response to: very acidic protein crystallization
Dear all, I'm sending a summary of useful advices which I received on my email concerning the crystallization of a very acidic protein. I would like to thank all the people who responded! Have a nice day! Kornelius There are an number (WT mutants) of X-ray structures published on xylose isomerase from A. missouriensis (see e.g. 1XIM). This is a highly negatively-charged protein with a pI of 3.2-3.5. RNase P protein is quite basic (20-25% Arg/Lys). Crystallization conditions (Stams et al., Science v 280 p 752, 1998) are not particularly informative for your problem, although notably it could only be crystallized at 3 or 24 mg/ml. DLS revealed that the protein was a monomer or dimer, respectively, in solution under these conditions. Before cocrystallization with another protein, I would be inclined to try crystallization from high [salt] or in the presence of polyamines. * This is a difficult problem. It reminds me of the opposite: when you have a protein with many positive charges and it is meant to interact with a negatively charged polymer known as DNA. When you omit the DNA, frequently you cannot crystallize the protein presumably because the repulsive positive charges keep the protein from assuming the correct conformation. Along that thought, you might try to find (more) positively charged particles to counteract your protein charges. I cannot think of positively charged polymers very quickly, but they must exist and/or it must be possible to make those. Maybe (arbitrary thought) you could try positively charged detergent molecules? You write that you have apparently decent CD data confirming you protein folding. Do you have information on the protein aggragation? (I would somehow not encourage dynamic light scattering, it is a pain in the neck.) Size exclusion chromatography or analytical ultracentrifugation could help in assessing this. I am asking about these things because, if you had confirmation of the aggragation state (notably the knowledge that the protein does NOT aggragate), then you could try to use SAXS to determine the global shape and perhaps the positions of the individual domains. It would also tell you if the protein is fully folded, or partially folded (which would be of great importance for crystallization). It would also tell you how these parameters change as function of environmental parameters (pH, ions present, additives), so you might experimentally determine which conditions/additives help your protein to be 'best behaved' for crystallization. * Have you run your protein sequence through the FoldIndex server (http://bip.weizmann.ac.il/fldbin/findex) to see if it is even predicted to be completely folded? When you have a protein with many charges, those charged areas are likely not to be folded, but just hanging out into solvent (since their interactions will be very favorable). * The problem with these highly negatively charged proteins is that they are extremely soluble. It is hard to get them out of solution. You mentioned you tried concentrations up to 50 mg/ml. This does not surprise me. Ten years ago we managed to crystallize a halophilic 2Fe-2S ferredoxin and determined its structure. The protein was crystallized from 4 M phosphate, pH 7. It was the only salt that brought the protein out of solution. The reference is F. Frolow, M. Harel, J.L. Sussman, M. Mevarech, and M. Shoham. (1996) Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin. Nature Structural Biology 3:451-457. * We have worked with a highly basic protein that refused to even precipitate at concentrations lower than 100 mg/ml. What finally worked was to co-crystallize it with monoclonal Fab that were available from collaborators. You might consider trying favorite additives for DNA crystallization, e.g., cobalt hexamine, spermine, spermidine, etc. * We managed to crystallize a halophilic protein (very acidic) in its presumably natural medium (3M NaCl) + around 2M ammonium sulfate. On the other hand, we completely failed (so far) with other halophilic protein around these conditions and many others. Have you checked the proteolytic digestion pattern of your protein ? Could there be some flexible regions the prevent crystallization ? -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
Re: Meeting Biophysics of l igand binding to drug targets ” . 1st February deadline for financial help for s tudents
Hi, what is intended as a student? Phd student? thanks Francesco 2007/1/22, alberto podjarny [EMAIL PROTECTED]: ***Announcement* The meeting Biophysics of ligand binding to drug targets will take place at the Holiday Inn Hotel, Illkirch/Strasbourg, France, from May 14 till May 16, 2007. There are still fellowships for students available. The deadline is 1st February. Detection of ligand binding is a critical process in the identification of leads during the drug design process. During this meeting, speakers will survey the different biophysical methods used to detect ligand binding to pharmaceutical targets, such as crystallography, differential calorimetry, mass spectrometry, nuclear magnetic resonance and plasmon resonance. They will also describe the modelling approaches which integrate this information into an atomic description of the ligand-target interactions. The emphasis of the meeting will be placed on the comparison of the