Re: [ccp4bb] השב: Re: [ccp4bb] Curious electron density associated with Asp sidechain
could this also be an alternative C-Terminus at lower occupancy? Regards Kornelius On Fri, Apr 26, 2013 at 10:45 AM, Boaz Shaanan bshaa...@exchange.bgu.ac.ilwrote: Hi, it looks like gly-asp not asp-gly in this case, doesn'it? Cheers, Boaz הודעה מקורית מאת: Jonathan Cooper bogba...@yahoo.co.uk תאריך: אל: CCP4BB@JISCMAIL.AC.UK נושא: Re: [ccp4bb] Curious electron density associated with Asp sidechain Hello Tony is that Asp-Gly? If so, it could be prone to succinimide formation. Check out this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323960/ and references therein! Good luck Jon.Cooper --- On *Thu, 25/4/13, Antony Oliver antony.oli...@sussex.ac.uk* wrote: From: Antony Oliver antony.oli...@sussex.ac.uk Subject: [ccp4bb] Curious electron density associated with Asp sidechain To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 25 April, 2013, 17:10 Dear CCP4 colleagues. I'm just finishing up a refinement, but am left with one little curio that I just can't seem to solve. One aspartic acid residue is associated with some extra, unexplained electron density. --please see: http://i.imgur.com/vCYOqam.png Where, the Fo-Fc map is contoured at 3.78 rsmd in Coot. I have tried a number of different modelling scenarios, but as yet can't reach a wholly satisfactory conclusion; waters, alternate conformers, really don't seem to cut it. I though about some radiation-induced phenomena, but this data set was collected on a home-source, so I guess this is unlikely. So, I would really appreciate some ideas and suggestions. Hopefully it is blindingly obvious to someone. Random Thought: could it be PEGylation of the side-chain? Some other hopefully useful background information: * I'm sure it is/was an ASP, because the same protein (made from the same construct) has been used in previous crystallisations, and the resultant structures have clear, unambiguous electron density for the side chain. * the crystallization condition is PEG 200, with some Na/K phosphate at pH 5.8, and NaCl. The protein itself contains HEPES buffer. With many thanks, Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.ukhttp://mc/compose?to=antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 -- *Kornelius Zeth* *Email: kornelius.z...@gmail.com* *Unidad de Biofisica (CSIC-UPV/EHU) Barrio Sarriena s/n 48940, Leioa, Vizcaya* *SPAIN*
Re: [ccp4bb] off-topic:a dataset from a polluted sample
Dear Tianlong, you may use Arcimboldo using e.g. a long helix (or b-hairpin) as starting model for de novo phasing. If you succeed to phase your structure, you should be able see if it was part of your protein. Best wishes Kornelius On Wed, Jul 18, 2012 at 6:46 AM, Tianlong Zhang ztlc...@gmail.com wrote: ** Dear all, Recently we obtained a dataset and processed it to 1.9 A in P6 (for a/b/c: 49.6, 49.6, 65.3) which suggested there were about 100 amino acids in per AU. Our protein is about 50kDa for a protein complex and expressed in Hi5 cell. Therefore, we dissolved several crystals for N-terminal sequencing and the sequence is totally different from the complex. We suggested that this data set was for a protein from Hi5 cell. Furthermore, the signals of the sequencing were a little weak for determining the protein sequence by blast. I'm looking forward for your suggestions. Enclosed pleased find the the sequencing result. Thanks very much! Best regards. -- Tianlong Zhang -- *Kornelius Zeth* *Unidad de Biofisica (CSIC-UPV/EHU) Barrio Sarriena s/n 48940, Leioa, Vizcaya* *SPAIN*
Re: [ccp4bb] PISA server
it seems that it's stuck since long time. I asked there for help six week ago but have not received any answer. Cheers Kornelius On Sat, 26 Mar 2011 16:58:26 + Daniel Bonsor bon...@bbri.org wrote: Is the PISA server having problems? I can not seem to upload any structure. Thanks Dan -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
[ccp4bb] Question regarding S-SAD
Dear all, we have collected S-SAD data (2x4000 Frames, 0.1 degrees, wedges of 40 Frames, Rf between 3-5% overall) and received a dataset to 2.5 A which is complete with a redundancy of 30-40. Space group is P6 and the AU 120 residues, 6 Mets. Native data are up to 1.9 A Sharp and Phenix both return the expected number of sites and given the phasing power to approx. 3.8 A after HA refinement I was hoping to get some maps which I could use for tracing the structure. However DM, Solomon and Resolve all fail to significantly improve the phases and densities look not as if they can be traced. There is some partial twinning of 15%. What I noticed was that the spot profiles in XDS looked not as the are supposed to, presumably because the mosaicity of the spots are 0.4-0.6 and spot integration may be hampered. Still, merging Rfactors are brilliant. Any hints + ideas are welcome! Best wishes Kornelius -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
