Re: [ccp4bb] unexplained density
Hi, You probably tried it but what happens if you fit in PMSF and refine? Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Annemarie Weber [annemarie.we...@uni-konstanz.de] Sent: Monday, February 03, 2014 6:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unexplained density Dear all, I am refining a 1.4 A resolution structure and found some well-defined but unfortunately unexplained density. The protein was purified in HEPES buffer with PMSF and protease inhibitor cocktail tablets (Roche) added. It was cocrystallized with AMPPNP in PEG2000MME and Tris. I modelled a molecule into the density, which fits quite well but I do not know what it is and how it got there. Attached are two pictures with the difference density and the molecule I modelled in there. Has anybody seen something like this before? Maybe some degradation product of the buffer/PMSF? Any suggestions will be highly welcome. Thanks a lot Annemarie
Re: [ccp4bb] unexplained density
What does anomalous difference Fourier look like? -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Annemarie Weber Sent: Monday, February 03, 2014 11:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unexplained density Dear all, I am refining a 1.4 A resolution structure and found some well-defined but unfortunately unexplained density. The protein was purified in HEPES buffer with PMSF and protease inhibitor cocktail tablets (Roche) added. It was cocrystallized with AMPPNP in PEG2000MME and Tris. I modelled a molecule into the density, which fits quite well but I do not know what it is and how it got there. Attached are two pictures with the difference density and the molecule I modelled in there. Has anybody seen something like this before? Maybe some degradation product of the buffer/PMSF? Any suggestions will be highly welcome. Thanks a lot Annemarie 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] unexplained density
It looks like a Hepes? Padayatti On Mon, Feb 3, 2014 at 8:37 AM, Annemarie Weber annemarie.we...@uni-konstanz.de wrote: Dear all, I am refining a 1.4 A resolution structure and found some well-defined but unfortunately unexplained density. The protein was purified in HEPES buffer with PMSF and protease inhibitor cocktail tablets (Roche) added. It was cocrystallized with AMPPNP in PEG2000MME and Tris. I modelled a molecule into the density, which fits quite well but I do not know what it is and how it got there. Attached are two pictures with the difference density and the molecule I modelled in there. Has anybody seen something like this before? Maybe some degradation product of the buffer/PMSF? Any suggestions will be highly welcome. Thanks a lot Annemarie -- P
Re: [ccp4bb] unexplained density
*** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Annemarie, This could be a reaction product of the PMSF with either tris or alanine (as you have modeled, but you should know the source). Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Annemarie Weber annemarie.we...@uni-konstanz.de To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 3 Feb 2014 17:37:00 +0100 Subject: [ccp4bb] unexplained density *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear all, I am refining a 1.4 A resolution structure and found some well-defined but unfortunately unexplained density. The protein was purified in HEPES buffer with PMSF and protease inhibitor cocktail tablets (Roche) added. It was cocrystallized with AMPPNP in PEG2000MME and Tris. I modelled a molecule into the density, which fits quite well but I do not know what it is and how it got there. Attached are two pictures with the difference density and the molecule I modelled in there. Has anybody seen something like this before? Maybe some degradation product of the buffer/PMSF? Any suggestions will be highly welcome. Thanks a lot Annemarie --- End of Original Message --- - Note: The information contained in the e-Mail message and/or attachments to it may contain confidential or privileged information. If you are not the intended recipient, any dissemination, use, review, distribute, prinitng or copying of the information contianed in this e-Mail message and/or attachments to it are strictly prohibited. If you have received this communication in error. Please notify us by reply e-Mail or telephone and immediately and permanently delete the message and any attachment. Thank you.
Re: [ccp4bb] Unexplained density near cobalt
Are the imidazole rings of the histidines distorted? If they are, it could be water/hydroxide. If not, it is probably a cobalt ion side show. Cheers, Rob Meijers EMBL Hamburg --- On Thu, 7/7/11, Artem Evdokimov artem.evdoki...@gmail.com wrote: From: Artem Evdokimov artem.evdoki...@gmail.com Subject: Re: [ccp4bb] Unexplained density near cobalt To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, July 7, 2011, 9:39 PM 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
Re: [ccp4bb] Unexplained density near cobalt
Hi MM, Co in its +2 oxidation state typically forms a tetrahedrally coordinated species in high pH environment; and pentagonally coordinated species with five ligands at low pH. A Co ion in its +3 oxidation state forms a octahedrally coordinated species with six ligands. Co +3 is unusual in biological molecules. However it is seen in crystal structures, sometimes with partial occupancy and is a likely product of radiation damage caused by the X-ray beam. In the past I have found that Co+2 is prone to oxidation in the beam if the crystallizing condition has a pH lower than 6.5. Here is a article you might enjoy. Comparison of solution and crystal properties of Co(II)-substituted human carbonic anhydrase II. Avvaru BS, Arenas DJ, Tu C, Tanner DB, McKenna R, Silverman DN. Cheers Balu
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
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
Re: [ccp4bb] Unexplained density
Dear Daniel Is the omit map for the model with and without the zinc ions possible to confirm its existence? I am curious. What is the resolution of dataset? stephen Quoting Daniel Bonsor bon...@bbri.org: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan -- Dr. Stephen Sin-Yin Chui Research Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)
Re: [ccp4bb] Unexplained density
