[ccp4bb] Protein aggregation and crystallization
Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume. But the proteins donot precipitate out !! Is it worth while to go ahead for crystallization trials?? Any other suggestion is most welcome. Thanks Anita
Re: [ccp4bb] Protein aggregation and crystallization
Hi, I do have 10% glycerol in my buffers, and still the constructs come in the void volume. and I have sarkosyl in the lysis buffer. but none in the elution or dialysis buffer. So do I still need detergents please suggest. reg. Anita On Sat, Aug 27, 2011 at 12:13 AM, Pius Padayatti wrote: > Answer is simply no. > aggregates are of no value > why would you try it? > you could try adding glycerol to 10 % during the > preparation( all the way from homogenization) itself and try detergents > like c8e4 and series of that. > i know one membrane associated protein need > detergent from the begining itself. > Please do not chop off the membrane part keep it > and chop some of the unstructured cytosolic part if you want. > all the best. > let us know if any of these worked > Padayatti > > On Fri, Aug 26, 2011 at 3:03 AM, anita p wrote: > > Hi All, > > I am working on a protein which has a membrane spanning region and as > > cytosolic domain.I have made various deletion constructs of the protein, > so > > that I can have a crystallizable fragment. There is no homologues > mentioned > > in the pdb for this protein. > > All of these constructs are purified successfully but when concentrated > and > > loaded on a gel filtration column Superdex-200, they elute in the void > > volume. But the proteins donot precipitate out !! > > Is it worth while to go ahead for crystallization trials?? > > Any other suggestion is most welcome. > > Thanks > > Anita > > > > > > > > -- > Pius S Padayatti,PhD, > Phone: 216-658-4528 >
Re: [ccp4bb] Protein aggregation and crystallization
Hi Yury, I have done dynamic light scattering and it shows its polydispersed. Please let me know if it is still ok for setting trays. reg. anita On Fri, Aug 26, 2011 at 10:21 PM, Yuriy Patskovsky < yuriy.patskov...@einstein.yu.edu> wrote: > Anita, > an assembly may be quite large - I would check it somehow, maybe by light > scattering or centrifugation > > Good luck > > Yury > -- > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anita p [ > crystals...@gmail.com] > *Sent:* Friday, August 26, 2011 3:03 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Protein aggregation and crystallization > > Hi All, > I am working on a protein which has a membrane spanning region and as > cytosolic domain.I have made various deletion constructs of the protein, so > that I can have a crystallizable fragment. There is no homologues mentioned > in the pdb for this protein. > All of these constructs are purified successfully but when concentrated and > loaded on a gel filtration column Superdex-200, they elute in the void > volume. But the proteins donot precipitate out !! > Is it worth while to go ahead for crystallization trials?? > Any other suggestion is most welcome. > Thanks > Anita > >
[ccp4bb] drops swelling
Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita
Re: [ccp4bb] drops swelling
Thanks a lot for your suggestions. Would it be worth while to try and remove glycerol at the last step which is gel filtration and then set trays in my case? I have never tried without glycerol. reg. Anita On Fri, Sep 9, 2011 at 7:54 PM, Enrico Stura wrote: > Anita, > > see the message I posted yesterday on the CCP4bb: > http://www.mail-archive.com/**ccp4bb@jiscmail.ac.uk/**msg22638.html<http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html> > > or google "stura glycerol" > Re: [ccp4bb] How to help crystal grow bigger > > In your case the sentence you need is: > "You need to have a higher (double) glycerol concentration in the reservoir > else you will risk finding that your drops will get biggger and not smaller. > This note of caution applies to vapour diffusion set ups as equilibration > can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, > V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. > Cryst. Growth & Des. 11 :2755–2762. " > > http://pubs.acs.org/doi/abs/**10.1021/cg101364m<http://pubs.acs.org/doi/abs/10.1021/cg101364m> > > Enrico. > > > > On Fri, 09 Sep 2011 13:22:53 +0200, anita p wrote: > > Dear Crystallographers, >> I have set hanging drop trays with 2ul of protein and 2 ul of resorvior >> solution. I have seen in some cases the drops are swelling. My protein >> buffer has 15% glycerol in it. >> This is happening mainly when I have peg 400 or peg MME or MPD or >> Jeffamine >> in the buffer condition. >> Could any one suggest a remedy for this. >> >> with regards >> Anita >> > > > -- > Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office > Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab > LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE > http://www-dsv.cea.fr/en/**institutes/institute-of-** > biology-and-technology-saclay-**ibitec-s/unites-de-recherche/** > department-of-molecular-**engineering-of-proteins-** > simopro/molecular-toxinology-**and-biotechnology-laboratory-** > ltmb/crystallogenesis-e.-stura<http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura> > http://www.chem.gla.ac.uk/**protein/mirror/stura/index2.**html<http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html> > e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 >