different methods, in particular on the reasons of their apparent discrepancy which occurs sometimes. Twenty two conferences will be given by international experts on the different techniques, coming both from the academic and industrial backgrounds. Attendance from EU students is strongly encouraged, and 20 studentships are available, covering all staying expenses as well as 200 € of travel expenses. Registration fees (covering all staying expenses) are 400€ for academic participants and 800 € for industrial participants. All details of the meeting are available at the URL http://biobinding.u-strasbg.fr On behalf of the local committee Looking forward to seeing you in Illkirch Alberto Podjarny Alberto D. Podjarny IGBMC UMR 7104 - U596 LGBS 1, rue Laurent Fries 67404 Illkirch TE: (33) 3 88 65 33 11 FAX: (33) 3 88 65 32 01
Re: ccp4bb on new site
As far as the subject header line is concerned, ye olde ListServ command: SET CCP4BB SUBJECTHDR would probably work if one emailed it to the server (i.e. [EMAIL PROTECTED] *not* CCP4BB@JISCMAIL.AC.UK) or you can do it via the web interface. It appears that the mail/web command interface will not let you change the Reply-To feature. Phil Jeffrey Kjeldgaard Morten wrote: Unfortunately, It appears that JISCMAIL is using the outdated LISTSERV software to run it's mailing lists, so there is not much hope of getting such things as the [ccp4bb] subject tag and reply to sender features back :-( Morten --Morten Kjeldgaard, asc. professor, MSc, PhD
crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Hello All, I am trying to soak some crystals with a small molecule that is quite hydrophobic. I am having trouble with solubilty of the small molecule. It will dissolve up to about 1 mM in 100 % DMSO, but precipitates at concentrations of less than 15 micromolar when the DMSO concentration is below 20 percent in my crystal growth solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Do you mean N,N-dimethylformamide, aka dimethylformamide, aka DMF, or do I need a chemistry lesson? On Monday 22 January 2007 02:49 pm, Parthasarathy, Gopalakrishnan wrote: Hi Todd, DMF (Dimethyl Fluoride) is a good alternative to DMSO. Sarathy -- Craig A. Bingman, Ph.D. Center for Eukaryotic Structural Genomics and Department of Biochemistry University of Wisconsin--Madison 433 Babcock Drive Madison, WI 53706 (608) 263-5923
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Sarathy (and Todd) DMF is usually used for dimethyl formamide. From the name I have no idea what dimethyl fluoride may be (methylene difluoride, though wrong, is used for difluor methane,but that's not mixable with water, and only a liquid at low temperature ;-) ) Todd, If your protein has a sufficiently high affinity for your small molecule, 15 uM and solid organic compound should work fine (if you wait long enough). In some cases, if there is residual solubility of a compound, just adding it as a solid works. Jens Parthasarathy, Gopalakrishnan wrote: Hi Todd, DMF (Dimethyl Fluoride) is a good alternative to DMSO. Sarathy *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *Green, Todd *Sent:* Monday, January 22, 2007 3:40 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* crystal friendly solvents that are useful for dissolving hydrophobic small molecules? Hello All, I am trying to soak some crystals with a small molecule that is quite hydrophobic. I am having trouble with solubilty of the small molecule. It will dissolve up to about 1 mM in 100 % DMSO, but precipitates at concentrations of less than 15 micromolar when the DMSO concentration is below 20 percent in my crystal growth solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green -- 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 (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - 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: advice
On Jan 22 2007, Eaton Lattman wrote: Will someone knowledgeable tell me what the present state of full 6 dimensional searches in molecular replacement? Presumably you're referring to systematic 6D searches, not stochastic ones like in EPMR or QoS. Do you mean can it be done on current hardware or is it worth doing? If the former, then it's doable, though slow. In Phaser, for instance, you can generate a complete list of rotations (using the fast rotation function with keywords to prevent clustering and to save all solutions), then feed that big list of rotations to the fast translation search. In a typical problem that would probably run on a single processor in significantly less time than the average PhD, and could be made reasonably quick with a cluster. If the latter, our feeling is that it isn't worth it. We've tried the full search option on a couple of monoclinic problems (where it's only a 5D search), and nothing came up with the full list of orientations that didn't come up with the first hundred or so orientations. We conclude that, even in the most recalcitrant cases, the rotation search gives a better than random indication of whether an orientation is correct, so it's not necessary to search through all possible orientations. However, we do feel that it can be worthwhile to try a reasonably large number of orientations in difficult cases. Best regards, Randy Read P.S. When we generate our list of orientations, we use Lattman angles to get reasonably even sampling of rotations.