[ccp4bb] SUMMARY: ROSETTA for MR model generation
Summary to answers to question about MR-based models generated by ROSETTA (thanks to all who replied so quickly): ***De novo methods*** 1. Arcimboldo (suggested by Isabel Uson Finkenzeller and Peter Grey) Nature Methods 6:651-3. Crystallographic ab initio protein structure solution below atomic resolution is based on PHASER, SHELXE and CONDOR. ***In fact this program did the job!!!*** http://chango.ibmb.csic.es/ARCIMBOLDO/ http://en.wikipedia.org/wiki/Giuseppe_Arcimboldo 2. ROSETTA and MD model comparison (by jonathan elegheert) - the smaller the protein the better. There really is an upper limit; every amino acid makes computation exponentially more intensive. - For some proteins it works better than for others. In literature, it has been suggested that the observed failures are not due to bad conformational sampling of the potential energy surface of proteins, but are rather due to the low-resolution scoring function, which sometimes fails to score the models resembling the native fold as the lowest energy structures. However, this observation is largely outweighted by the number of succesfull protein structure predictions, which make Rosetta one of the best structure prediction algorithms available to date. - In most publications, 1 models are generated and scored. I believe something like 10 is rather necessary. I don't know if you have access to multinode machines, but a cluster is definitely necessary. Installing the software to work with MPI is also no trivial task. - The tricky part is the scoring of the clusters of models you will generate; if you don't have the x-ray structure to score and benchmark against (since you want to use it for MR in the first place), you have to score against the most represenative structures of the largest low-energy clusters. And exactly this can be a problem (see 2nd point). - Say you'd get a model with 1.8 A rmsd with the 'real' structure (would be a very good result); this still would translate to a sequence identity of +- 30%, so already marginal. 3. ROSETTA and MRBumb (by Martyn Winn) We also had a look at this problem: D.J Rigden, R.M Keegan and M.D Winn Acta Cryst. D64 1288-1291 (2008) - Molecular Replacement using ab initio polyalanine models generated with ROSETTA Our approach is to use Rosetta to generate a large set of models, and feed these to MrBUMP to process. 4. Recent literature about ROSETTA (by Francois Berenger ) High resolution protein structure prediction and the crystallographic phase problem http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504711/ Prospects for de novo phasing with de novo protein models http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631639/ ***Different servers and model preparation tools*** 1. Alternative servers for model preparation (Colin Levy): FFAS in conjunction with either ElNEMO or CASPR 2. MR models generated by ROSETTA and other web server tools(Phyre and I-TASSER)in conjunction with MR program REM and OEDM-DEDM phase refinement tool, both included into IL MILIONE (Acta Cryst (2009) D65 477-484) (Rocco Caliandro). www.ba.ic.cnr.it 3. PYRE-SERVER (Eike Schulz) http://www.sbg.bio.ic.ac.uk/~phyre/. 4. Balbes (Edward Snell) http://www.ysbl.york.ac.uk/~fei/balbes/index.html -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
[ccp4bb] ROSETTA for MR model generation
Dear all, I was wondering if anybody has used the ROSETTA software to generate a MR model that could subsequently being used successfully for a MR solution case. The sequence of the protein we work with is relatively small, ~ 85 residues. Crystallization is not very reproducible. Resolution is 1.9 A. Crystals are extremely rare. I would be grateful for any hints and will send a summary of all personally sent comments to the list. Thank you and have a nice day Kornelius -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] precipitation of protein in elution buffer
