Re: [ccp4bb] protein quasicrystals?
Hi Yu, Have you tried using the Additive Screen (e.g. from Hampton Research) on top of your crystallization condition, and varying the ratio between protein complex and crystallization solution? From the diffraction pattern, it looks you are almost there. Salt crystals don't show spots close to the beam stop usually, so the chance of them being salt crystals is negligible. A little bit of tweaking in crystallization conditions should do the trick. If all this fails, probably, you will have to remove any floppy ends from the proteins in the new constructs. I had a similar experience where the protein with His-tag showed crystals in a lot of conditions but all spherical and cylindrical crystals diffracting to 3.5 A. The protein after removing the His-tag gave crystals that diffracted to 2.4 A. Good luck! -Gyan On Tue, Feb 13, 2018 at 9:09 AM, Yu Qiu <yu@sanofi.com> wrote: > Hi, > > > > I have been trying to crystallize a protein complex and keep getting > sphere shape crystals. The diffraction is around 3 angstrom, but looks like > multiple lattices. I am wondering if it could be a quasi crystal? Is there > anyone has such experience? > > > > Thanks, > > Yu > -- Gyanendra Kumar, PhD Associate Scientist, St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Cell: 631-875-9189 https://www.linkedin.com/in/gyanendrakumar https://scholar.google.com/gyanendrakumar <https://scholar.google.com/citations?user=lnil0IQJ=en> https://www.researchgate.net/profile/Gyanendra_Kumar3 http://stjude.academia.edu/GyanendraKumar ---
Re: [ccp4bb] Protein or DNA crystals
Hi Joseph, Are you having trouble getting bigger crystals. You said you shot them, and they are not salt. Do they diffract at all? If they are diffracting to any extent, you could optimize the crystallization condition to get better, bigger crystals and shoot them. A simple way of growing fewer, bigger crystals in the same condition is to set up bigger drops and dilute the well solution by water to 95%, 90%, 85%... And if that doesn't work, you can try additive screen on this current crystallization condition. As a control for determining if these are complex crystals or just DNA crystals, you can set up a crystallization in the same crystallization condition with just DNA, no protein. -Gyan On Mon, Jun 19, 2017 at 9:20 AM, Joseph Ho <sbddintai...@gmail.com> wrote: > Dear all: > > I would like to seek your opinion on our crystal hits. We are working > on protein/dsDNA complex. By changing different protein and DNA > (14-22bp) constructs, we recently got some hits from commercial > screens using sitting drop vapor diffusion (very small xtals). The > precipitant is PEG and the picture of crystals are attached. In this > particular condition, it is 30%PEG3350, sodium succinate pH5.5 and > 100mM NaCl. The crystal seems floating and sit in the bottom. We do > some test shot from other conditions and it is not salt crystals. The > crystals can suck in izit dye. I do some google and it seems izit dye > also turns dsDNA crystal into blue. We also do UV/Vis microscope but > no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp. > > This is our first time to work on protein/DNA complex crystals and we > are not certain if this is just DNA or protein/DNA crystals. Can you > provide your comments on our hits? > > Thank you for your help > > Joseph > -- Gyanendra Kumar, PhD Associate Scientist, St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Cell: 631-875-9189 https://www.linkedin.com/in/gyanendrakumar https://scholar.google.com/gyanendrakumar <https://scholar.google.com/citations?user=lnil0IQJ=en> https://www.researchgate.net/profile/Gyanendra_Kumar3 http://stjude.academia.edu/GyanendraKumar ---
[ccp4bb] Display issues with CCP4-7 and Coot on Zalman with MacBook Pro
Dear All, Yesterday I started using the new MacBook Pro running on Mac OS 10.12.2, that has only USB-C ports. I also use a Zalman 3D monitor as an additional display with it attached through an adapter. Somehow CCP4 and Coot are not displaying on the Zalman Monitor (the ccp4 and coot windows disappear, other windows like Chrome browser or microsoft office windows appear fine). When I disconnect the monitor and use only the MacBook display, the two programs work fine. I must mention here, I did not have this issue with my previous MacBook Pro that had all the different ports like ethernet, USB, thunderbolt etc, running on Mac OS 10.7. Has anyone faced a similar issue and found a solution? thanks! -Gyan -- Gyanendra Kumar, PhD Associate Scientist, St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Cell: 631-875-9189 https://www.linkedin.com/in/gyanendrakumar https://scholar.google.com/gyanendrakumar <https://scholar.google.com/citations?user=lnil0IQJ=en> https://www.researchgate.net/profile/Gyanendra_Kumar3 http://stjude.academia.edu/GyanendraKumar ---
Re: [ccp4bb] Ambiguous metal ion at active site.
