[ccp4bb] Dependency of theta on n/d in Bragg's law
Dear all, I am shocked by my own ignorance, and you feel free to do the same, but do you agree with me that according to Bragg's Law a diffraction maximum at an angle theta has contributions to its intensity from planes at a spacing d for order 1, planes of spacing 2*d for order n=2, etc. etc.? In other words as the diffraction angle is a function of n/d: theta=arcsin(lambda/2 * n/d) several indices are associated with diffraction at the same angle? (I guess one could also prove the same result by a number of Ewald constructions using Ewald spheres of radius (1/n*lambda with n=1,2,3 ...) All textbooks I know on the argument neglect to mention this and in fact only n=1 is ever considered. Does anybody know a book where this trivial issue is discussed? Thanks! Ciao Pietro Sent from my Desktop Dr. Pietro Roversi Oxford University Biochemistry Department - Glycobiology Division South Parks Road Oxford OX1 3QU England - UK Tel. 0044 1865 275339
Re: [ccp4bb] Dependency of theta on n/d in Bragg's law
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Pietro, You may take a textbook into account which deals with Laue diffraction. If you search for the keyword Laue and the author Helliwell at the IUCR journals, you will get a large number of hits, indicating that this is by no means a 'trivial' issue (e.g. http://dx.doi.org/10.1107/S0909049599006366 for an overview or http://dx.doi.org/10.1107/S0108767387098763 for the treatment of harmonics). As far as I understand, certain scaling programs take the lambda/2 contribution of monochromators into account. Regards, Tim Gruene On 08/20/2013 04:36 PM, Pietro Roversi wrote: Dear all, I am shocked by my own ignorance, and you feel free to do the same, but do you agree with me that according to Bragg's Law a diffraction maximum at an angle theta has contributions to its intensity from planes at a spacing d for order 1, planes of spacing 2*d for order n=2, etc. etc.? In other words as the diffraction angle is a function of n/d: theta=arcsin(lambda/2 * n/d) several indices are associated with diffraction at the same angle? (I guess one could also prove the same result by a number of Ewald constructions using Ewald spheres of radius (1/n*lambda with n=1,2,3 ...) All textbooks I know on the argument neglect to mention this and in fact only n=1 is ever considered. Does anybody know a book where this trivial issue is discussed? Thanks! Ciao Pietro Sent from my Desktop Dr. Pietro Roversi Oxford University Biochemistry Department - Glycobiology Division South Parks Road Oxford OX1 3QU England - UK Tel. 0044 1865 275339 - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.14 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFSE4EgUxlJ7aRr7hoRAomhAJ94WrXRCTx8gevMAzrhenVri2EkhwCghyC1 UuckhqtUEG0uB9hheG1uxz0= =+cAo -END PGP SIGNATURE-
Re: [ccp4bb] Dependency of theta on n/d in Bragg's law
Dear Pietro, Ladd Palmer book does explain it, just first example that springs to mind. HTH D -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pietro Roversi Sent: 20 August 2013 15:37 To: ccp4bb Subject: [ccp4bb] Dependency of theta on n/d in Bragg's law Dear all, I am shocked by my own ignorance, and you feel free to do the same, but do you agree with me that according to Bragg's Law a diffraction maximum at an angle theta has contributions to its intensity from planes at a spacing d for order 1, planes of spacing 2*d for order n=2, etc. etc.? In other words as the diffraction angle is a function of n/d: theta=arcsin(lambda/2 * n/d) several indices are associated with diffraction at the same angle? (I guess one could also prove the same result by a number of Ewald constructions using Ewald spheres of radius (1/n*lambda with n=1,2,3 ...) All textbooks I know on the argument neglect to mention this and in fact only n=1 is ever considered. Does anybody know a book where this trivial issue is discussed? Thanks! Ciao Pietro Sent from my Desktop Dr. Pietro Roversi Oxford University Biochemistry Department - Glycobiology Division South Parks Road Oxford OX1 3QU England - UK Tel. 0044 1865 275339 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
[ccp4bb] Postdoc Position in Structural Biology of Membrane Proteins
