[ccp4bb] Ligand Refinement
Hello Everyone, I am attempting to fit NAD to a 2.1 A lactate dehydrogenase structure I've built and I'm running into several problems: 1. I loaded NAD from the COOT monomer library and was able to fit roughly 2/3 of it into the density using the real space refine zone function but the nicotinamide moiety flies apart as a result of the refinement. My work around for this was to load another ligand and roughly fit just the nicotinamide ring then cut and paste the coordinates from the second ligand into the file for the first. I then merged the ligand with the LDH structure using the merge molecules function in COOT. I fit NAD to four different chains this way. Attempting to refine the ligands after merging also results in the ring coming apart. 2. I was hoping that a round of refmac would then refine the entire ligand and thereby neatly fit the crudely placed nicotinamide ring. When I run refmac it fits the first NAD fairly rigidly such that while it does fit the nicotinamide ring it knocks the adenine moiety out of place. The other three NAD's look like they imploded. The atoms are too close together resulting in a structure which looks nothing like NAD and which contains multiple carbons with greater than four bonds. I was advised to create a new ligand dictionary and attempt refmac refinement with it. I have created files using refmac in review mode, loading the NAD coordinates into Sketcher and running libcheck, the PRODRG server and downloading from the HIC-UP server. Running refmac with any of these libraries produces the exact same result. Trying to use the real space refine zone on the one ligand which didn't come apart gives me the error message No Restraints Found!. I am completely stumped as to what to try next. Any suggestions would be greatly appreciated. Thanks! Larry Grant
Re: [ccp4bb] Ligand Refinement
Larry Grant wrote: I am attempting to fit NAD to a 2.1 A lactate dehydrogenase structure I've built and I'm running into several problems: 1. I loaded NAD from the COOT monomer library and was able to fit roughly 2/3 of it into the density using the real space refine zone function but the nicotinamide moiety flies apart as a result of the refinement. My work around for this was to load another ligand and roughly fit just the nicotinamide ring then cut and paste the coordinates from the second ligand into the file for the first. I then merged the ligand with the LDH structure using the merge molecules function in COOT. I fit NAD to four different chains this way. Attempting to refine the ligands after merging also results in the ring coming apart. https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0905L=COOTP=R62001=COOT9=AJ=ond=No+Match%3BMatch%3BMatchesz=4 2. I was hoping that a round of refmac would then refine the entire ligand and thereby neatly fit the crudely placed nicotinamide ring. When I run refmac it fits the first NAD fairly rigidly such that while it does fit the nicotinamide ring it knocks the adenine moiety out of place. The other three NAD's look like they imploded. The atoms are too close together resulting in a structure which looks nothing like NAD and which contains multiple carbons with greater than four bonds. I was advised to create a new ligand dictionary and attempt refmac refinement with it. That should not be necessary. I have created files using refmac in review mode, loading the NAD coordinates into Sketcher and running libcheck, the PRODRG server and downloading from the HIC-UP server. Running refmac with any of these libraries produces the exact same result. The devil's in the detail. Trying to use the real space refine zone on the one ligand which didn't come apart gives me the error message No Restraints Found!. If you are talking about Coot, then you need to make sure that the new dictionary you read in matches the comp_id of the residue/monomer you are trying to refine. Paul.
