[ccp4bb] A new detector for my HKL2000 package
Dear all, I have collected a dataset on a Saturn 944HG CCD detector, and I want to process the data on HKL2000. The detector is not found in the list of the HKL2000 pop-up window in the beginning, what should I do to make the HKL2000 package recognize my dataset? I'm a computer dummy, so would you please tell me the way for dummies? Sincerely, Pattis ~(He's a junior crystallographer!)~
[ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment
Dear All, 3D structure modeling server I-TASSER predicts a binding site for NADPH and I want to test this prediction. What would be the nice quick way to tell whether this protein bind NADPH or not, when I have a lot of recombinant protein? Sincerely, Xuan Yang
Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment
Dear Jacob, Nanodrop ultrafiltration sounds really fancy to me. I am afraid that I have no access to such equipment yet. Thanks for letting me know about this new technology. Sincerely, Xuan Yang 2010/8/4 Jacob Keller j-kell...@fsm.northwestern.edu I like nanodrop ultrafiltration: concentrate your protein to the highest stable concentration possible figure out what is the lowest possible robustly-detectable nadph signal on your nanodrop combine the two in such a way in the top of a microcon of appropriate MWCO to acheive the highest possible protein concentration with the lowest possible nadph concentration. Take a baseline spec reading before spinning. spin long enough to get enough flowthrough to measure on the nano (~10uL is plenty.) Flowthrough should be the free nadph concentration L. Total L should be known, as well as total P, so you can figure out bound concentrations PL easily. you should probable do this in triplicate or so, with appropriate controls. I found 50uL/microcon to be a good balance of pipette-ability and economy of protein. If you want to get fancier, you can do more samples varying concentrations (or do some other more sophisticated method.) Jacob On Wed, Aug 4, 2010 at 3:10 AM, Xuan Yang pattisy...@gmail.com wrote: Dear All, 3D structure modeling server I-TASSER predicts a binding site for NADPH and I want to test this prediction. What would be the nice quick way to tell whether this protein bind NADPH or not, when I have a lot of recombinant protein? Sincerely, Xuan Yang
Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment
Dear Jürgen, Thanks for the feedback. Differential Scanning Fluorimetry is a nice method but my current goal is to identify the nature of the cofactor if any. I was wondering whether MassSpec could be of help. Sincerely, Xuan 2010/8/4 Jürgen Bosch jubo...@jhsph.edu Check the thermal stability with and without your ligand. You could do this via CD or with the help of Sypro Orange in a RT-PCR machine. Jürgen .. 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.mac.com/bosch_lab/ On Aug 4, 2010, at 4:10, Xuan Yang pattisy...@gmail.com wrote: Dear All, 3D structure modeling server I-TASSER predicts a binding site for NADPH and I want to test this prediction. What would be the nice quick way to tell whether this protein bind NADPH or not, when I have a lot of recombinant protein? Sincerely, Xuan Yang
[ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Active aggregates?
Dear James, Could you provide the reference of your success story? My protein also formed large soluble aggregates and I am desperate for such successful stories! By the way, have your performed DLS to your protein? What about the polydispensity? Is it lower than 20%? Thanks in advance! Sincerely, Xuan Yang 2009/8/27 James Stroud xtald...@gmail.com Just try crystallizing it. What is a crystal but a massive aggregate? That they are still soluble and active is great news. As a grad student, I had a similar phenomenon with an early project. I showed a gel in group meeting where both activity and aggregation were obvious, said the aggregate was no problem, got ridiculed when I said I was going to throw it in trays despite what anyone said, had giant crystals after a few trays, and solved the structure with miras. Get the structure and then worry about why it's aggregating. The structure will probably provide you with the clues you need. On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote: Dear all, This is a question on how to cope with the protein that seems to form massive aggregates in solution but enzymatically active. I'm working on a protein whose molecular weight is around 70kDa and can be divided into two domains (say A and B domains). We expressed this protein in E.coli fused with GST and purified using some chromatography. The GST affinity chromatography works well and proteinase digestion to remove the tag does wonders too. The purified protein was confirmed to be active enough, we can detect both activities from these two domains. But the retention time from the gel filtration clearly shows it is awfully aggregated (comes out at the void region). DLS measurement indicates the averaged diameter is around 45 nm, which I feel is a bit too long. Analytical ultracentrifuge result implies that the distribution of the molecular species is wide, some portion got precipitated with 1K rcf (means the molecular weight is more than 5MDa) and the rest is ranging from 1MD to 5MDa with a peak at 1MDa. I made new two constructs covering the A and B domains respectively, both of them are active again, but only the A domain has got the same symptom as the intact protein. The B domain seems to exist as a monomer in solution. Here come my questions, (I) How can I interpret this phenomenon? (II) Is there anything we can try to change the situation? (III) Does it make sense to try crystallization? (probably not).(IV) Has anyone got such experience? I tried the methylation on lysine side chains, I also tried the buffer with 0.2M arginine or 10% glycerol but the all the results just seem hopeless. The protein before the proteinase treatment also comes out at the void region from the gel filtration. cheers toyoyuki --? Toyoyuki Ose o...@sci.hokudai.ac.jp Graduate School of Life Science Hokkaido University N21W11 Kita-ku, Sapporo 001-0021 Japan
Re: [ccp4bb] Active aggregates?