Postdoc in Structural Biology of Membrane Proteins A postdoc position is available immediately at the Skirball Institute, New York University School of Medicine, to study the mechanism of membrane transporters using X-ray crystallography in combination with various biochemical and biophysical techniques (http://skirball.med.nyu.edu/faculty/da-neng-wang/wang-lab). The Skirball Institute, located in the middle of Manhattan, provides a great environment for research. Our laboratory is equipped with state-of-the-art facilities. Strong background in cell culture, eukaryotic protein expression, protein biochemistry, or structural biology is required. Send CV, statement of research interest and names of three references to Da-Neng Wang at da-neng.w...@med.nyu.edu. Da-Neng Wang, Ph.D. Professor Structural Biology Program Skirball Institute of Biomolecular Medicine New York University School of Medicine 540 First Avenue New York, NY 10016 USA Phone: (212) 263-8634 Fax: (212) 263-8951 E-mail:da-neng.w...@med.nyu.edu Web:http://skirball.med.nyu.edu/faculty/da-neng-wang/wang-lab ---
Re: [ccp4bb] non-diffracting xtals
Hi Evgeny I do have a few free cysteine residues but i am not sure if this is the problem in my case, I say that because i have crystallized this protein with another ligand without any issues and if oxidation of cysteine residues is a problem, i would imagine that it would have happened in the case where it crystallized properly. furthermore, i have other hits with the protein which gives very mozaic and twinned crystals which diffract till 2.3 Å. I am having lots of trouble processing that data and for the work i am trying to do i need to be able to produce and reproduce well diffracting crystals in a robust manner. My protein elutes as a single peak on an s-200 once i got rid of aggregates. Thanks Mahesh On Tue, Aug 20, 2013 at 11:09 AM, Evgeny Osipov e.m.osi...@gmail.comwrote: It is very similar with my situation: we are trying to crystallize Fab fragments. All we got is spherulites. After rMMS-seeding we got better shaped crystals with only 5 A resolution. So now I think that this caused by free cysteine near Fab-Fc knee. Oxidation of them leads to uncontrolled aggregation and I am trying to figure out how to protect free cysteine without reduction of disulphide bridges between heavy and light chains. So, here is the question: do you have free cys residues and are your protein is monodisperse (SE-chromatography or DLS)? P.S.: I am sorry for any mistakes in my letter because Thunderbird does not provide any grammar checking tool. 20.08.2013 04:13, Mahesh Lingaraju пишет: Hi Jürgen you are right, I did not try any major optimization yet. I only tried to vary PEG and protein concentration. That did not really improve things too much. The protein mostly forms spherulites beyond 25% PEG. I am also thinking that these crystals are poorly diffracting/not diffracting as they might be growing from sub-microscopic spherulites ? Thanks for the insights Mahesh On Mon, Aug 19, 2013 at 8:00 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Well said Petri, also how much PEG3350 do you have in your conditions ? More than 25% ? I'm going after cryo-conditions at this point, you might want to replace your PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350. Almost sounds as if no optimization of the original conditions was performed yet. Plenty to do for you, also since you have some crystal use them for seeding into your new screens. Jürgen On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote: Hi Petri They are non-diffracting at the home source and they are cryo cooled. Like david suggested I guess ill try introducing a buffer as my condition does not have a buffer. it is ammonium acetate and PEG 3350. Thanks for the encouragement ! Mahesh On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula petri.kurs...@gmail.comwrote: Hi, non-diffracting on the home source or state-of-the-art synchrotron? Cryocooled or room-temperature? What happens if you change the buffer but keep your pH? etc etc... For an important project, one should never ever give up. Petri --- Petri Kursula, PhD project leader, adjunct professor Department of Biochemistry Biocenter Oulu, University of Oulu, Finland Department of Chemistry, University of Hamburg/DESY, Germany www.biochem.oulu.fi/kursula www.desy.de/~petri/research http://www.desy.de/%7Epetri/research petri.kurs...@oulu.fi --- On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju mxl1...@psu.edu wrote: Hello people I recently obtained hexagonal rod like crystals (150x50x20 um) which turned out to be non diffracting. What is the usual convention for cases like this ? do people usually give up on the condition or still try to optimize it ? The crystals are also not very reproducible. I believe it is because of ammonium acetate in the condition causing fluctuations in the pH because of its volatility. Is there any way to work around such a problem ? Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] non-diffracting xtals