[ccp4bb] smeared bacteria pellets in protein expression
Dear ccp4bb, We used to routinely overexpress a number of mammalian proteins to inclusion bodies in E.coli. Lately however we get from time to time smeared and small or no pellets after spinning down the cultures (lysed I assume). I have read (with some horror) previous threads pointing at phage contamination and toxic proteins. I am doubtful about the phages as the problem is not expanding and very frustratingly it appears seemingly random, coming and going. Finally the one time I checked I did not see any plaque formation on a e.coli plate tested with a lysed culture. We have expressed the proteins before many times to inclusion bodies without any problems. I still could imagine that they could be toxic when soluble. But why would suddenly happen since we did not change any conditions that we are aware of? It would be really great to get some of your ideas on what else could cause this kind of problem (IPTG, aeration, leftovers from glassware cleaning, media) or if I missed something with respect to the phages. Many thanks for your time! hannes uchtenhagen -- Hannes Uchtenhagen Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden Office: +46-(0)8-524 86981 Mobile: +46-(0)7-36901461
Re: [ccp4bb] smeared bacteria pellets in protein expression
Hi Hannes, We have recently had a batch of phage contamination - one of the symptoms was exactly as you described, when pelleting the cultures, a smeary, fluffy pellet appeared. The other symptoms we had were that during growth, the OD would rise up until ~0.5, then drop, a foam would develop on top of the media (that remained upon standing) and if you examine the flask by eye there are ~2 mm strands floating around in the (almost clear) media. We found that this occurred (apparently) randomly within batches of the same media (using the same starter culture, same aliquot of antibiotics, IPTG etc), which ruled out a problem with the central media kitchen. After asking around, we found that other people in our building had experienced similar problems. The general consensus was that there was probably some airborne phage floating around in the incubator, which was getting in when flasks were open. At that point we made sure that we never opened our flasks in the incubator, so every time we do an OD or need to open the flask, we remove it, bring it back to our lab (a different room) and open it under a flame. Since then we have not had a single flask die. Although it is a bit of a pain to have to remove flasks every time you take an OD or induce, it is less of a pain than watching your media slowly die (especially if you like doing NMR, and have expensive multiple-labelled media...) Best Regards, Tom On Wed, May 12, 2010 at 9:29 AM, Hannes Uchtenhagen hannes.uchtenha...@ki.se wrote: Dear ccp4bb, We used to routinely overexpress a number of mammalian proteins to inclusion bodies in E.coli. Lately however we get from time to time smeared and small or no pellets after spinning down the cultures (lysed I assume). I have read (with some horror) previous threads pointing at phage contamination and toxic proteins. I am doubtful about the phages as the problem is not expanding and very frustratingly it appears seemingly random, coming and going. Finally the one time I checked I did not see any plaque formation on a e.coli plate tested with a lysed culture. We have expressed the proteins before many times to inclusion bodies without any problems. I still could imagine that they could be toxic when soluble. But why would suddenly happen since we did not change any conditions that we are aware of? It would be really great to get some of your ideas on what else could cause this kind of problem (IPTG, aeration, leftovers from glassware cleaning, media) or if I missed something with respect to the phages. Many thanks for your time! hannes uchtenhagen -- Hannes Uchtenhagen Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden Office: +46-(0)8-524 86981 Mobile: +46-(0)7-36901461 -- Skype: tom.murray.rust +44 7970 480 601 (UK Number) +61 41535 4486 (Aussie Mobile)
Re: [ccp4bb] smeared bacteria pellets in protein expression
Dear Hannes, I am afraid it can still be phage problems. There are many kinds of phages that infect E. coli and some of them behave like you describe: not always lysed cells but from time to time and coming and going. The phage may anyway be present but in its lysogenic state and only entering the lytic pathway under stressful conditions. Try some strains that lack or have mutated form of a common phage receptor: fhuA. I know that for example NEB sells a BL21(DE3) alternative (T7 Express) with a mutated fhuA (fhuA2). This strain (T7 express) also has the benefit of lacking the lambda prophage so that if your expressed proteins (esp. the inclusion bodies) trigger the SOS response you will not have lambda phage-caused lysis at least. Best regards, Martin PS. I am not affiliated to NEB in any way On May 12, 2010, at 10:29 AM, Hannes Uchtenhagen wrote: Dear ccp4bb, We used to routinely