Hi Peter, we had a four similar cases recently. What really helped us was to add urea in amounts of 1-2 M. We tested the possible influence on secondary structure by limited proteolysis and CD/Trp-fluorescence and it seemed the protein maintained the same overall conformation as in the absence of urea. This compound also stabilized proteins for crystallization and allowed nicely diffracting crystals in three cases while in the absence we had nothing than precipitate etc. Good luck Kornelius On Thu, 22 Apr 2010 10:49:38 +0530 peter hudson peter.hudson.pe...@gmail.com wrote: Hello all I apolozize for a off topic question on BB. I am working with small proetin-protein complex, each have molecular weight 10kDa. I elute this N-terminal His-tagged complex through Ni-NTA resin in 50mM of NaH2Po4, 0.3M of NaCl, 5% glycerol and 250mM of Imidazole.Similarly, Lysis buffer and wash buffers contains same component without and with 20mM of imidazole respectively. His tag is present on the N-terminus of one partner of the complex. I am able to purify this protein in small scale and eluted fraction contains 1-2mg/ml of protein complex. But, when i scale up the purification volume to 1-2 lit of culture i get a really good yield like 1or 2 fraction will have 10mg/ml of proetin complex.I store the eluted fraction both at 4 degree as well as 25 degree. The fraction contains higher amount proteins get precipitated and settles on the bootom of microfuge tube while the fractions with less amount of proetin(1-2mg/ml) remain soluble. can anyone suggest any way to keep this protein complex solube without breaking the complex? Whether there is any other way to screen the solubility of protein with different buffers without using dynamic light scattrer? please suggest any reference which suggest protocols to check the solubility. I would appreciate the help. Thanks in advance Peter -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Lost my protein
Hi Yanming, you can try adding 1 M of urea, 1% detergent (OG,DM) that often helps to keep proteins in solution. Best wishes Kornelius On Tue, 5 May 2009 09:06:09 -0700 yanming Zhang shanma...@yahoo.com wrote: Hi all, during concentrating my protein, using Amicon Ultra centrifugal filter devices, 5000g, 4C, I lost large amount of my protein (75%). I heard the same story from one of my colleagues too. It seems the membrane of the Amicon tube ate my protein. Why this happen and how to recover my protein? Thank you very much. Yan -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
[ccp4bb]
Unless you want a ligand free molecule it might be a good idea to add the substance already to the growing Ecoli (?) culture. That has proven to be successful for such molecules. Best wishes Kornelius On Wed, 1 Apr 2009 16:59:05 + KUMARASWAMI MUTHIAH megun...@hotmail.com wrote: Anybody tried to cocrystallize the protein-progesterone or estrogen complexes, if so how do you go about the solubility of these compounds? Progesterone is only soluble in 50% chloroform or 100% DMSO and the dilution of this stock is not possible as chloroform falls out of solution. Lots of papers out there used soaking with progesterone or expressed the protein in the presence of progesterone. Any suggestions would be appreciated.Thanks _ Rediscover Hotmail®: Now available on your iPhone or BlackBerry http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobile1_042009 -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Design Constructs
Hi - we normally use: http://toolkit.tuebingen.mpg.de/hhpred together with: http://toolkit.tuebingen.mpg.de/quick2_d Both routines have a nice interface and a very sensitive and novel CS-Blast algorithm which can be used/integrated into the search. Best wishes Kornelius On Mon, 30 Mar 2009 14:11:02 +0100 Haridasan Namboodiri vnambood...@locuspharma.com wrote: Hello I am designing a protein construct for structural biology. It is a protein kinase which has not been crystallized earlier. I was comparing the kinase domains of other closely related family members characterized biochemically vs crystallization constructs. For crystallography constructs, there are different stretches of amino acid residues particularly at the N-terminus (some contain extra 2-5 residues while others have 15-20 residues. My question: Is there a rational way of designing exact constructs one would propose to make, eg., by a sequence alignment showing nearest homology neighbors that guided construct design etc.. Sincerely Hari Haridasan V. M. Namboodiri, PhD Scientist-Structural Biology Locus Pharmaceuticals, Inc. Four Valley Square 512 Township Line Road Blue Bell, PA 19422 email: vnambood...@locuspharma.com Ph: 215-358-2012 Fax: 215-358-2020 -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
[ccp4bb] Job opening at Max Planck / Friedrich Miescher Institute Tübingen
