.99 30.00 -62.39 150.00 1450. 0.72 Sol_RF 17 142.61 30.00 180.00 30.00 -74.78 150.00 1439. 0.72 Sol_RF 1872.25 -150.00 179.99 30.00 -144.50 150.00 1422. 0.71 Sol_RF 1947.42 -120.00 179.98 60.00 -94.83 120.00 1417. 0.71 Sol_RF 20 153.12 30.00 179.98 30.00 -53.75 150.00 1292. 0.64 Sol_RF 21 155.99 30.00 179.98 30.00 -48.01 150.00 1274. 0.63 Sol_RF 22 138.05 30.00 179.99 30.00 -83.91 150.00 1258. 0.63 Sol_RF 2354.12 120.00 89.15 60.00 69.320.00 1254. 0.62 Sol_RF 24 129.04 -136.48 179.99 43.52 101.93 136.48 1193. 0.59 Sol_RF 25 138.65 -164.29 179.99 15.71 82.69 164.29 1185. 0.59 Sol_RF 2684.16 51.37 179.98 51.37 -168.32 128.63 1176. 0.59 Sol_RF 2790.00 150.00 59.82 60.00 59.82 -60.00 1162. 0.58 Sol_RF 2843.66 -133.74 179.98 46.26 -87.31 133.74 1161. 0.58 Sol_RF 29 127.85 -169.33 179.99 10.67 104.29 169.33 1153. 0.57 Sol_RF 30 124.15 30.00 179.99 30.00 -111.71 150.00 1147. 0.57 My question is: 1. For a 70 kDa protein compelx, is it common to have such a large cell with a dimention as long as 480 angstrom? 2. Is it possible that the longest dimention of cell is doubled? If it is, how to divide it? 3. How to interpret the self-rotation results. The results from CNS and Molrep differs so much. 4. Any other suggestions on the molecular replacement are appraciated. Thanks. Wei Zhang PKU
[ccp4bb] Ph.D. Fellowship - University of Copenhagen
Dear supervisors and potential candidates, There is a vacant Ph.D. student fellowship available in the Biostructural Research Group, University of Copenhagen. Please, follow the links for further information. http://www.farma.ku.dk/index.php?id=6811 http://www.farma.ku.dk/index.php/Project-9/6839/0/ Best regards, Ole Kristensen -- Asc. Prof. Ole Kristensen Biostructural Research Department of Medicinal Chemistry University of Copenhagen Universitetsparken 2, DK-2100 Copenhagen, Denmark. Phone: +45 35336121 Mobile: +45 21463724
Re: [ccp4bb] Van der Waals contacts
I may have missed the original question, but the significance of VdW interactions in general should hugely depend on the context. Let's say, I would expect VdW in protein interfaces to have little effect, because it gets compensated, on average, by VdW with water molecules in dissociated state. This effect is known for hydrogen bonding as well, there was once a discussion whether HBs in interfaces are actually stabilizing or not. Eugene. On Wed, 15 Jul 2009, Nadir T. Mrabet wrote: Totally agree with what you write. Yet, I was only answering the question asked (that dealt mostly with van Der Waals contacts and effective contacts). At a time of crisis (DeltaG = only a few kcal/mol), I would say 0.6 kcal/mol would be a little more than negligible. Question of balance. Nadir Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Bernhard Rupp wrote: It might be worthwhile to consider the energy column in the pdf: At RT we have about 0.6 kcal/mol thermal energy, so a *single attractive* vdW interaction has little impact - it is generally the sum of many of those contributing to notable and important attractive forces. For a *single repulsive* (steep branch) vdW this is quite different - your quickly get energetically significant repulsions even for one bad contact. So the vdW repulsion are both useful (nearly independent) stereochemical restraints and a good indicator for improbable conformations (see molprobity clash score, rama, etc). BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nadir T. Mrabet Sent: Wednesday, July 15, 2009 7:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Van der Waals contacts This issue is far from being trivial. To address the question, I recommend looking at this simple example (attached pdf file) which shows that even when the atom distance is twice the vdw diameter, there is still attraction between atoms (unified type, with implicit H atoms). Some, but not all, consider that water screens out interactions, that is, the vdw interaction is attractive provided that R Rm + 2 x R(H2O). But then, one may also need to consider the special case of water-mediated H-bonds, where the heavy atoms being considered are polar. HTH, Nadir Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Clayton, Gina Martyn wrote: Hi CCP4ers Perhaps I am hashing over old news...but We are having a discussion about Van Der Waals contacts and effective contacts i.e. the real distance of a VDW bump between say a CH and a CH group which sometimes is described as between a C and a C as i.e. 2x 1.6A and ending about 4A but not including hydrogen. Some programs list contacts, to say a ligand, as far as 6A apart and some of the simulation programs use that distance too for contacts for protein protein interactions. Does anyone know of a good paper that discusses the effective distance or has a comment on where a VDW force may begin and end or it's effective distance - though some say it never truly ends just approaches zero... G Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] mtz2hkl: New Version 0.3 available.