Dear Dilip, I was wondering if the problem was more basic, just about setting the occupancy in your input pdb file. Try setting the occupancy of Mg to 1 in your input coordinate file (pdb file) and refine it. If its Mg, it should turn blue. If its not Mg, the density should turn red instead of staying green, or may be partially green. Next, try Mn with occupancy 1 in the input file. Did you have Calcium chloride in your crystallization condition? You could try Ca as well. -Gyan On Thu, Jul 9, 2015 at 4:35 AM, Dilip Kumar dku...@igib.in wrote: Dear All I have solved a structure of a metal-ion dependent exonuclease enzyme. In homologous structures, two or three Manganese ions are present at catalytic center. However, I have used 2 mM MgCl2 in protein purification buffer. I tried to fit both of these metal ions at catalytic center but in both cases it still shows green density (Sigma level ~ 7) in difference map and low b-factor (10) for these metal ions. For better understanding I have attached the screenshot of metal ions with difference map on. Please suggest me the possible reasons or methods to validate the presence of any other metal ions at catalytic center. Thanks in advance. Regards Dilip Kumar Research Associate Chemical and Systems Biology Unit CSIR-Institute of Genomics Integrative Biology Delhi-110025 -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Skin on drops
Hi Adam, The diffraction improved from 8 Angstrom to 2.6 Angstrom. But, I had also removed the His-tag from the protein, so I am not sure how much was the role of this step and how much was it because of the addition of DTT. Two variables in one experiment :). -Gyan On Wed, Feb 25, 2015 at 4:18 PM, Adam Brummett adam-brumm...@uiowa.edu wrote: Gyan, With the addition of DTT to remove the skin, did you see an increase in the resolution with the skin no longer present compared to with it still there? -Adam On Feb 25, 2015, at 11:15 AM, Gyanendra Kumar gyanendr...@gmail.com wrote: Adding DTT in your protein buffer or crystallization solution may also help. You could try increasing amounts of DTT/BME/TCEP in your crystallization solution and find a balance between reduction of skin formation vs getting crystals. Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on the drop in which the crystals were tightly embedded. -Gyan On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut han_c...@yahoo.co.uk wrote: Dear Ulrike, you could try avoid the drop-air interface by overlying sitting drops with silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will alter the kinetics with which your drops reach equilibrium, and hence may alter your ability to get crystals of the protein. Batch crystallization under oil is another option of course. Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO for example) in your crystallization conditions may also be something to consider. If none of these work, I'd concentrate on harvesting the crystals from the skin. I tend to cut these open from the side, flip over the skin so that one has better access to the crystals that generally are associated with the inner face of the skin. You can try peel the crystals off the skin, or cut out a piece of skin surrounding a crystal. That will not hurt diffraction quality of the crystals. Hope that helps. Han On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: Dear crystallographers, I am trying to crystallize a soluble protein which tends to form aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the crstallization process a thick skin is formed on top of the sitting-drops. As well the crystals are buried in precipitate. Before I start harvesting I try to remove the skin but still it is hardly possible to get any crystals out of these drops. Any suggestions how to avoid the formation of skin on crystallization drops ? Cheers, Ulrike Han Remaut, PhD Laboratory of Structural Molecular Microbiology VIB / Vrije Universiteit Brussel Building E4, Pleinlaan 2 1050 Brussel han.rem...@vib-vub.be tel. +32-2-629 1923 / +32-499 708050 http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 --- -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] Skin on drops
Adding DTT in your protein buffer or crystallization solution may also help. You could try increasing amounts of DTT/BME/TCEP in your crystallization solution and find a balance between reduction of skin formation vs getting crystals. Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on the drop in which the crystals were tightly embedded. -Gyan On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut han_c...@yahoo.co.uk wrote: Dear Ulrike, you could try avoid the drop-air interface by overlying sitting drops with silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will alter the kinetics with which your drops reach equilibrium, and hence may alter your ability to get crystals of the protein. Batch crystallization under oil is another option of course. Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO for example) in your crystallization conditions may also be something to consider. If none of these work, I'd concentrate on harvesting the crystals from the skin. I tend to cut these open from the side, flip over the skin so that one has better access to the crystals that generally are associated with the inner face of the skin. You can try peel the crystals off the skin, or cut out a piece of skin surrounding a crystal. That will not hurt diffraction quality of the crystals. Hope that helps. Han On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: Dear crystallographers, I am trying to crystallize a soluble protein which tends to form aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the crstallization process a thick skin is formed on top of the sitting-drops. As well the crystals are buried in precipitate. Before I start harvesting I try to remove the skin but still it is hardly possible to get any crystals out of these drops. Any suggestions how to avoid the formation of skin on crystallization drops ? Cheers, Ulrike Han Remaut, PhD Laboratory of Structural Molecular Microbiology VIB / Vrije Universiteit Brussel Building E4, Pleinlaan 2 1050 Brussel han.rem...@vib-vub.be tel. +32-2-629 1923 / +32-499 708050 http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] skin on crystal
Try adding b-ME/DTT/TCEP in your crystallization conditions, if its not there already in your protein buffer. On Mon, Jan 13, 2014 at 2:02 PM, Debasish Chattopadhyay debas...@uab.edu wrote: We crystallized a protein at 4 and 22 deg C in different conditions: from ammonium sulfate in acetate buffer pH 5 and PEG4000 in Hepes buffer at pH 7.5 In both cases the drops have a slimy skin (almost feels like DNA). We therefore think that the skin is generated from the protein. I am sure some of you have had similar experiences. I would like your suggestions about how to avoid the skin. Please note that we are *not* asking for suggestions on how to handle the skin (such as using various tools) *we are only interested in knowing if there is a way to prevent the formation of the skin*. Thank you so much Debasish Ph: (205)934-0124; Fax: (205)934-0480 -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] noncovalent interactions
You could try LPC CSU Server: http://bip.weizmann.ac.il/oca-bin/lpccsu -Gyan On Wed, May 12, 2010 at 1:02 PM, gauri misra kamga...@gmail.com wrote: Hi, Which free online servers are best for calculation of non covalent interactions present in several pdb structures? Thanks for any insights provided by the community members... Cheers GM