Dear Ose, I think we have met similar problems. My protein is relatively small, ~17kD, but it could form aggregates larger than 100nm. A classic Protein Science paper in 2003 by DR Smith titled Crystal structures of fusion proteins with large-affinity tags offered me great help. With MBP tag and optimized linker my protein became significantly less aggregated (~10nm). However, this is still far from a guarantee for success since it is still quite difficult to obtain crystals, although Jovine's group reported a recent success about ZP-N. By the way, can you observe a monomer peak during ultracentrifuge? In terms of DLS, how about the polydispensity? HPV E6 also aggregates to similar size, but the granules were relatively homogeneous. More importantly, it was observed that in cell the protein could still form such large aggregates. Maybe for some proteins, they were just meant to be like this. And based on my very limited knowledge, most structures of such proteins remained as the higher-hanging fruit. Last but not the least, J Jancarik, et al. designed a elegant protocol (2004，Acta Cryst. D60. 1670-1673.) to find the optimized buffer that could increase the solubiltiy and homogeneity, whichmight be helpful too. Good luck! Xuan Yang 2009/8/27 ose toyoyuki o...@castor.sci.hokudai.ac.jp Dear all, This is a question on how to cope with the protein that seems to form massive aggregates in solution but enzymatically active. I'm working on a protein whose molecular weight is around 70kDa and can be divided into two domains (say A and B domains). We expressed this protein in E.coli fused with GST and purified using some chromatography. The GST affinity chromatography works well and proteinase digestion to remove the tag does wonders too. The purified protein was confirmed to be active enough, we can detect both activities from these two domains. But the retention time from the gel filtration clearly shows it is awfully aggregated (comes out at the void region). DLS measurement indicates the averaged diameter is around 45 nm, which I feel is a bit too long. Analytical ultracentrifuge result implies that the distribution of the molecular species is wide, some portion got precipitated with 1K rcf (means the molecular weight is more than 5MDa) and the rest is ranging from 1MD to 5MDa with a peak at 1MDa. I made new two constructs covering the A and B domains respectively, both of them are active again, but only the A domain has got the same symptom as the intact protein. The B domain seems to exist as a monomer in solution. Here come my questions, (I) How can I interpret this phenomenon? (II) Is there anything we can try to change the situation? (III) Does it make sense to try crystallization? (probably not).(IV) Has anyone got such experience? I tried the methylation on lysine side chains, I also tried the buffer with 0.2M arginine or 10% glycerol but the all the results just seem hopeless. The protein before the proteinase treatment also comes out at the void region from the gel filtration. cheers toyoyuki --? Toyoyuki Ose o...@sci.hokudai.ac.jp Graduate School of Life Science Hokkaido University N21W11 Kita-ku, Sapporo 001-0021 Japan
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Dear Marius, I checked out the references, and it was mentioned that The zinc EXAFS spectra of P. shermanii superoxide dismutase have shown that zinc can be incorporated in the active center instead of the iron. (Eur. Biophys. J. * 24* , 243-250). And this example further aroused my concern about the possibility of non-specific metal binding. Especially when the SOD showed full activity regardless of Mg or Fe being incorporated (J. biol. inorg. chem. 1 , 532-541). I think the best solution available was in-cell NMR if in vitro experiments could not tell the difference. Thanks for sharing your experience. Sometimes it just seemed that anything was possible in protein science. Sincerely, Xuan 2009/8/6 Marius Schmidt marius.schm...@ph.tum.de Long time ago I had a superoxide dismutase that was active with iron as well as manganese. No matter what functional metal (Fe or Mn) was bound a substantial fraction up to 1/3 of the molecules had the (non-functional) Zinc in their metal binding site (found by AAS and EXAFS). Zinc is everywhere, even in plastic bottles for your distilled water, it is extremely hard to get rid of. And it seems to fit to many iron binding sites. When I recall correctly a fairly stable source of Fe(II) is Mohr's salt. However, Fe binds also unspecifically to the protein, so it is very hard to quantify iron binding to their specific site because eg. EPR spectra change with each washing step. Best Marius Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn We will either find a way or make one. Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
Re: [ccp4bb] Zinc or Iron binding protein, that is a question!
Dear Mr. Fritz, Yes, the protein is not an E.coli protein! Instead, it was cloned from a virus. And since it was a nonstructural viral protein, I thought it might be appropiate to treat it as eukaryotic proteins. E.coli system was quite different from eukaryotic ones, hence I was quite cautious about the ICP-ES result and trying to confirm it via alternative method. Thanks very much for mentioning the examples which suggested that Fe might be contaminants. Indeed, when I cut the protein in two parts (still with MBP) and test them via ICP-ES again, Fe became negligible in both and Zn stoichiometry increaed to 1:1 in the C-terminal part. The result lead me to focus on Zn instead of Fe. But I still want to confirm the idea. Matallo biochemistry was exactly what I dreamed to do. Sincerely, Xuan Yang 2007/8/6 Guenter Fritz guenter.fr...@uni-konstanz.de Hi Xuan, I guess your protein is not an E.coli protein. There are several examples that eukaryotic Zn-proteins expressed in E.coli contain Fe instead of Zn. I am sceptic whether IMAC with different metal ions will give the solution of the problem. If you really want to get information on the metal ion binding properties you will have to do some matallo biochemistry: preparing apo protein, reconstitution with metal ions, UV-Vis spectroscopy, EPR would be great, ... Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn mailto:ya...@moon.ibp.ac.cn We will either find a way or make one.
[ccp4bb] Zinc or Iron binding protein, that is a question!
Dear Sir or Madam, The ICP-ES results indicated that 1 molar my protein purified from E.coli Origami(DE3) contained about a half molar Zinc and nearly a quarter molar Iron (whether II or III was not available). The protein carried a MBP tag on the N-terminal and the situation was similar with or without His tag at the C terminal. I want to determine whether my protein really bind Zinc or Iron. Does anyone have any experience about such problems? Specifically, now I want to compare the binding efficiency on various IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control). However, considering the instability of Fe(II) in solution, the design still seemed problematic. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 Academic email: ya...@moon.ibp.ac.cn We will either find a way or make one.