PhD-thesis/Doktorarbeit at the Max Planck Institute for Developmental Biology and Friedrich Miescher Laboratory Structural Study of How Cohesin Ring Is Loaded on the DNA Project description: Cohesin is a protein complex comprised of Smc1, Smc3 and Scc1 proteins that assemble into a gigantic trimeric ring and function to hold sister chromatids together inside the ring. One of the mysteries of the ring function is the mechanism of how DNA gets inside it. According to the current model ATP hydrolysis by the Smc heads triggers a long-distance structural change at the other end of the protein, in the so called hinge domain, resulting in the opening of the gate for DNA. A cohesin loader complex comprised of Scc2 and Scc4 proteins is required for cohesin loading on DNA in vivo. The proposed project involves characterization of the interactions between the cohesin loader and cohesin and between the parts of the cohesin complex with a goal of deciphering the DNA loading mechanism. Techniques to be used: Molecular and cell biology, biochemistry (cloning, expression, purification), X-ray crystallography (using home source and synchrotron radiation), electron microscopy (home EM). We are seeking a highly motivated biology/chemistry/physics student who has ideally acquired one of the above techniques during his/her diploma/master thesis. The project is a collaboration between the groups of Drs Kornelius Zeth (protein structure) and Dmitri Ivanov (chromosomal biology). For further information or applications please contact: Dmitri Ivanov, Dr. Friedrich Miescher Laboratory Spemannstr. 39, 72076 Tübingen Germany [EMAIL PROTECTED] Kornelius Zeth, Dr. MPI Developmental Biology Spemannstr. 35, 72076 Tübingen Germany [EMAIL PROTECTED]
[ccp4bb] asymmetric oligomers
Dear all, I'm searching for examples of crystal structures that show a clear asymmetry in the dimeric/oligomeric state. This asymmetry should not have been induced by the crystal packing (e.g. two domains connected by a long linker packing different, termini/loops which interact differently with the surrounding) rather than by an internal asymmetry (which may be confirmed by other techniques e.g. SAXS). The reason I'm asking: I have solved a couple of crystal structures of a chimera protein (110 residues, dimeric, 1.2 A resolution) and mutants of the same fragment consisting of basically two four helix bundles and a short connector fragment and these are highly asymmetric (although the H-bond pattern of the helix residues doesn't change) while the same structures solved by NMR are symmetric and largely different. PISA gives a variety of contacts (H-bonds, salt bridges) between the two chains, interface seems ok, stable, low B-factors). Any comments and suggestions are appreciated Best wishes and thanks! Kornelius -- 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: [ccp4bb] Protein Color
you can do a simple wavelength scan at the synchrotron of the protein solution frozen in a loop. Best wishes Kornelius On Fri, 5 Sep 2008 12:21:29 -0400 Matthew Alan Bratkowski [EMAIL PROTECTED] wrote: Hello. I am working with a protein that turns a yellowish-brown color when it is concentrated to around 2 mg/ml or higher in a small volume (a few hundred uL). I was wondering if the protein bound a metal or other prosthetic group that would give it this color? The protein's color somewhat resembles iron binding proteins, but there is no peak in the 400 nm range that would suggest heme, and an iron sulfur cluster is not that likely since there are only five cysteines in the protein. Proteins with structures homologous to the one I am studying bind magnesium, but are not know to bind other metals. Any information about what this color might suggest about the protein or how I could analyze possible bound metals or prosthetic groups using only a small amount of protein would be helpful. Matt -- 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: [ccp4bb] Anion binding sites in proteins
There is a number of 'defined' anions known from structures of halorhodopsin and the cores of coiled-coil proteins often show them as well. -- 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: [ccp4bb] Sequence of crystallised protein fragment
ESI-MS at a precision of +-2 Da should work alone. Best wishes Kornelius On Tue, 1 Jul 2008 19:32:56 +0100 Matthew Chu [EMAIL PROTECTED] wrote: N-terminal sequencing / MS for intact mass analysis are the only ways that I can think of. Matt 2008/7/1 Klaus Futterer [EMAIL PROTECTED]: We have a 150 kDa protein that reproducibly crystallises at one of the Hampton Screen conditions. However, we know from SDS gel analysis that the crystals contain only a 45 kDa fragment, that forms through proteolytic cleavage over time. We would like to determine the sequence boundaries of the fragment. We believe a combination of N-terminal sequencing plus MS analysis might give us the information we need, but I was wondering whether the ccp4bb community has other suggestions. Klaus - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ - Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- 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: [ccp4bb] Right terminal residues for constructs.