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear all, a new version of mtz2hkl is available. Previous versions only wrote HKLF3 format if the data were amplitudes. The option '-2' now converts amplitudes into intensities (and takes care of the standard deviations, too). The conversion of the standard deviations is not paricularly sophisticated and does not revert the process truncate carries out, so the advice remains to not loose intensities from the mtz-file in the first place. A binary for Linux as well the source code for compilation for other platforms is available from my homepage http://shelx.uni-ac.gwdg.de/~tg/mtz2x/mtz2hkl/mtz2hkl.php Tim Gruene - -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.9 (GNU/Linux) iD8DBQFKXx+ZUxlJ7aRr7hoRAminAJ0W5i6cAGwkBrswKTEpyVHswSouOACfSys4 SlMkYs6Zxcx6O1A/RvQ/2xc= =WT1G -END PGP SIGNATURE-
[ccp4bb] APPLICATION PROCEDURE FOR PROPOSALS FOR BEAMTIME ON THE ESRF Bio-SAXS BEAMLINE ID14-3
REMINDER: APPLICATION PROCEDURE FOR PROPOSALS FOR BEAMTIME ON THE ESRF Bio-SAXS BEAMLINE ID14-3 The new Bio-SAXS beamline at ID14-3 at the ESRF ( http://www.esrf.fr/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-3 ) was officially inaugurated on the 15th June 2009 (http://www.esrf.fr/news/general/inauguration_ID14-3/inauguration_ID14-3/). Robotic sample loading is now available on the beam-line using a device constructed in a collaboration between the EMBL (Grenoble and Hamburg outstations) and the ESRF. Automated data analysis will also be implemented following the model of the SAXS facility at X-33, EMBL Hamburg. Eventually the end-station will be fully automated (sample loading, data collection, processing and preliminary analysis) and will rapidly provide users standard data concerning the size (radius of gyration, maximum dimension and volume) and molecular weight of samples and allow on-the fly /ab-inito/ shape reconstruction in order to provide feedback enabling the data collection strategies to be optimized. Future plans extend to allowing remote access to the new Bio-SAXS facility, based on the system currently in use on the ESRF's MX end-stations. Application for beam-time on this facility can be made via the rolling access mechanism at ANY time. Those who wish to apply (including members of ESRF MX BAG Groups) should use the mechanism at: http://www.esrf.fr/UsersAndScience/UserGuide/Applying/ProposalGuidelines/MXnon-BAGproposal where it must be clearly indicated in the title of the proposal that the application is for Bio-SAXS beamtime on ID14-3. begin:vcard fn:David FLOT n:FLOT;David org:ESRF;Macromolecular Crystallography group adr:6 rue Jules Horowitz;;BP220;Grenoble;;38043;France email;internet:david.f...@esrf.fr title:Beam-Line Operation Manager tel;work:+(33) 4 76 88 17 63 tel;fax:(+33) 4 76 88 26 24 x-mozilla-html:FALSE url:www.esrf.eu version:2.1 end:vcard
[ccp4bb] tips on crystallizing a Protein-DNA complex
I was hoping people could give some tips on the best way to go about crystallizing a protein-DNA complex. I have a large amount of experience in protein crystallisation but have never tried co-crystallisation with DNA until I started this project. If you want to reply to me personally I will then post a summary. My protein is a dimer and has been shown by several methods to bind to DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have several questions: 1. Do people routinely try different lengths of DNA? 2. Do you start with blunt or sticky ends? 3. Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? 4. Which screens would you try first? 5. Where do you order the DNA from as there is a large difference in price depending on supplier. What scale do you go for and what purification? 6. We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? 7. Any other useful tips? Thanks in advance for the suggestions and advice Clare Dr. Clare E. M. Stevenson John Innes Centre, Department Biological Chemistry Colney Lane Norwich Norfolk NR4 7UH
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
Hi Clare, Two papers that might be worth checking out that address some of your questions - 1. Tan et al, Crystallization of the Yeast MATa2/MCM1/DNA Ternary Complex: General Methods and Principles for Protein/DNA Cocrystallization J. Mol. Biol. (2000) 297, 947±959 2. Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed for proteinnucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409417 cheers, Kushol Kushol Gupta, Ph.D. Mathilde Krim Fellow in Basic Biomedical Research Van Duyne Laboratory - Univ. of Pennsylvania School of Medicine BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 I was hoping people could give some tips on the best way to go about crystallizing a protein-DNA complex. I have a large amount of experience in protein crystallisation but have never tried co-crystallisation with DNA until I started this project. If you want to reply to me personally I will then post a summary. My protein is a dimer and has been shown by several methods to bind to DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have several questions: 1.Do people routinely try different lengths of DNA? 2.Do you start with blunt or sticky ends? 3.Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? 4.Which screens would you try first? 5.Where do you order the DNA from as there is a large difference in price depending on supplier. What scale do you go for and what purification? 6.We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? 7.Any other useful tips? Thanks in advance for the suggestions and advice Clare Dr. Clare E. M. Stevenson John Innes Centre, Department Biological Chemistry Colney Lane Norwich Norfolk NR4 7UH