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Toluenes and derivatized benzenes may absorp into your plastic tray? Or into the tape covering your tray? Just few other destinations. It would make sense that if the binding of the 'drug' to the protein is tight, then you do not need much in immediate contact, it will get there. The method below sounds very promising to me. Mark -Original Message- From: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 22 Jan 2007 3:57 PM Subject: Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules? So I've never actually tried this proposed extension to the idea, but: I (and many others) have gotten small hydrophobics (toluene, iodobenzene etc.) into proteins, and these things typically have very small partition coefficients, and they aren't horribly volatile (that's why I am a little partial to iodobenzene). Why not saturate a small solution of your small molecule in iodobenzene and just add a few microliters on top; if the binding is tight enough you can pull it through and not bug your protein with a denaturing co-solvent. I've noticed that the iodobenzene does largely disappear overnight (in hanging drop), I don't know if this is because of evaporation or the iodobenzene just falls into the reservoir. Maybe stick to sitting drop. -Original Message- From: CCP4 bulletin board on behalf of Green, Todd Sent: Mon 1/22/2007 12:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: crystal friendly solvents that are useful for dissolving hydrophobic small molecules? Hello All, I am trying to soak some crystals with a small molecule that is quite hydrophobic. I am having trouble with solubilty of the small molecule. It will dissolve up to about 1 mM in 100 % DMSO, but precipitates at concentrations of less than 15 micromolar when the DMSO concentration is below 20 percent in my crystal growth solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green Check Out the new free AIM(R) Mail -- 2 GB of storage and industry-leading spam and email virus protection.
Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
On Monday 22 January 2007 04:57 pm, Maneesh Yadav wrote: I've noticed that the iodobenzene does largely disappear overnight (in hanging drop), I don't know if this is because of evaporation or the iodobenzene just falls into the reservoir. Maybe stick to sitting drop. Or it partitions through the vapor phase into the plastic crystallization tray. -- Craig A. Bingman, Ph.D. Center for Eukaryotic Structural Genomics and Department of Biochemistry University of Wisconsin--Madison 433 Babcock Drive Madison, WI 53706 (608) 263-5923
: misbound ligand examples?
A biochemist friend asked for examples of cases were a protein was co-crystallized with or soaked in a ligand that bound in the wrong place - say, because the ligand used wasn't quite the right one or because other important ligands were absent. I'm sure such examples are out there, especially when soaks were done at high concentrations, but I'm having trouble thinking of concrete examples. Help? thanks, Phoebe Rice --- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: advice
I'd like to add that the value of a molecular replacement solution tends to be inversely correlated with the effort needed to find the solution. In other words, the harder you have to work to find the MR solution the less informative the phase information you tend to get. When you have very high resolution and/or NCS you may still be able to solve the structure. However, in cases were the search model is only distantly related to the protein of interest and Phaser can't find the solution, the solution may not be worth finding and you're better of focussing on getting experimental phases. Bart Randy J. Read wrote: On Jan 22 2007, Eaton Lattman wrote: Will someone knowledgeable tell me what the present state of full 6 dimensional searches in molecular replacement? Presumably you're referring to systematic 6D searches, not stochastic ones like in EPMR or QoS. Do you mean can it be done on current hardware or is it worth doing? If the former, then it's doable, though slow. In Phaser, for instance, you can generate a complete list of rotations (using the fast rotation function with keywords to prevent clustering and to save all solutions), then feed that big list of rotations to the fast translation search. In a typical problem that would probably run on a single processor in significantly less time than the average PhD, and could be made reasonably quick with a cluster. If the latter, our feeling is that it isn't worth it. We've tried the full search option on a couple of monoclinic problems (where it's only a 5D search), and nothing came up with the full list of orientations that didn't come up with the first hundred or so orientations. We conclude that, even in the most recalcitrant cases, the rotation search gives a better than random indication of whether an orientation is correct, so it's not necessary to search through all possible orientations. However, we do feel that it can be worthwhile to try a reasonably large number of orientations in difficult cases. Best regards, Randy Read P.S. When we generate our list of orientations, we use Lattman angles to get reasonably even sampling of rotations.