You can try: http://toolkit.tuebingen.mpg.de/hhpred this gives you a nice domain border prediction based on analog. structures. Best wishes Kornelius On Tue, 20 May 2008 19:34:39 +0200 Jayashankar [EMAIL PROTECTED] wrote: Dear friends and scientists, (A pre-Structural biological question.) I Have a multidomain protein , I know the domain boundaries, But am still not that rational to what residues a construct should start or end? But I have learned from people that changing one residue changes the fate of the construct. I will be greatful for the insights from the scientific community. I dont want to do any Directed evolution approach. -- S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany -- 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: [ccp4bb] Setting drops with proteins that are hard to concentrate
can't you try different volumes: i.e. 360 nl protein solution in very little buffer and salt + 40 nl precipitant. Should give roughly 10 mg/ml after equilibration. Best wishes Kornelius On Thu, 24 Apr 2008 12:42:42 +0100 Roberto Steiner [EMAIL PROTECTED] wrote: I have a similar problem Matthew. Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see. Regards, Roberto On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome 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. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED] -- 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
[ccp4bb] POSTDOC POSITION
POSTDOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY OF MITOCHONDRIAL OUTER MEMRBANE COMPLEXES The objective of this position is to determine the atomic structure of complexes from the outer mitochondrial membrane. Our goal is to understand the crosstalk of mitochondria with the cytosol via VDAC-dependent complexes. The positions is initially financed for one year with a possible extension for two more years. Interested candidates with a good background in protein cloning, purification and crystallization should send a letter and CV, together with names, addresses and telephone numbers of three scientists from whom letters of recommendation can be obtained to: -- 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
[ccp4bb] Thermofluor experiment
Dear all, a question very related to the discussion before. I have been reading the papers about the thermofluor experiment with great interest. I wonder what people think about the underlying principles/ideas and the success that the method yielded in their own labs for crystallization or related purposes? Has anybody used this method with membrane proteins in order to find out the stability of the protein in the presence of a second detergent? Is the method limited to this certain dye (sypro orange)? Have a nice day Kornelius P.S.: I will make a summary of all opinions. -- 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: [ccp4bb] Plz suggest me
could you please be more specific how you refined the structure, what program have you been using and which restraints did you set for the two independent molecules. Best wishes Kornelius On Tue, 1 Jan 2008 11:42:40 + Md. Imtiyaz Hassan [EMAIL PROTECTED] wrote: Dear freinds We have crystallized a cyanobacterial phycoerythrin which having two (alpha subunit) in an assymetric unit and held together by only three hydrogen bonds. When we compare the A and B molecule the RMSD is 1.32. because they same sequences then how it is possible to that two molecules are deviated at such extent. now the question is are both the molecules are crystallographic dimer? are two molecules are structural subunit as alpha 1 and alpha2? Is there any problem in refinement ? The structure was refined at 2.6 A with R 0.23/0.28 I hope a possible suggestion with warm regards Have a nice day.. Md. Imtiyaz Hassan, Ph.D. Young Scientist CIRBSc, JMI Telefax: +91 11 26983409 Mob-9868311151 __ Sent from Yahoo! Mail - a smarter inbox http://uk.mail.yahoo.com -- 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: [ccp4bb] Malonate Crystals?