[ccp4bb] native gels
Hi All, Has anyone run a native gel for proteins at pI>8 . I want to pour my own native gel. Do I run a discontinuous page or a continuous one?? Please help with regards to the buffer system to be used, and the dye to be used. With regards Rashmi
[ccp4bb] Na acetate as purification buffer
Hi all, Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein on Histrap column (AKTA) followed by SEC? My protein has a pI of 9. I tried pH7.4 but it has precipitation problems. While doing buffer screening using 24 well hanging drop I found that lower pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole purification??? Thanks in advance Anita
[ccp4bb] granular precipitate
Hi All, I have set up initial screen in hanging drop trays with a protein of theoritical pI of 8.5. The protein is in acetate buffer 10mM, KCl 100mM and 2% glycerol pH 5 . In 85-90% of the conditions I see granular precipitate in 1 day. I tried to open the coverslip, and touch few drops, They had a skin layer and the granular looking thing are part of the skin. The protein was set up at 10 and 5 mg/ml concentration. Is this normal? I have tried to change the protein buffer to Hepes at pH 7 with other constituents same,and tried to set up few screen trays. I cant see granular precipitate again. The precipitates look light brown and amorphous in nature. Is Na acetate forming some granular precipitate instead of protein? Kindly suggest me ways to move forwards. Thanks in advance Anita
[ccp4bb] detergent or protein
Hi All, I would like to have your expert advice on crystals. I am using detergents as 5% (w/v) of DDM 0.4ul in a 4ul (protein + condition + detergent). The precipitant is 28% peg 20K After 1 day I am able to see little plates of irregular shape . I am able to see some needles if I change into MEGA-10. I am able to see some hexagonal 3D crystals when I change the precipitant to Ammonium sulphate and the detergent to Anapoe-C10E6 Is there a possibility to check whether this crystals are protein or of detergent, even before shooting it ? Does the control drop test help ?? awaiting for feedback Anita
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[ccp4bb] Tev Cleavage issue !!
Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could* highlight a bit more on this issue it would be helpful for me.* I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov wrote: > For starters, you could re-clone the protein with e.g. just a His tag or > move the tag to another end, or put some distance between the end of TEV > site and the protein; or perhaps use no tag at all -- or a different one? > > Is it possible that the tag is messing you up - yes. Is it 'probable' - I > can't say that I know because I've crystallized literally dozens of proteins > with His-tags attached, and more than a few with His-tag and cleavage site. > I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick > to blame the tag (since there are so many other possible things to blame). > > Based on the behavior of your protein after cleavage, it may be that you > have oligomer(s) forming in solution such that cleaved and uncleaved > proteins do not segregate. You may wish to explore other kinds of > chromatographic separation e.g. ion exchange of HIC - they may or may not > work out. You can also consider cleaving your protein at lower > concentration, in the presence of detergents or polyols, etc. > > Cheers, > > Artem > > On Thu, Apr 7, 2011 at 9:37 PM, anita p wrote: > >> Hi Crystallographers, >> I am working of 23 Kda protein with a Nterminal His tag and a TEV >> cleavage site. >> I am getting crystals with the his tag and tev site intact, but they dont >> diffract. >> *Is it probable that they dont diffract because of the extra his tag and >> the tev site?* >> >> I am trying to get rid of this tag but the reaction is optimum at 10:1 >> protein to TEV ratio in micrograms overnight incubation without shaking. >> I tried to run it on histrap column after this reaction but I am not able >> to purify cleaved protein from TEV and uncleaved. >> I have tried several times but I get 3 bands ie., the TEV, uncleaved and >> Cleaved. >> I have also tried to use the Nibeads instead of the histrap column, but >> no difference is seen. >> * Is there a possible way to approach this problem?* >> >> Suggestions awaited >> Anita >> > >
[ccp4bb] Detergents
Hi, I had set up crystallization with a bicine as buffer and peg 400 as precipitant. I used the detergent DDAO/LDAO as an additive to the crystallization drop (one of the hampton additive screen condition, it says 5% on the vial) I have a clear drop and in the centre there is a shiny precipitate (looks like granules attached to eah other). Then I opened the drop to touch those granules with a needle, but suprisingly its like a skin and the skin surrounded my needle, and then some how I was able to but back the skin in the ppt. orelse the drop is clear if I remove the skin from the drop. I couldnot see the same on the control drop with no protein and just buffer. Does anyone have any experience with such kind of of shiny skins on crystallization drops?? Is it protein? Can I go forward and use it for seeding?? Please suggest with regards Rashmi
[ccp4bb] reproducibility of protein crystals
