[ccp4bb] ITC workshop [Off-topic]
Dear All, In the spirit of complementary methods (e.g. see Acta Cryst.D Vol 71), I would like to bring to your attention an upcoming bootcamp on“Isothermal Titration Calorimetry Analysis”, to be held 10-11 September, 2015at the NIH in Bethesda, MD, USA. Topics will include basic principlesand advanced topics like multi-site binding models, experimental design,multi-method global analysis, and use of cutting-edge analytical softwareincluding NITPIC, ITCsy, and GUSSI. The bootcamp is organized by theFoundation for Advanced Education in the Sciences. You can sign up andget more information at http://www.faestraining.org/index.php/conferences-bootcamps Sincerely, Chad
Re: [ccp4bb] First stucture of FCFV
I once encountered mold-dependent crystallization of a protein. Wouldn't that have made for a lively Methods section? Luckily, we determined the structure from crystals derived from a different, non-moldy condition. Whew. Chad From: Artem Evdokimov artem.evdoki...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 3, 2014 7:55 AM Subject: Re: [ccp4bb] First stucture of FCFV Common molds like aspergillus or penicillium. After a while you sometimes get sporangia, then you can tell with more certainty. .. A. On Apr 3, 2014 3:50 AM, Bernhard Rupp hofkristall...@gmail.com wrote: Several people were asking what this FCFV tentacles actually might be. I think it is some fungus/yeast growing out of nutritious drops. Does resemble fungus/mushroom mycelium. I have also some that look like huge bacteriophages with nice heads on them, probably yeast buds. There is also a yeast lab next to the Xtallization facility :-/ … feel free to speculate. Best, BR
Re: [ccp4bb] question on charge charge interactions
Tom, How about Magalhaes et al., J. Protein Chem., Vol. 13, p 195? Chad From: Tom Peat tom.p...@csiro.au To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, March 27, 2014 8:09 PM Subject: Re: [ccp4bb] question on charge charge interactions Hello Joel, I like the example of HIV protease, but in this case these Asp residues are found in the active site of the protein, and unless there is substrate (or inhibitor) in the active site, these would be solvent exposed (unless I'm looking at the wrong pair of Asp residues). In the particular case I'm looking at, I have a buried pair with no other charged residues around- no waters/ metals, just Phe, Leu, etc. Which is why I think it might be slightly more rare than most of the examples I've heard about so far. Thanks for the help. Cheers, tom -Original Message- From: Joel Tyndall [mailto:joel.tynd...@otago.ac.nz] Sent: Friday, 28 March 2014 12:02 PM To: Peat, Tom (CMSE, Parkville); CCP4BB@JISCMAIL.AC.UK Subject: RE: question on charge charge interactions Tom, I would think the case can be made for sharing a proton (one ionised and one not) in either case but more so for acidic residues. See HIV protease Asp-Asp as a well-established example Hope this helps J -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tom Peat Sent: Friday, 28 March 2014 12:12 p.m. To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] question on charge charge interactions Hello All, I am appealing to the community as I don't seem to be able to find through Google what I am looking for, and I just don't have the ability to look through every structure in the PDB to find this. I have what I think is an interesting case: a two domain protein structure with a mostly hydrophobic interface between the two domains- the kicker is that I have two charged residues buried in this domain and they are identical. That is, I'm looking for an Arg-Arg or Asp-Asp type interaction (not Arg-Asp) where these residues are less than 4 Angstroms apart. So I was wondering if anyone had seen this in any other structure(s)? The really interesting bit is that this interaction actually regulates the activity of the enzyme domain although the domain interface isn't that close to the catalytic site. Thanks in advance to all those who can find a reference to this kind of interaction. Cheers, tom
[ccp4bb] Off-Topic: Hydrodynamic/Thermodynamic Workshop in Dallas, TX
Dear All, I am very pleased to announce that a workshop entitled “Methods for the Analysis of Thermodynamic and Hydrodynamic Properties of Macromolecules and their Complexes” will be held on May 19-23, 2014 in Dallas, TX, at The University of Texas Southwestern Medical Center. I post here because, in the past, we have had a very strong level of participation from the Structural Biology community. The workshop will feature lectures on the practical aspects of performing AUC, ITC, DLS, and fluorescence spectroscopy experiments. There will also be demonstrations and step-by-step instruction on how to analyze your data using SEDFIT and SEDPHAT, and lectures covering the theoretical underpinnings of the analyses. I think we can all agree that these methods and analytical techniques can add significant value to our structures. The instructors will be experts with many years of experience with these technologies and analytical methods. They include Drs. Peter Schuck, Rodolfo Ghirlando, Joy Zhao, and myself. You can find more information about the workshop and registration at the following links: http://www.utsouthwestern.edu/education/cme/symposia/macromolecules/index.html http://www.utsouthwestern.edu/education/cme/symposia/macromolecules/registration.html Best regards, Chad
[ccp4bb] Off-Topic: NITPIC ITC software available
Dear All, Apologies for the off-topic post, but I think there might be a number of people subscribed to this list who also use ITC and would find this useful: I am pleased to announce the immediate availability of the software program NITPIC. You may download it from http://biophysics.swmed.edu/MBR/software.html. What does NITPIC do, you ask? The software is designed to read and integrate thermograms generated by GE, MicroCal, MCS, and TA isothermal titration calorimeters. It calculates the baseline in an unbiased way, integrates the power peaks, generates error estimates for the integrations, and outputs the data for analysis. It interfaces seamlessly with Peter Schuck’s SEDPHAT analysis program, which features a large variety of interaction models. When used with SEDPHAT, NITPIC also outputs data that are compatible with the high-quality graphics renderer GUSSI. We find that NITPIC generally outperforms the manufacturers’ analysis programs, and it removes the tedious step of manual baseline adjustment. The program is documented in a pdf file that accompanies the downloaded executable. It runs only under Windows operating systems; it has been tested under Windows XP, 7, and 8. The algorithms underlying the NITPIC program can be found in Keller et al. (2012), Anal. Chem., Vol. 84, pp. 5066-5073. If you find any bugs, please report them to me: chad.brauti...@utsouthwestern.edu Enjoy, Chad
[ccp4bb] postdoctoral opportunity in Dallas, TX
Posted on behalf of David Chuang, Ph.D.: Postdoctoral Position in X-ray Crystallography is available in the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas. The incumbent will: 1) investigate the structure and function of mitochondrial protein kinases (for references see: EMBO J. 24:1763, 2005; EMBO J. 25:5983, 2006; Structure 16:1849, 2008), and 2) work with a multi-disciplinary team comprising biochemists, synthetic chemists, pharmacologists and structural biologists to structure-based design and produce novel small-molecule inhibitors specific for these protein kinases. The inhibitors will be studied in cell culture and animal models as new strategies to metabolically target cancer, type 2 diabetes and disorders in branched-chain amino acid catabolism. Applicants must have a Ph.D. degree with training and first-author publications in X-ray crystallography. Send CV with cover letter and names of three references to Dr. David Chuang david.chu...@utsouthwestern.edu. UT Southwestern is an Equal Opportunity, Affirmative Action employer.
Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation
Dear Kushol Jerry, I have to take exception to Kushol's contention about SV. As long as the buffer and protein parameters are correct and the sample is well behaved (i.e. not undergoing dynamic rearrangement on the time scale of the SV experiment, not aggregated, homogeneous), one can derive very accurate molar masses from SV, and it is far superior in this respect than SEC. However, as you aptly point out, the problem may lie in the sample's behavior. If the protein is populating multiple oligomeric states, or if it is undergoing a fast interconversion of such states, or if it is not pure, spurious masses may be calculated. If such pathologies are observed, it's not clear to me that a sedimentation equilibrium experiment would help. For a complicated interaction model, SE data (and AUC data in general) can be difficult to analyze, and some model assumptions are almost always present. So, the questions are: 1. Did Jerry run SV or SE? 2. If the former, then did he see a nice, single boundary, or a big smear? 3. If the latter, how were the data analyzed to come to his conclusion? Hopefully he can comment on these aspects. Cheerio, Chad From: Kushol Gupta kushol.gu...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, May 27, 2011 9:51:04 AM Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Hi Jerry – By AUC, do you mean sedimentation velocity (SV)? Both gel filtration and SV are not terribly great ways to determine precise molecular mass, especially if the macromolecule of interest is anisotropic in shape. In your SV values, do you see a large f/fo, or a broad distribution? Can you run a sedimentation equilibrium experiment? If you run HYDROPRO on your prospective oligomer structure, do you arrive at theoretical S and Rs values that jive with your solution data? A nice crosscheck you could do with the data in hand (if your measurements from both approaches were performed in the same buffer at the same temperature) is calculate the mass using the Siegel and Monty equation (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346–362), where the mass of the particle is calculated from Rs (from gel filtration) and the S(t,b) value from sedimentation velocity. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From:CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state. Could anyone give some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully
Re: [ccp4bb] itc equipment
Hey, Engin, I saw this instrument at the Protein Society meeting this summer. Looks impressive. Be aware that even though the volume will be significantly smaller, the concentration needed to get a measurable amount of heat will probably be higher (the Microcal rep I talked to confirmed this to me). It won't have to be 7-fold higher, but maybe 2- or 3-fold higher. Best of luck, Chad Engin Ozkan [EMAIL PROTECTED] wrote: Fellow crystallographers and biochemists, This will be off topic. Does anyone have any experience with the new ITC Microcal is selling (itc200)? They claim that itc200 requires one seventh the sample volumes. We're especially interested in an equipment that would require less sample without sacrificing signal. We've had difficulty obtaining such information from MicroCal or other sources. Engin - Stanford University School of Medicine Molecular and Cellular Physiology - Looking for last minute shopping deals? Find them fast with Yahoo! Search.
Re: [ccp4bb] : misbound ligand examples?
Hi, Phoebe, Sorry to jump in late on this one- but I second Stefan's note here. When soaking dinucleotides (which are poor substrates) into Klenow Fragment xtals, I noted binding both at the active site and at a crystal interface site that is likely nonphysiological. The adventitious site is just big enough to accommodate a dinucleotide- no binding was observed here with longer oligos. Alas, that dinucleotide-containing structure is not deposited. Chad Stefan Knapp [EMAIL PROTECTED] wrote: we see quite frequently ligands sitting in crystal interfaces in addition to the described binding site for example - STK16, a S/T kinases pdb-code: 2BUJ, the ATP competitive ligand staurosporine binds to ATP site as well as to a symmetry related molecule forming a nice aromatic stacking interaction also quite interesting CK1 gamma 1 (S/T kinase) pdb-code 2CMW, ATP competitive ligand binds also to upper kinase lobe Stefan Stefan Knapp Structural Genomics Consortium Oxford UK 22/01/2007 23:29 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 - Be a PS3 game guru. Get your game face on with the latest PS3 news and previews at Yahoo! Games.