we have had the same case recently - it turned out to be Ca-malonate. It seems that malonate has similar properties as oxalate being insoluble at higher concentrations. Best wishes Kornelius On Fri, 16 Nov 2007 13:48:49 -0600 Jacob Keller [EMAIL PROTECTED] wrote: Dear Crystallographers, I have just found some crystals in a 1 year old screen, and, having been repeatedly trained to be very skeptical, think that probably they are salt. So the question: has anybody seen malonate crystals before? I was wondering whether they could possibly be birefringent, given that the molecular structure is not chiral. The crystals I see are very birefringent. None of the other ingredients is chiral either. In general, my understanding had been that the birefringence was due to the chirality of the molecules/crystals...is this misguided? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] *** -- 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
[ccp4bb] sealed tube estimations
Dear all, we are in the progress of 'collecting data' on a sealed tube machine which we want to buy. I know that this issue was handled several times here and I also looked through older mails. However, my impression is that the generators that are offered change quite quickly according to recent developments and the ECM2007 and therefore I would kindly ask people for their opinions. We don't need a high brilliance source but rather something for pre-screening crystals and data collection of potential derivatives. Everyone is welcome to send opinions to me directly so that the majority of list people is not affected. One of the important requirements for us is: We are a small team of crystallography people and only little additional manpower for maintenance is available. Best wishes and have a nice day Kornelius -- 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: [ccp4bb] MAD screen pattern
The probably more elegant way is to perform ESI-MS of the protein. Best wishes Kornelius On Fri, 20 Jul 2007 12:22:27 +0800 劉家欣(OZ) [EMAIL PROTECTED] wrote: Dear all: A dataset of Sel-Met crystal was collected in synchotron. However, I am not sure whether the sel-Met is incoperated in the protein. The screen pattern is decribed as below. Anybady had the experiences about this? Best wish jaishin -- 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: [ccp4bb] How to replace 2.45M K/Na phosphate buffer - 2
at around 4 molar P-buffer freezes clean. Best wishes Kornelius On Fri, 22 Jun 2007 14:42:00 +0200 Claudia Scotti [EMAIL PROTECTED] wrote: Sorry: forgotten the pH: the crystals grow between pH 6.5 and 6.7. Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Date: Fri, 22 Jun 2007 14:15:47 +0200 From: [EMAIL PROTECTED] Subject: [ccp4bb] How to replace 2.45M K/Na phosphate buffer To: CCP4BB@JISCMAIL.AC.UK Dear list, I've crystals in a condition including 2,45 M K/Na phosphate buffer for the well condition, and 1 microl protein + 0.8 microl Na/K phosphate buffer + 0.25 microl 30% 1,6-hexanediol in the drop condition. These crystals dissolve in several cryoprotectant solutions and survive only in malonic acid, with which they give diffraction at 7-8 A. Among the several different possibilities, I'd like to try to optimize the crystals, and one possiblity among others would be to change the buffer. Any suggestions, please, about how to replace K/Na phosphate? At these concentrations it is acting as a buffer but also as a precipitant and I was wondering if anybody has experienced nice buffer-salt combinations that proved to be useful starting from a similar condition. Thanks a lot, Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 _ Make every IM count. Download Windows Live Messenger and join the i’m Initiative now. It’s free. http://im.live.com/messenger/im/home/?source=TAGWL_June07 _ Make every IM count. Download Windows Live Messenger and join the i’m Initiative now. It’s free. http://im.live.com/messenger/im/home/?source=TAGWL_June07 -- 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
[ccp4bb]
you can derivatize the protein before crystallization. By mass you can check the amount as most derviatives bind covalent. Moreover you can remove metal that doesn't bind. Best of luck Kornelius On Tue, 17 Apr 2007 13:49:33 -0400 [EMAIL PROTECTED] wrote: Hi, Without knowing exactly what you did, it's hard to guess however here are a few suggestions, in addition to the ideas already given by others: a. try iodination. Simply place an iodine crystal near the drop (glue it to the cover slip using a tiny bit of grease) and let the vapors penetrate the drop gradually. Also you can add 0.1 mM of KI to the solution then add a speck of iodine - the KI will slowly transition the iodine via the KI3 complex to the protein. You have to experiment with timing - what worked for me was 1 day exposure. The crystals may or may not turn yellow - depends on your protein and on conditions. b. I am surprised that mercury derivatives did not work. Have you tried increasing amounts of ethyl mercury phosphate? c. Have you tried my favorite platinum derivative - KPt(NO2)3? You can add a LOT of this stuff - it's very mild on the protein. Artem Hey there, I have crystallised a protein in 30% MPD and it it gives nice native data. However, it seems impossible to get heavy metal derivatives and I read that MPD does chelate heavy metals. Did any of you make similar experiences with xtals in MPD conditions? If so did you find a good solution (introduction of selmet makes the protein ustable) to make heavy metal derivatives? Best Regards Daniel -- 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
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
