Re: [ccp4bb] How to evaluate Fourier transform ripples
Hi Conan, Specifically, the reviewers question whether ripples may affect on the electron density around heavy metal center which has a Mo-S-As connection. From which angle or in which way this problem should be addressed most convincingly ? The maximum entropy estimate of the map should be insensitive to series termination errors, and would thus answer the referee's concerns. You could give it a try with the program 'graphent' which can read mtz files and produce ccp4 map files. Whether the referee considers maximum entropy methods 'convincing' remains to be seen ... ;-) My twocents, Nicholas -- Dr Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
Re: [ccp4bb] Map Using Both Bijvoet and Dispersive Differences with Model Phases
Dear Jacob, Andrea Thorn and I will be demonstrating such a program (called AnoDe) at the Software Fayre at the IUCr Meeting in Madrid and it is already available from my beta-test site on email request. AnoDe requires only two input files: a PDB file of the refined structure and the name_fa.hkl file from SHELXC that includes the phase shift alpha that is added to the heavy atom phase to get a starting value for the native phase in experimental phasing by SAD, MAD, SIRAS etc. Anode determines the heavy atom substructure phase by subtracting alpha from the native phase that it calculates from the PDB file. The resulting 'heavy atom density' is summarised in a variety of useful ways and a file is written for Coot to display it. The advantage of this extremely simple approach is that it can be used for SAD, SIR, SIRAS, MAD and RIP phase information and that apart from SHELXC no other program, library, environment etc. are required. Best wishes, George On Wed, Jul 06, 2011 at 11:46:14AM -0500, Jacob Keller wrote: Dear Crystallographers, it seems to me that for clearly identifying/characterising anomalous scatterers for a solved structure, one could make a map using two datasets: one at the f peak, one low energy remote. One would then use the signal both from the Bijvoet differences in the peak dataset plus the differences between the peak and low-energy datasets, which I think I have seen called dispersive differences. I guess this would be like a MAD map, but using pre-existing model phases--is there such an animal in the software, or would it even be helpful? Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] Map Using Both Bijvoet and Dispersive Differences with Model Phases
I forgot to say in my email about the program AnoDe that of course it also uses the FA values from the SHELXC output file name_fa.hkl as the amplitudes for the 'heavy atom map' with phases (native-alpha). This procedure is remarkably simple and effective. George On Thu, Jul 07, 2011 at 09:46:37AM +0200, George M. Sheldrick wrote: Dear Jacob, Andrea Thorn and I will be demonstrating such a program (called AnoDe) at the Software Fayre at the IUCr Meeting in Madrid and it is already available from my beta-test site on email request. AnoDe requires only two input files: a PDB file of the refined structure and the name_fa.hkl file from SHELXC that includes the phase shift alpha that is added to the heavy atom phase to get a starting value for the native phase in experimental phasing by SAD, MAD, SIRAS etc. Anode determines the heavy atom substructure phase by subtracting alpha from the native phase that it calculates from the PDB file. The resulting 'heavy atom density' is summarised in a variety of useful ways and a file is written for Coot to display it. The advantage of this extremely simple approach is that it can be used for SAD, SIR, SIRAS, MAD and RIP phase information and that apart from SHELXC no other program, library, environment etc. are required. Best wishes, George On Wed, Jul 06, 2011 at 11:46:14AM -0500, Jacob Keller wrote: Dear Crystallographers, it seems to me that for clearly identifying/characterising anomalous scatterers for a solved structure, one could make a map using two datasets: one at the f peak, one low energy remote. One would then use the signal both from the Bijvoet differences in the peak dataset plus the differences between the peak and low-energy datasets, which I think I have seen called dispersive differences. I guess this would be like a MAD map, but using pre-existing model phases--is there such an animal in the software, or would it even be helpful? Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] How to delete loops from Protein for crystallization
Hi Obayed, I would start with doing some limited proteolysis with a few different proteases followed by Mass Spec analysis of the fragments to identify loops that are readily cut. There is lots of info and published protocols, see e.g. http://biowww.net/protocols/proteomics/mass_spectrometry/protein_digestion/ Flip Op 7/7/2011 00:51, Obayed Ullah schreef: Hi all I am thinking to delete flexible loop for crystallization of my protein. But i am not sure how to decide which area i should delete. This protein have around 20% sequence identity with other solved structure. Can anybody suggest me how to proceed for that. Is there any established strategy for that one? Obayed Ullah
Re: [ccp4bb] How to evaluate Fourier transform ripples
Dear Thierry (and others) Perhaps a bit confusing to say a sharp feature such as an heavy atom. Heavy atoms are, well, heavy (lots of electrons) rather than sharp. Any ripples around them in a Fourier truncated map could swamp the lighter atoms nearby. If Zbyszek's criteria A (and his other criteria apply), there will be no sharp cut off in the data and no significant ripples. If the data had a sharp cut off like a box function (an extreme case), each atom would have ripples around it like a sinc (sin(x)/x) function. This is (or at least used to be) a problem in small molecule crystallography if the data did not gradually fade to zero at the resolution of the calculation. I am sure this is just rewording what you are saying! Regards Colin PS Following Nicholas's suggestion, it would be interesting to compare maximum entropy and Fourier maps particularly at different resolution cut offs. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Fischmann, Thierry Sent: 06 July 2011 18:59 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to evaluate Fourier transform ripples Dear Zbyszek Wouldn't ripples be the results of calculating maps with truncated Fourier summations (unavoidably), and, consequently, be more obvious around a sharp feature such as an heavy atom metal center? The mathematic basis can be found here: http://en.wikipedia.org/wiki/Gibbs_phenomenon With best regards, Thierry Note: sent a 2nd time as it seems that it did not reach the BB the first time. Apologies if the message reaches you twice. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zbyszek Otwinowski Sent: Wednesday, July 06, 2011 1:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to evaluate Fourier transform ripples The question about Fourier transformation ripples has a straightforward answer in a fairly typical situation: A) data are collected to the resolution limit of diffraction, B) phases are uniform in quality across the resolution range, which is equivalent to R-free being uniform with respect to resolution within a factor of 2 or so, C) maps are not sharpened. The ripples originate from not including unobserved structure factors. The intensity of diffraction decreases rapidly past the measurability limit, so, in the above situation, the unobserved diffraction contributes very little. Consequently, the answer is that typically one should not see ripples. Ripples should not be confused with the effect of electron density maps being smoothed by vibrations and other forms of disorder. Zbyszek Otwinowski Dear All, Hi. I was asked in a manuscript revision to discuss about the possible effects of Fourier transformation ripples on the crystallographic results. Specifically, the reviewers question whether ripples may affect on the electron density around heavy metal center which has a Mo-S-As connection. From which angle or in which way this problem should be addressed most convincingly ? Thank you for any suggestion.Best,Conan Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 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 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.
Re: [ccp4bb] How to evaluate Fourier transform ripples
There is a relatively instructive figure to what Colin describes available from the gallery: http://www.ruppweb.org/garland/gallery/Ch9/pages/Biomolecular_Crystallography_Fig_9-05_PART1.htm For B=10, at 2.5 cutoff, ripples expected; for B=100 and 2.5 A, no ripples expected. One can also generate what Colin says using the FT calculator on my web site: Calculate first SFs with a Metal and a C and low B-factors, but cut the resolution at 2.5 or so. http://www.ruppweb.org/cgi-bin/webfac.exe?cell=20anode=Curesolution=2.5E1=Cx1=0.1N1=1.0B1=5.0E2=FEx2=0.7N2=1.0B2=5.0A1=NoOUTFILE=webfac.phsanoweb=GoGoGo ! Then generate the FT on a fine grid (for esthetics, not information conservation - see Shannon) and observe the big ripples. http://www.ruppweb.org/cgi-bin/webmap.exe?grid=100resolution=2.5INFILE=webfac.phsanoweb=GoGoGo ! (you need to select unique file names or the webserver will overwrite simultaneous submissions) An extreme case of ripples is in the Uranium atom figure. They look similar but not as excessive around real heavy atoms or even Se. http://www.ruppweb.org/garland/gallery/Ch10/pages/Biomolecular_Crystallography_Fig_10-10.htm BR On Thu, Jul 7, 2011 at 2:17 AM, Colin Nave colin.n...@diamond.ac.uk wrote: Dear Thierry (and others) Perhaps a bit confusing to say a sharp feature such as an heavy atom. Heavy atoms are, well, heavy (lots of electrons) rather than sharp. Any ripples around them in a Fourier truncated map could swamp the lighter atoms nearby. If Zbyszek's criteria A (and his other criteria apply), there will be no sharp cut off in the data and no significant ripples. If the data had a sharp cut off like a box function (an extreme case), each atom would have ripples around it like a sinc (sin(x)/x) function. This is (or at least used to be) a problem in small molecule crystallography if the data did not gradually fade to zero at the resolution of the calculation. I am sure this is just rewording what you are saying! Regards Colin PS Following Nicholas's suggestion, it would be interesting to compare maximum entropy and Fourier maps particularly at different resolution cut offs. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Fischmann, Thierry Sent: 06 July 2011 18:59 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to evaluate Fourier transform ripples Dear Zbyszek Wouldn't ripples be the results of calculating maps with truncated Fourier summations (unavoidably), and, consequently, be more obvious around a sharp feature such as an heavy atom metal center? The mathematic basis can be found here: http://en.wikipedia.org/wiki/Gibbs_phenomenon With best regards, Thierry Note: sent a 2nd time as it seems that it did not reach the BB the first time. Apologies if the message reaches you twice. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zbyszek Otwinowski Sent: Wednesday, July 06, 2011 1:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to evaluate Fourier transform ripples The question about Fourier transformation ripples has a straightforward answer in a fairly typical situation: A) data are collected to the resolution limit of diffraction, B) phases are uniform in quality across the resolution range, which is equivalent to R-free being uniform with respect to resolution within a factor of 2 or so, C) maps are not sharpened. The ripples originate from not including unobserved structure factors. The intensity of diffraction decreases rapidly past the measurability limit, so, in the above situation, the unobserved diffraction contributes very little. Consequently, the answer is that typically one should not see ripples. Ripples should not be confused with the effect of electron density maps being smoothed by vibrations and other forms of disorder. Zbyszek Otwinowski Dear All, Hi. I was asked in a manuscript revision to discuss about the possible effects of Fourier transformation ripples on the crystallographic results. Specifically, the reviewers question whether ripples may affect on the electron density around heavy metal center which has a Mo-S-As connection. From which angle or in which way this problem should be addressed most convincingly ? Thank you for any suggestion.Best,Conan Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 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 Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally
Re: [ccp4bb] how to distinguish between phase separation, spherulite, and microcrystalline?