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
1. Do people routinely try different lengths of DNA? Yes, its usually the most important variable because the ends like to stack against each other to form a pseudo-continuous helix. 2. Do you start with blunt or sticky ends? Both. Plan on a dozen or so different duplexes to start (you can usually mix-n-match top bottom strands to get more variety at the ends). 3. Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? Probably wouldn't hurt, but probably not necessary. You can screen a lot more DNAs if you start out with mix-n-pray. 4. Which screens would you try first? PEG/ion AND hand-made PEG vs. salt, +/- Mg++, with concentrations that suit our complexes' personalities. 5. Where do you order the DNA from as there is a large difference in price depending on supplier. What scale do you go for and what purification? We've had good luck with IDT. For screening, even for lengths in the low 30s, we just use the unpurified stuff. Its much faster and cheaper that way! Then try purifying the oligos that give you hits. Sometimes it matters, sometimes it doesn't. 6. We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? Usually a bit of extra DNA. 7. Any other useful tips? Protein-DNA cocrystals are often (at least in my lab) fragile and a pain to freeze. Do at least some of your screens with enough glycerol around to act as a cryoprotectant, so that you won't have to do extra crack-inducing manipulations later. Thanks in advance for the suggestions and advice Clare Dr. Clare E. M. Stevenson John Innes Centre, Department Biological Chemistry Colney Lane Norwich Norfolk NR4 7UH Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
Dear Clare et al: The absolute classic paper in the field is this: Schultz, S. C., Shields, G. C. Steitz, T. A. (1990). Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. J. Mol. Biol. 213, 159–166. I've learned more from this than from reading probably any other single crystallization paper. It describes many of the things you ask about, especially trying different lengths/ends. (Last time I had heard from Steve Schultz he had ditched the rat race and was teaching on the Navajo res. He is my hero.) Bill On Jul 16, 2009, at 7:09 AM, clare stevenson (JIC) wrote: I was hoping people could give some tips on the best way to go about crystallizing a protein-DNA complex. I have a large amount of experience in protein crystallisation but have never tried co-crystallisation with DNA until I started this project. If you want to reply to me personally I will then post a summary. My protein is a dimer and has been shown by several methods to bind to DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have several questions: 1. Do people routinely try different lengths of DNA? 2. Do you start with blunt or sticky ends? 3. Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? 4. Which screens would you try first? 5. Where do you order the DNA from as there is a large difference in price depending on supplier. What scale do you go for and what purification? 6. We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? 7. Any other useful tips? Thanks in advance for the suggestions and advice Clare Dr. Clare E. M. Stevenson John Innes Centre, Department Biological Chemistry Colney Lane Norwich Norfolk NR4 7UH
[ccp4bb] postdoctoral position
Postdoctoral position available: Applications are invited for a postdoctoral position to investigate the structure and biochemistry of proteins involved in Gram-negative bacterial cell wall recycling. Peptidoglycan metabolites produced during cell wall turnover are found to regulate an increasing number of bacterial processes, including the induction of antibiotic resistance in a number bacterial species that cause opportunistic infections in humans. We are focused on understanding the biology of the cell wall recycling pathway, with the goal of developing small molecule based strategies to disrupt the pathway and suppress antibiotic resistance mechanisms that are coupled to it (J. Biol. Chem. 2007; 282:21382-91, 2009 Antimicrob Agents Chemother. 2009 53:2274-82, Protein Sci. 2009;18:1541-51). A number of projects are well advanced, some with crystals already in-hand. More project information can be found at: http://umanitoba.ca/faculties/science/microbiology/staff/mark/ We seek enthusiastic applicants who hold a PhD in biochemistry or related field. Candidates should have a strong background in molecular biology, protein purification/crystallization and protein X-ray crystallography. Our group is located in the Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada. The laboratory includes state-of- the-art Rigaku instrumentation (Micromax 007 HF, X-stream cryosystem and R-axis 4++ detector) and has access to the newly commissioned Canadian Light Source Synchrotron, which is a short drive away. The University of Manitoba (http://umanitoba.ca/) is the largest University in the province (over 30,000 students, faculty, and staff) and hosts a large, interactive biomedical research community. Winnipeg is a vibrant, multicultural city with an outstanding performing arts scene, numerous cultural events and festivals, and great dining (http://www.destinationwinnipeg.ca/). With over 110,000 fresh water lakes in our province alone, there is also much to explore for those who love the outdoors. Salary will be in accordance with the Canadian Institutes of Health Research (CIHR) (http://www.cihr.ca/ ) standards. Fellowship funding is also available through agencies such as the CIHR and the Manitoba Health Research Council (MHRC) (http://www.mhrc.mb.ca/). The University of Manitoba encourages applications from qualified women and men, including members of visible minorities, Aboriginal peoples, and persons with disabilities. All qualified candidates are encouraged to apply. Please direct inquiries and CV's (including names of three referees) to the following email address: brian_m...