Dear Crystallographers, I have got my protein crystallized once and then it is not reproducing, though I am using the same batch of protein and same condition. What are the reasons behind nonreproducibility of protein crystals? I am very new to this field hence I apologize if it is a lame question. With regards Anita
[ccp4bb] detergents
Hi all, I have been trying to purify cytosolic fraction of membrane protein whose domain boundries are unknown. hence I have made a series of deletion constructs. The expression and purification is not a problem. I get good yields of the proteins. But on a gelfiltration column, they run in the void volume, which suggest that they are huge aggregates. I have tried different salts, DTT bME etc but no change. I use sarkosyl in my lysis buffer. But could any one tell me if I can use any detergents as (bOD) in the purification protocol, and how much should I use. with regards Anita
[ccp4bb] ITC with unfolded proteins
Hello everyone, I have a query for the scientists working on protein-protein interaction. It is known that some proteins exist in unfolded or molten globule state and attain structure on interaction with other folded proteins. Many a times, it is difficult to obtain the structure of these complexes. Is it possible to quantitatively determine the thermodynamics of interaction between an unfolded protein and a folded protein using ITC? Later may be perform an alascan to determine the residues of the unfolded partner involved in the interaction. Please share your ideas cheers Anita
Re: [ccp4bb] ITC with unfolded proteins
That is a very interesting question, which I would request the seniors out there to give their insights on. I was imagining that a recombinant purification of an unfolded partner would aggregate which would cause trouble in ITC. Am I correct in this theory? Would love to have more insights. thanks in advance Anita On Fri, Mar 14, 2014 at 7:18 PM, wrote: > Hi, > > I think the experiment is doable, but how would you decouple > protein-protein interaction from folding of the unfolded > protein due to protein interaction? > > Reza > > Reza Khayat, PhD > Assistant Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > > > Original message > >Date: Fri, 14 Mar 2014 18:07:48 +0530 > >From: CCP4 bulletin board (on behalf > of Anita P ) > >Subject: [ccp4bb] ITC with unfolded proteins > >To: CCP4BB@JISCMAIL.AC.UK > > > > Hello everyone, > > I have a query for the scientists working on > > protein-protein interaction. > > It is known that some proteins exist in unfolded or > > molten globule state and attain structure on > > interaction with other folded proteins. > > Many a times, it is difficult to obtain the > > structure of these complexes. > > Is it possible to quantitatively determine the > > thermodynamics of interaction between an unfolded > > protein and a folded protein using ITC? Later may be > > perform an alascan to determine the residues of the > > unfolded partner involved in the interaction. > > Please share your ideas > > cheers** > > Anita >
[ccp4bb] Guard columns from FPLC
Hi All, Sorry for this off topic. I have heard that there are these little columns called guard columns which can be attached to AKTA purifiers. These columns prevent the incoming huge aggregates to be deposited and blocking of the gel filtration columns. Can any one advice me regarding where to purchase these columns. I could not find them in GE website. We have Superdex 16/60 on AKTA purifier. Thanks in advance. Have a good day Anita
[ccp4bb] hydrophilic protein going to aggregate
Hello Crystallographers, I am trying to express and purify a soluble domain of a membrane protein for crystallization. The amino acid content is as below Ala (A) 12 13.8% Arg (R) 10 11.5% Asn (N) 2 2.3% Asp (D) 8 9.2% Cys (C) 1 1.1% Gln (Q) 1 1.1% Glu (E) 4 4.6% Gly (G) 16 18.4% His (H) 3 3.4% Ile (I) 0 0.0% Leu (L) 3 3.4% Lys (K) 1 1.1% Met (M) 1 1.1% Phe (F) 2 2.3% Pro (P) 6 6.9% Ser (S) 11 12.6% Thr (T) 1 1.1% Trp (W) 1 1.1% Tyr (Y) 1 1.1% Val (V) 3 3.4% I could purify the protein using IMAC to 95% purity, how ever, I had to elute with very high concentration of imidazole 2 M, still some protein is attached to the beads as I could observe on the SDS PAGE. I concentrated the protein to 25 mg/ml of 3 mls and on performing SEC, I found the protein to be in the Void fraction of SEC 200. Any expert advice on how to optimize this purfication and why it is still attached to the beads? thanks in advance Anita
[ccp4bb] off topic Thermal shift assay
Hi All, I want to use a thermofluor for the thermal shift assay. My proteins are cytoplasmic truncations of membrane protein. I have read about ANS, sypro-orange and CPM. Which is the once that is popularly used by the crystallographers for condition optimization for crystallization ?? I have read that it sypro orange is not good for hydrophobic proteins and CPM can't be used with DTT or bME in the buffer. I am a bit confused . Please help thanks in advance Anita
[ccp4bb] generating symmetry related
Dear All, I have a small molecule structure file coordinates in CSD CIF format, I would like to analysis inter-molecular interaction between them by generating symmetry related nearest neighbor structures. I want to store the coordinates of the generated structures and further analysis it using in-house tools. Since i would like to do this for multiple structure, can anyone let me know of the a method or tool (command-line) without the use of GUI to get these structure. with regards Anita