jobey, the last two are kind of phase separation. In some cases the oil like droplets are more in number througout the drop. First two are microcrystalline but the second one is more looks like made from protein precipitate. hope this helps if needed please refer to Terese Bergfors website at here http://xray.bmc.uu.se/terese/ hope this helps Padayatti PS On Wed, Jul 6, 2011 at 6:35 PM, joybeiyang joybeiy...@gmail.com wrote: Dear All, I am having difficulties in distinguishing phase separation, spherulite and microcrystalline in some of my drops. I have attached the photos of these drops, would someone kindly help me with this? (The names of the photos already show my uncertainty, so just skip the description here) Thank you so much in advance. Best, Bei 2011-07-06 joybeiyang -- Pius S Padayatti,PhD, Phone: 216-658-4528
[ccp4bb] Postdoctoral Research Associate in X-ray and neuron protein crystallography
Postdoctoral Research Associate in X-ray and neuron protein crystallography at Oak Ridge National Laboratory, USA Reference Code ORNL11-93-NSSD Eligibility Requirements Degree: Doctoral Degree received within 60 months or currently pursuing. Description The main research objective of this project is to re-engineer enzymes involved in the xylose sugar pathway to improve their activity and stability under conditions involved in the efficient production of biofuels from lignocellulosic biomass. This is a multi-task, multi-disciplinary effort involving molecular biology, X-ray and neutron crystallography, rational design using computer modeling techniques, and enzyme activity assays. The successful candidate for this position will be involved in the protein crystallography component of the project which will be based at the DOE BER funded Center for Structural Molecular Biology at ORNL. Specifically, the postdoc will optimize crystal growth crystallographic data collection. X-ray data will be collected using an in-house X-ray system and neutron data will be collected using neutron beam lines that are becoming available at the two world-leading neutron scattering facilities run by ORNL (HFIR and SNS). Protein structure refinement using both X-ray and neutron data will be carried out using CNS and PHENIX software packages. From the mechanistic insights arising from the crystallographic structures, specific amino acid residues will be targeted for mutagenesis. Model building studies will be used to predict consequences of mutating residues with varying sizes and charge properties and mutant proteins will be assayed for improved properties. Qualifications The successful candidate will have a PhD in protein crystallography or a related field and have experience in molecular biology techniques associated with plasmid-based expression systems, protein purification, and protein crystallization. Furthermore, the successful candidate will also have experience with crystallographic data collection and analysis, and protein structure determination and refinement. An ability to work in a collaborative team that includes molecular biologists, enzyme scientists, beam lines scientists, computational crystallographers, and collaborators from other institutes is essential. Please apply at https://www3.orau.gov/ORNL_TOppS/Posting/Details/155 For informal questions please contact Leighton Coates and Paul Langan at leighton0coa...@gmail.com
Re: [ccp4bb] Unexplained density near cobalt
hey Mischa I would guess that is a split cobalt/metal site occupancy 0.1 and 0.9 or something like that. If you calculate an anomalous difference map you may be able to confirm/reject that suggestion, depending on the strength of the anomalous signal. cheers Preben On 07.07.2011, at 17:07, Machius, Mischa Christian wrote: Y'all, I was wondering if anyone had any thoughts about a feature we observe with a metal-binding site: we have a cobalt that is bound by four histidines and one carboxyl group. There is extra density near the cobalt. See pictures below. The extra density spans the NE2 atoms from two histidines. The Fo-Fc peak (green) has a height of up to 10 sigma (eight molecules in the asymmetric unit, all showing the same feature). I placed a water molecule into the density to get some distances: the distances between the peak and the neighboring histidine NE2 atoms is ~1.8Å and ~2.0Å, resp. The distance between the peak and the cobalt is ~1.7Å. The resolution is 1.24Å. Any input would be greatly appreciated. Many thanks in advance! Cheers! MM Screen shot 2011-07-07 at 9.44.43 AM.pngATT1.cScreen shot 2011-07-07 at 9.44.55 AM.png J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] Unexplained density near cobalt