@umanitoba.ca = Brian L. Mark, MSc, PhD Assistant Professor Department of Microbiology Room 418, Buller Building University of Manitoba Winnipeg, Manitoba CANADA R3T 2N2 Phone (204) 480-1430 Fax (204) 474-7603 Web: http://www.umanitoba.ca/science/microbiology/staff/mark/
[ccp4bb] MAD wavelength
Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Thanks a lot for your comments. All the best, Jerry McCully _ Insert movie times and more without leaving Hotmail®. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutorial_QuickAdd_062009
Re: [ccp4bb] MAD wavelength
I think scans are better than theory, and there can also be white lines which are often dramatically higher than predicted. Just make sure that you do not roast your best crystal when you do your scan! Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Jerry McCully To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 16, 2009 12:15 PM Subject: [ccp4bb] MAD wavelength Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Thanks a lot for your comments. All the best, Jerry McCully -- Insert movie times and more without leaving Hotmail®. See how.
Re: [ccp4bb] MAD wavelength
Always take the scan results ahead of the typical values unless they are obviously wrong. Only use the predicted values if the scan is broken or too weak (e.g. very small crystals) and in that case I'd be tempted to add 10-20 eV to the typical peak wavelength to make sure you weren't actually collecting the inflection point since they are typically very close in SeMet. In my NSLS X29-dominated data collections, I find I end up using something like this for non-oxidized SeMet: Peak: 12664 eV, 0.9790 Angstrom (usually in range 12662-12664) Infl: 12662 eV, 0.9792 Angstrom (usually in range 12660-12662) I also typically use high energy remote: 12860 eV, 0.964 Angstrom give or take a few eV. This tends to translate well between the relatively small number of beamlines that I personally end up using. But I always prefer to take the results from the Chooch analysis of the scan from the actual crystal. Cheers (and good luck) Phil Jeffrey Princeton Jerry McCully wrote: Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results?
Re: [ccp4bb] MAD wavelength
Just checking my oxidized Se-Met experiments, I have 12658 to 12661 eV for my peak energies, and 3 eV lower for the inflection. As others have said, do the fluorescence scan. Use your experimentally determined values. Engin On 7/16/09 11:54 AM, Phil Jeffrey wrote: Always take the scan results ahead of the typical values unless they are obviously wrong. Only use the predicted values if the scan is broken or too weak (e.g. very small crystals) and in that case I'd be tempted to add 10-20 eV to the typical peak wavelength to make sure you weren't actually collecting the inflection point since they are typically very close in SeMet. In my NSLS X29-dominated data collections, I find I end up using something like this for non-oxidized SeMet: Peak: 12664 eV, 0.9790 Angstrom (usually in range 12662-12664) Infl: 12662 eV, 0.9792 Angstrom (usually in range 12660-12662) I also typically use high energy remote: 12860 eV, 0.964 Angstrom give or take a few eV. This tends to translate well between the relatively small number of beamlines that I personally end up using. But I always prefer to take the results from the Chooch analysis of the scan from the actual crystal. Cheers (and good luck) Phil Jeffrey Princeton Jerry McCully wrote: Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? -- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
Re: [ccp4bb] MAD wavelength
I second Phil's opinion - it is better to scan and be sure - as long as the scan results are not hideously abnormal. If you cannot scan for whatever reason but are sure that the X-ray optical system is properly calibrated - then use Phil's numbers below :) Artem Always take the scan results ahead of the typical values unless they are obviously wrong. Only use the predicted values if the scan is broken or too weak (e.g. very small crystals) and in that case I'd be tempted to add 10-20 eV to the typical peak wavelength to make sure you weren't actually collecting the inflection point since they are typically very close in SeMet. In my NSLS X29-dominated data collections, I find I end up using something like this for non-oxidized SeMet: Peak: 12664 eV, 0.9790 Angstrom (usually in range 12662-12664) Infl: 12662 eV, 0.9792 Angstrom (usually in range 12660-12662) I also typically use high energy remote: 12860 eV, 0.964 Angstrom give or take a few eV. This tends to translate well between the relatively small number of beamlines that I personally end up using. But I always prefer to take the results from the Chooch analysis of the scan from the actual crystal. Cheers (and good luck) Phil Jeffrey Princeton Jerry McCully wrote: Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results?