[ccp4bb] off topic: protein peptide binding
Hi All, I wanted some advice regarding mapping out Protein-peptide interaction. The peptide is a 12 mer and the protein is 15kDa. Invivo studies suggest that the peptide is binds the protein and helps in transport. Hence I feel it would perhaps transient binding. I know that I should do ITC or BIAcore to show binding, but before going to those techniques, I feel, running a native gel would perhaps help. So the native gel can have lane1: protein, lane 2: peptide, lane 3: protein+peptide. If the protein binds to the peptide then I should not see a band corresponding to the peptide in lane 3. But before I start this experiment, I wonder if any body has run 12 mer peptide on native gel, How long should I run... How much quantity of peptide I should for the gel. I wont be able to do western or pull-down, Equipment for native gel is available to me. kindly advice, regards Anita
[ccp4bb] off topic: rmsf in simulation
Hi All, I am trying to understand the mechanism of protein-peptide interaction in two complexes (protein-pepA and protein-pepB). While trying to perform some simulation experiments, I find that the* root mean square fluctuation (RMSF) by residues of protein in the complex is higher than that of the protein alone.* Please refer the figure attached to this email. pepA binds with higher affinity (in uM-range) than pepB according to invitro studies. Does this happen normally?? Please advice. Thanks in advance Anita <>
Re: [ccp4bb] off topic: rmsf in simulation
Hi All thanks for your detailed reply. A higher RMSF(as I showed in the png.) *doesnot *mean that the RMSD for Calpha backbone showed be high. Am I correct ?? Because in my case the backbone RMSD for the receptor of the peptide bound structure is lower than the receptor alone. Because I wanted to know if my simulations have gone fine. thanks again in advance. Anita On Sun, Dec 9, 2012 at 6:44 PM, Chandra Verma wrote: > to complement the very nice description by jeremy, you may wish to try and > decompose the vibrational modes to get this sense by focussing on the > origins of the "red shift" in the vibrational spectrum and this accounts > largely for the increased vibrational entropies upon complexation. This > paper may be used as a guide > (Dissecting the vibrational entropy change on protein/ligand binding: > burial of a water molecule in bovine pancreatic trypsin inhibitor J Phys > Chem B 2001 105 8050-8055) > & > > There is some very nice work by Olano & Rick in > > JACS 2004 126:7991 on Hydration free energies and entropies for water in > protein interiors. > > and by carol post on how the increased entropies upon complexation are the > origin of the mechanism of some drugs. (for example Influence of an > Antiviral Compound on the Temperature Dependence of Viral Protein > Flexibility and Packing: a Molecular Dynamics Study J. Mol. Biol. (1998) > 276: 331-337) > > > > > Quoting Jeremy Tame > > >: > > Different proteins do different things. Some adopt fewer conformations >> and a more rigid structure after binding >> a ligand, and others do the opposite. Haemoglobin is a nice example of a >> protein that becomes a lot more flexible >> after picking up ligands. For any reaction of the kind P + L -> PL there >> is an entropy cost of making one molecule >> from two. For the protein to activate low frequency modes in the complex >> is one way to compensate for this by >> increasing the entropy of the bound form. The paper by Sturtevant (PNAS >> 74, 2236, 1977) is worth a read, as is >> Cooper and Dryden (Eur Biophys J, 11, 103, 1984), if you are interested >> in relating fluctuations to thermodynamics. >> All too often people attempt direct comparisons of structural models and >> affinities without realising that the so-called >> "angstroms to calories" problem often frames the question in a form that >> cannot be answered sensibly. For >> example, imagine a protease which is produced as a zymogen. Both forms >> may have essentially identical crystal >> structures even though the zymogen is more flexible. The protease can be >> activated by loss of vibrational modes >> in the unbound state which are re-awakened in the complex with substrate; >> hence the zymogen will have lower >> substrate binding and activity. You might be interested in a review by >> Homans (ChemBioChem 6, 1585, 2005) which >> discusses the use of NMR to look at entropy changes in protein-ligand >> binding reactions. It is by no means unusual >> for a residue's entropy to increase in the bound state, although in your >> case it seems to be the whole protein! >> >> On Dec 9, 2012, at 1:05 PM, anita p wrote: >> >> Hi All, >>> I am trying to understand the mechanism of protein-peptide interaction >>> in two complexes (protein-pepA and protein-pepB). >>> While trying to perform some simulation experiments, I find that the >>> root mean square fluctuation (RMSF) by residues of protein in the complex >>> is higher than that of the protein alone. >>> Please refer the figure attached to this email. pepA binds with higher >>> affinity (in uM-range) than pepB according to invitro studies. >>> >>> Does this happen normally?? Please advice. >>> Thanks in advance >>> Anita >>> >>> >>