Hi, Anomalous map might give you some idea if it can be zinc. ~L~ __ Lari Lehtiö Pharmaceutical Sciences, Department of Biosciences Åbo Akademi University, BioCity, FIN-20520 Turku Finland +358 2 215 4270 http://www.users.abo.fi/llehtio/ __ Quoting Dr. STEPHEN SIN-YIN, CHUI chui...@hkucc.hku.hk: Dear Daniel Is the omit map for the model with and without the zinc ions possible to confirm its existence? I am curious. What is the resolution of dataset? stephen Quoting Daniel Bonsor bon...@bbri.org: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan -- Dr. Stephen Sin-Yin Chui Research Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)
Re: [ccp4bb] Unexplained density
The figures would be more helpful if you told us what each map contour represented, and at what contour level. I assume blue is 2fo-fc and green+red is fo-fc, positive and negative. It would be even more helpful to have the maps to turn over in 3D, but I understand you would probably be reluctant share that. The density looks much too strong to be mere water. Acidic surface residues may bind zinc. The metals might appear at multiple alternate locations, if the occupancies are less than 1.0. Synchrotron anomalous maps would be helpful in seeing Zinc, whose K edge is at 1.28 A. Multiple alternate locations are likely to show up at special symmetry positions. I have the impression this may be a special position in your map, but I can't be certain (2-fold axis?). Zinc and cacodylate may form cage complexes which can be bound between multiple surface acid resides (personal observation, not published). The complexes I have seen have one zinc at each pole with three cacodylates around the equator. Your density does not look especially similar to the complexes I have seen. Cacodylate also has a pretty good peak on anomalous maps, and since it's peak is at 1.04 A, anomalous maps at multiple wavelengths could distinguish between arsenic and zinc. On 04/29/10 17:38, Daniel Bonsor wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Unexplained density
I think cacodilate is less likely because it's negatively charged as the carboxy groups that surround the density. I think it's Zn or it may be another (endogenous) metal ion like Ca. You need to look at the coordination. Maia David Schuller wrote: The figures would be more helpful if you told us what each map contour represented, and at what contour level. I assume blue is 2fo-fc and green+red is fo-fc, positive and negative. It would be even more helpful to have the maps to turn over in 3D, but I understand you would probably be reluctant share that. The density looks much too strong to be mere water. Acidic surface residues may bind zinc. The metals might appear at multiple alternate locations, if the occupancies are less than 1.0. Synchrotron anomalous maps would be helpful in seeing Zinc, whose K edge is at 1.28 A. Multiple alternate locations are likely to show up at special symmetry positions. I have the impression this may be a special position in your map, but I can't be certain (2-fold axis?). Zinc and cacodylate may form cage complexes which can be bound between multiple surface acid resides (personal observation, not published). The complexes I have seen have one zinc at each pole with three cacodylates around the equator. Your density does not look especially similar to the complexes I have seen. Cacodylate also has a pretty good peak on anomalous maps, and since it's peak is at 1.04 A, anomalous maps at multiple wavelengths could distinguish between arsenic and zinc. On 04/29/10 17:38, Daniel Bonsor wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan
Re: [ccp4bb] Unexplained density
It could be a cacodylate bridging between two zincs. I would want to know if anomylous maps show a third peak in the middle. On 04/30/10 12:41, Maia Cherney wrote: I think cacodilate is less likely because it's negatively charged as the carboxy groups that surround the density. I think it's Zn or it may be another (endogenous) metal ion like Ca. You need to look at the coordination. Maia -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Unexplained density
It's hard to see clearly the density, but judging from the abundance of carboxy groups, it may be a metal. Maia Daniel Bonsor wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan
Re: [ccp4bb] Unexplained density
The close approach of the two Zinc atoms may not be too implausible if the charges are being quenched by coordination of the negatively charged residues. William Ho On Thu, Apr 29, 2010 at 5:38 PM, Daniel Bonsor bon...@bbri.org wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan
Re: [ccp4bb] Unexplained density
I think it could be zinc ion. On Fri, Apr 30, 2010 at 5:38 AM, Daniel Bonsor bon...@bbri.org wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan -- Best regards, Xianhui
Re: [ccp4bb] Unexplained density
While it would not be unreasonable for zinc ions to be coordinated to the asp residues (perhaps with bridging waters or hydroxides), I am a little troubled by the lack of electron density around the coordinating residues. If there are zinc ions tightly coordinated at full occupancy, I would normally expect the coordinating residues to be more well-defined, with fairly low b-factors. If you want to play with zincs, I would start by placing two zincs in the blob ends of the density, and constraining the Zn-O links in refmac so the metals don't wander. (The Zn-O bond should be about 2.0 A, and refmac gets this right when you generate the constraints cif file.) Zn(II) typically likes to be four-coordinate, approximately tetrahedral, and usually likes at least one or two softer ligands, like His or Cys. Looks like that can't be the case here--all hard ligands, carboxylates and maybe waters. Cheers. On 4/29/2010 5:38 PM, Daniel Bonsor wrote: Hello again I currently have some unexplained density in my structure. As you can hopefully see from the images (see file), the density is dumbbell shaped. Whatever it is, it is coordinated by Asp and Glu residues. To me it looks like each lobe is a ring structure. The crystallization condition was: 6.5% PEG 8K, 10mM ZnSO4, 100mM sodium cacodylate pH 6.5, 100mM Am2SO4, 1% glycerol, with 20% glycerol as cryo. Protein was originally in 50mM Tris, 50mM NaCl pH 7.5. I originally placed a single Zn at the center of each lobe. Though after refmac, the Zn was displaced to one side. Two zincs in each dumbbell may have worked, but I am dubious about two zinc atoms being 4A apart and there is still some unexplained density. Are there any possible cyclization reactions of Tris, cacodylate or glycerol may have undergone to explain the density? Or is it simply a highly ordered water network? Or is there some other explanation? Thanks in advance Dan -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