Re: [ccp4bb] MAD wavelength
On Thursday 16 July 2009 10:15:51 Jerry McCully wrote: Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Yes. You should definitely trust the scanning results over any literature value. Let's leave aside for the moment that people may deliberately choose to collect data at 50-100 eV above the peak, and consider only the question of how accurately we can determine what beamline setting corresponds to where the peak is. A fluorescence scan tells you that the fluorescence peak was observed when the monochromator motor readout was N steps (where each step is typically less than 0.001 degree). This is reproducible over the short term, as you should be able to confirm by running the scan again. The nominal energy readout comes from converting the steps to degrees, and then converting the Bragg angle in degrees to an acceptance energy in eV. But these conversion assume a known setting at 0 motor steps, and if this zero point is off by even 0.001 degree that shifts the nominal energy readout near the Se edge by roughly 5 eV as I recall (depends on the monochromator crystal being used). The calibration of the beamline optics is always going to be imperfect, and is subject to minor drift over the course of time. You can ask your friendly neighborhood beamline support people to explain what factors are the most likely to affect calibration of their particular beamline. The usual culprits include heat load on the monochromator or upstream mirrors, and tunable steering of the stored particles in the ring. Not that you can do much about any of this, other than to scan again before collecting your next data set :-) -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] MAD wavelength
On a different note,it seems as if SeMet is not a regular experiment in your lab. So I would recommend not to burn your crystals and rather sacrifice the resolution versus loosing your SeMet anomalous signal after couple of degrees into the data collection. It would also be wise to run Mosflm STRATEGY ANOMALOUS to figure out where you should start collecting your data. Good luck, Jürgen On Jul 16, 2009, at 1:15 PM, Jerry McCully wrote: Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Thanks a lot for your comments. All the best, Jerry McCully Insert movie times and more without leaving Hotmail®. See how. - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.me.com/bosch_lab/
Re: [ccp4bb] MAD wavelength
My 2 cents: Cent #1 But they are slightly different ( may be 50 ev) in different papers. This statement itself contains the answer - always scan, but it was already suggested by many. I'd like to get to slightly different issue here. No oxidation or any other interaction in protein crystals moves the Se edge 50 ev from its theoretical value. It is obvious that in that particular experiment the monochromator was not calibrated properly. Ethan already touched on this but I wanted to emphasize again: when a scan identifies the edge to be way out of reasonable, the beamline support staff should be asked to check the monochromator calibration before publishing such exotic results. On our beamlines at GM/CA-CAT at APS, we do calibration at the start of each user group. It's easy, fast, and Se edges from sample to sample, project to project, user to user, are remarkably stable. Cent #2 Often orientation of your sample crystal has far greater effect on the way your scan as well as f' and f curves look than oxidation or other issues in the protein molecule. Check out Bricogne's publications few years back. Cheers, Nukri Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully Sent: Thursday, July 16, 2009 12:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] MAD wavelength Dear All: Next week we are going to try some seleno-Met labeled crystals. We checked the literature to try to find out the peak wavelength that has been used for SAD or MAD data collection. But they are slightly different ( may be 50 ev) in different papers. I guess this is due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Thanks a lot for your comments. All the best, Jerry McCully Insert movie times and more without leaving Hotmail(r). See how. http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_T utorial_QuickAdd_062009
[ccp4bb] OpenGL Stereo 3D on 120 Hz LCDs, at last!
FYI, for folks not subscribed to pymol-users: nVidia today released beta drivers which at last enable OpenGL-based stereo 3D visualization on 120 Hz LCDs using Quadro graphics cards. So long as you are willing to put up with Windows, you can finally abandon those old CRTs without spending a fortune and without sacrificing quality of the stereo 3D effect. Details posted at http://www.pymol.org http://www.pymol.org/ Cheers, Warren