We have a manganese binding protein that binds two Mn ions in a binuclear complex. It turns out that one of the metal ions can move about 2.0 Å depending on crystallization data collection conditions (check out PDB files 1ON1 and 2F5D for the alternate conformations). In some instances we could see both positions occupied within the same crystal (it looked a lot like what you are seeing under those circumstances). Peaks in the anomalous difference Fourier maps were the clearest evidence that it was manganese in each of the positions. Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA On Jul 7, 2011, at 8:07 AM, Machius, Mischa Christian wrote: Y'all, I was wondering if anyone had any thoughts about a feature we observe with a metal-binding site: we have a cobalt that is bound by four histidines and one carboxyl group. There is extra density near the cobalt. See pictures below. The extra density spans the NE2 atoms from two histidines. The Fo-Fc peak (green) has a height of up to 10 sigma (eight molecules in the asymmetric unit, all showing the same feature). I placed a water molecule into the density to get some distances: the distances between the peak and the neighboring histidine NE2 atoms is ~1.8Å and ~2.0Å, resp. The distance between the peak and the cobalt is ~1.7Å. The resolution is 1.24Å. Any input would be greatly appreciated. Many thanks in advance! Cheers! MM Screen shot 2011-07-07 at 9.44.43 AM.pngATT1.cScreen shot 2011-07-07 at 9.44.55 AM.png
[ccp4bb] PDB40 - Celebrating the 40th birthday of the PDB
The Worldwide Protein Data Bank (wwPDB; wwpdb.org) is organising a scientific symposium in Cold Spring Harbor this autumn to celebrate the 40th anniversary of the inception of the PDB and the many scientific contributions it archives. The meeting (PDB40) will kick off with an evening reception and plenary session on Friday 28 October and conclude after lunch the following Sunday. For details and registration, please see the meeting website at: http://meetings.cshl.edu/meetings/pdb40.shtml The program will showcase the scientific impact made by structural biology during the past 40 years with a distinguished panel of scientists who have been instrumental in the development of the PDB and structural biology. A list of all confirmed speakers and the titles of their presentations is now on the website. There are limited funds available to help a number of students and early-career scientists to attend PDB40. To apply for an award, please follow the instructions on the website. Applications are due by August 1, 2011. We hope to see many of you at the symposium! On behalf of wwPDB, --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] Unexplained density near cobalt
Could be a hexacoordinated cobalt with a water molecule (or a hydroxyl ion) depending on the chemical environment... Artem On Thu, Jul 7, 2011 at 10:07 AM, Machius, Mischa Christian mach...@med.unc.edu wrote: Y'all, I was wondering if anyone had any thoughts about a feature we observe with a metal-binding site: we have a cobalt that is bound by four histidines and one carboxyl group. There is extra density near the cobalt. See pictures below. The extra density spans the NE2 atoms from two histidines. The Fo-Fc peak (green) has a height of up to 10 sigma (eight molecules in the asymmetric unit, all showing the same feature). I placed a water molecule into the density to get some distances: the distances between the peak and the neighboring histidine NE2 atoms is ~1.8Å and ~2.0Å, resp. The distance between the peak and the cobalt is ~1.7Å. The resolution is 1.24Å. Any input would be greatly appreciated. Many thanks in advance! Cheers! MM
[ccp4bb] Potential Space Group Issue
Hello Everyone, I have a 3.1 Ang dataset for which I'd like to get to the bottom of what the correct space group is. The current unit cell in p212121 is 98.123 101.095 211.20190.000 90.00090.000 I fed the reflection data into Xtriage to look for twinning and pseudotranslational NCS and there is no indication for either issue in the Xtriage output. Also, all odd 00h, 00k, 00l reflections are systematically absent as they should be for p212121. However, my colleague who is also working on the same dataset recently reprocessed the data in P21. Here's the cell in p21: 98.010 100.940 210.470 90.00 90.04 90.00 p21 I am not sure if BETA=90.04 is significant enough to treat as p21 (0.04% deviation of beta angle from ideal lattice for p212121). I don't think so but I could be wrong. Could someone please clarify? Also, what kind of twinning and twinning operators can relate a p212121 cell to a p21 cell with almost identical unit cell parameters as that of the p212121 cell and leave all systematic absences intact? Thanks much. Raji --- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