Wow! Unexpected advices! Thank you very much, for addon and Fab! Ed, you are right in both cases: about my name and protease. I think it is good idea to try to screen for crystallization conditions with bME. Mahesh, I know nothing about your object, as well as buffer conditions. May be you would try bME too? Best wishes, 2013/8/20 Mahesh Lingaraju mxl1...@psu.edu Hi Evgeny I do have a few free cysteine residues but i am not sure if this is the problem in my case, I say that because i have crystallized this protein with another ligand without any issues and if oxidation of cysteine residues is a problem, i would imagine that it would have happened in the case where it crystallized properly. furthermore, i have other hits with the protein which gives very mozaic and twinned crystals which diffract till 2.3 Å. I am having lots of trouble processing that data and for the work i am trying to do i need to be able to produce and reproduce well diffracting crystals in a robust manner. My protein elutes as a single peak on an s-200 once i got rid of aggregates. Thanks Mahesh On Tue, Aug 20, 2013 at 11:09 AM, Evgeny Osipov e.m.osi...@gmail.comwrote: It is very similar with my situation: we are trying to crystallize Fab fragments. All we got is spherulites. After rMMS-seeding we got better shaped crystals with only 5 A resolution. So now I think that this caused by free cysteine near Fab-Fc knee. Oxidation of them leads to uncontrolled aggregation and I am trying to figure out how to protect free cysteine without reduction of disulphide bridges between heavy and light chains. So, here is the question: do you have free cys residues and are your protein is monodisperse (SE-chromatography or DLS)? P.S.: I am sorry for any mistakes in my letter because Thunderbird does not provide any grammar checking tool. 20.08.2013 04:13, Mahesh Lingaraju пишет: Hi Jürgen you are right, I did not try any major optimization yet. I only tried to vary PEG and protein concentration. That did not really improve things too much. The protein mostly forms spherulites beyond 25% PEG. I am also thinking that these crystals are poorly diffracting/not diffracting as they might be growing from sub-microscopic spherulites ? Thanks for the insights Mahesh On Mon, Aug 19, 2013 at 8:00 PM, Bosch, Juergen jubo...@jhsph.eduwrote: Well said Petri, also how much PEG3350 do you have in your conditions ? More than 25% ? I'm going after cryo-conditions at this point, you might want to replace your PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350. Almost sounds as if no optimization of the original conditions was performed yet. Plenty to do for you, also since you have some crystal use them for seeding into your new screens. Jürgen On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote: Hi Petri They are non-diffracting at the home source and they are cryo cooled. Like david suggested I guess ill try introducing a buffer as my condition does not have a buffer. it is ammonium acetate and PEG 3350. Thanks for the encouragement ! Mahesh On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula petri.kurs...@gmail.comwrote: Hi, non-diffracting on the home source or state-of-the-art synchrotron? Cryocooled or room-temperature? What happens if you change the buffer but keep your pH? etc etc... For an important project, one should never ever give up. Petri --- Petri Kursula, PhD project leader, adjunct professor Department of Biochemistry Biocenter Oulu, University of Oulu, Finland Department of Chemistry, University of Hamburg/DESY, Germany www.biochem.oulu.fi/kursula www.desy.de/~petri/research http://www.desy.de/%7Epetri/research petri.kurs...@oulu.fi --- On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju mxl1...@psu.edu wrote: Hello people I recently obtained hexagonal rod like crystals (150x50x20 um) which turned out to be non diffracting. What is the usual convention for cases like this ? do people usually give up on the condition or still try to optimize it ? The crystals are also not very reproducible. I believe it is because of ammonium acetate in the condition causing fluctuations in the pH because of its volatility. Is there any way to work around such a problem ? Thanks Mahesh .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme
[ccp4bb] Why MAD didn't work but SAD works well
Hi All, I have three datasets of SeMet-incorporated protein at peak, infl and high wavelength respectively. SAD with peak dataset works well to solve the phase problem. However, MAD with all three datasets didn't work at all. The completeness of all three datasets are more than 99%. So I think radiation damage should not be a problem. Does anyone have any idea about the possible reasons that MAD didn't work in this case? Thank you so much for any of your help! Best, Yafang -- Yafang Chen Graduate Research Assistant Mesecar Lab Department of Biological Sciences Purdue University Hockmeyer Hall of Structural Biology 240 S. Martin Jischke Drive West Lafayette, IN 47907
Re: [ccp4bb] Why MAD didn't work but SAD works well
Dear Yafang, If radiation damage is not a major problem, MAD should give you more phase information than SAD, i.e. better maps, especially at low resolution. If SAD works but MAD doesn't, there are several possible explanations: 1. (most likely) your datasets are inconsistently indexed. This can hapen in various ways depending on the Laue group and the unit-cell. If you are using hkl2map the plots of the anomalous CC between the different datasets are a good quick check. Some programs (e.g. the current shelxc) will try to detect this and correct it automatically. 2. You have mixed up the wavelengths or labels of the datasets and so the dispersive difference comes out with the wrong sign. 3. You have significant radiation damage and the RIP and dispersive differences have opposite signs. Always measure the inflection dataset last so that they reinforce each other rather than canceling. 4. You have severe radiation damage and only the first (peak) dataset is usable. Best wishes, George On 08/20/2013 11:05 PM, Yafang Chen wrote: Hi All, I have three datasets of SeMet-incorporated protein at peak, infl and high wavelength respectively. SAD with peak dataset works well to solve the phase problem. However, MAD with all three datasets didn't work at all. The completeness of all three datasets are more than 99%. So I think radiation damage should not be a problem. Does anyone have any idea about the possible reasons that MAD didn't work in this case? Thank you so much for any of your help! Best, Yafang -- Yafang Chen Graduate Research Assistant Mesecar Lab Department of Biological Sciences Purdue University Hockmeyer Hall of Structural Biology 240 S. Martin Jischke Drive West Lafayette, IN 47907 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] Why MAD didn't work but SAD works well
Dear George, if #4 is correct, shouldn't he be able to get good SIRAS using the peak dataset as HA and the last collected dataset as native Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Aug 20, 2013, at 5:42 PM, George Sheldrick wrote: Dear Yafang, If radiation damage is not a major problem, MAD should give you more phase information than SAD, i.e. better maps, especially at low resolution. If SAD works but MAD doesn't, there are several possible explanations: 1. (most likely) your datasets are inconsistently indexed. This can hapen in various ways depending on the Laue group and the unit-cell. If you are using hkl2map the plots of the anomalous CC between the different datasets are a good quick check. Some programs (e.g. the current shelxc) will try to detect this and correct it automatically. 2. You have mixed up the wavelengths or labels of the datasets and so the dispersive difference comes out with the wrong sign. 3. You have significant radiation damage and the RIP and dispersive differences have opposite signs. Always measure the inflection dataset last so that they reinforce each other rather than canceling. 4. You have severe radiation damage and only the first (peak) dataset is usable. Best wishes, George On 08/20/2013 11:05 PM, Yafang Chen wrote: Hi All, I have three datasets of SeMet-incorporated protein at peak, infl and high wavelength respectively. SAD with peak dataset works well to solve the phase problem. However, MAD with all three datasets didn't work at all. The completeness of all three datasets are more than 99%. So I think radiation damage should not be a problem. Does anyone have any idea about the possible reasons that MAD didn't work in this case? Thank you so much for any of your help! Best, Yafang -- Yafang Chen Graduate Research Assistant Mesecar Lab Department of Biological Sciences Purdue University Hockmeyer Hall of Structural Biology 240 S. Martin Jischke Drive West Lafayette, IN 47907 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