overexpress a number of mammalian proteins to inclusion bodies in E.coli. Lately however we get from time to time smeared and small or no pellets after spinning down the cultures (lysed I assume). I have read (with some horror) previous threads pointing at phage contamination and toxic proteins. I am doubtful about the phages as the problem is not expanding and very frustratingly it appears seemingly random, coming and going. Finally the one time I checked I did not see any plaque formation on a e.coli plate tested with a lysed culture. We have expressed the proteins before many times to inclusion bodies without any problems. I still could imagine that they could be toxic when soluble. But why would suddenly happen since we did not change any conditions that we are aware of? It would be really great to get some of your ideas on what else could cause this kind of problem (IPTG, aeration, leftovers from glassware cleaning, media) or if I missed something with respect to the phages. Many thanks for your time! hannes uchtenhagen -- Hannes Uchtenhagen Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden Office: +46-(0)8-524 86981 Mobile: +46-(0)7-36901461
[ccp4bb] Structural Biology Position at Astex Therapeutics
Structural Biologists (Ref SB-10) 10/05/2010 Cambridge Science Park Astex Therapeutics is a world leader in fragment-based drug discovery. The company has a strong commitment to X-ray crystallography - all our projects are based on a detailed understanding of protein: ligand interactions and thus structural biology plays a major role in drug design within the company. We have opportunities for structural biologists to join Astex and to influence our discovery and development projects. The successful applicants will have a PhD in Structural Biology, and will have experience in the areas of molecular biology, protein production and crystallography. A knowledge of, and an interest in protein-ligand interactions and drug design would be advantageous, as would be an interest in method development for high throughput crystallography. Kindly send your CV and a cover letter outlining your experience relevant to this role by email to the below address or by post. Human Resources Astex Therapeutics Ltd 436 Cambridge Science Park Milton Road Cambridge CB4 0QA UK Or apply by e-mail to: h...@astex-therapeutics.com Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing m.orei...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] problems with hydrogen names in PDB file from Shelx refinement
Usual apologies for non-CCP4 question. I have been working on two high resolution protein structures using Shelx for refinement. I included idealised riding C-H and mainchain amide N-H hydrogens in the refinement. In the PDB output by Shelxl the hydrogen atom names for valine, threonine, isoleucine and leucine are truncated from four characters (in the .res) to three. This results in duplicate names for atoms in these residues which is causing difficulties in depositing the structures. Each structure has over 1000 residues in the asymmetric unit so manually altering the names without introducing other errors is difficult. Does anyone know of any workaround? cheers Alice Alice Dawson Biological Chemistry and Drug Discovery College of Life Sciences The Welcome Trust Building University of Dundee Dow Street Dundee DD1 5EH Tel. 01382 385744 a.x.daw...@dundee.ac.uk
Re: [ccp4bb] smeared bacteria pellets in protein expression
We have had problems with phage contamination, either through contaminated plasmid stocks or more sporadic infections. Even if the cultures don't look clear, you can usually spot the contamination if the E. coli pellets are slimy and spread all the way up the centrifuge bottles. We have had good experiences with T7 Express cells for cleaning up contaminated plasmids. The last phage contamination was sporadic for several weeks before blowing up into an epidemic, so maybe give all your shakers a good scrub with bleach and be sure to decontaminate affected glassware as some phages can still survive through glass washing and autoclaving. If you think there might be a problem with inefficient glassware washing (it can happen and can also cause cell lysis if your glass ware is contaminated with detergent), try shaking your clean flasks for a few hours just with water at 37 degrees before autoclaving. Atlanta On 12 May 2010, at 11:16, Martin Hallberg wrote: Dear Hannes, I am afraid it can still be phage problems. There are many kinds of phages that infect E. coli and some of them behave like you describe: not always lysed cells but from time to time and coming and going. The phage may anyway be present but in its lysogenic state and only entering the lytic pathway under stressful conditions. Try some strains that lack or have mutated form of a common phage receptor: fhuA. I know that for example NEB sells a BL21(DE3) alternative (T7 Express) with a mutated fhuA (fhuA2). This strain (T7 express) also has the benefit of lacking the lambda prophage so that if your expressed proteins (esp. the inclusion bodies) trigger the SOS response you will not have lambda phage-caused lysis at least. Best regards, Martin PS. I am not affiliated to NEB in any way On May 12, 2010, at 10:29 AM, Hannes Uchtenhagen wrote: Dear ccp4bb, We used to routinely overexpress a number of mammalian proteins to inclusion bodies in E.coli. Lately however we get from time to time smeared and small or no pellets after spinning down the cultures (lysed I assume). I have read (with some horror) previous threads pointing at phage contamination and toxic proteins. I am doubtful about the phages as the problem is not expanding and very frustratingly it appears seemingly random, coming and going. Finally the one time I checked I did not see any plaque formation on a e.coli plate tested with a lysed culture. We have expressed the proteins before many times to inclusion bodies without any problems. I still could imagine that they could be toxic when soluble. But why would suddenly happen since we did not change any conditions that we are aware of? It would be really great to get some of your ideas on what else could cause this kind of problem (IPTG, aeration, leftovers from glassware cleaning, media) or if I missed something with respect to the phages. Many thanks for your time! hannes uchtenhagen -- Hannes Uchtenhagen Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden Office: +46-(0)8-524 86981 Mobile: +46-(0)7-36901461
[ccp4bb] Fwd: regarding purification of DNA binding protein
-- Forwarded message -- From: Arpit Mishra ar...@igib.in Date: Wed, May 12, 2010 at 6:20 PM Subject: regarding purification of DNA binding protein To: ccp...@jiscmail.ac hi all i am trying to purify DNA binding protein, but i couldnt get rid of the DNA after performing hi-trap heparin column, i have also tried high conc salt buffer (upto 1 M NaCl) and also tried o.2 M lithium sulphate, please suggest me some way to get rid of undesired DNA.. thanks Regards Arpit
Re: [ccp4bb] Fwd: regarding purification of DNA binding protein
Hello Arpit, don't you use DNAse during lysation of the cells? Or do you mean that you want to get rid of unspecific DNA and want to add some specific DNA after purification? In that case I'd understand you don't want to use DNAse. Tim On Wed, May 12, 2010 at 06:23:56PM +0530, Arpit Mishra wrote: -- Forwarded message -- From: Arpit Mishra ar...@igib.in Date: Wed, May 12, 2010 at 6:20 PM Subject: regarding purification of DNA binding protein To: ccp...@jiscmail.ac hi all i am trying to purify DNA binding protein, but i couldnt get rid of the DNA after performing hi-trap heparin column, i have also tried high conc salt buffer (upto 1 M NaCl) and also tried o.2 M lithium sulphate, please suggest me some way to get rid of undesired DNA.. thanks Regards Arpit -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Fwd: regarding purification of DNA binding protein
Hi Arpit, You can use PEI (polyethyleneimine). Adding PEI at low conc. (e.g. 0.25%) after cell lysis should precipitate most of the DNA. Note, it is best to do the addition step-wise at 4 deg with gentle steering over a period of time 10-15 min or so. Ibrahim On 5/12/10 10:20 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hello Arpit, don't you use DNAse during lysation of the cells? Or do you mean that you want to get rid of unspecific DNA and want to add some specific DNA after purification? In that case I'd understand you don't want to use DNAse. Tim On Wed, May 12, 2010 at 06:23:56PM +0530, Arpit Mishra wrote: -- Forwarded message -- From: Arpit Mishra ar...@igib.in Date: Wed, May 12, 2010 at 6:20 PM Subject: regarding purification of DNA binding protein To: ccp...@jiscmail.ac hi all i am trying to purify DNA binding protein, but i couldnt get rid of the DNA after performing hi-trap heparin column, i have also tried high conc salt buffer (upto 1 M NaCl) and also tried o.2 M lithium sulphate, please suggest me some way to get rid of undesired DNA.. thanks Regards Arpit
[ccp4bb] Electron Microscopy Position
Dear All The following position is available: Research Scientist Texas AM University Microscopy Imaging Center The MIC is looking to fill a permanent position (competitive salary and comprehensive benefit package included) with an energetic and passionate electron microscopist well versed in macromolecular cryo-TEM and tomographic applications in the Life Sciences. The ideal candidate would have a biology or biochemistry background and have a penchant for digital image processing and specimen preparation. A PhD is a necessary prerequisite for this position. The successful candidate will (i) conduct scholarly and professional RD, (ii) support the up keeping of a cryo FEI TF20 with energy filter, (iii) interact with undergraduate, graduate students, staff, faculty and visiting scientists in TEM through personal training and in a tutorial/lecture type environment including assessments, (iv) provide technical/research support for TEM in the interdisciplinary life sciences arena (including consultation in initiation of studies, scheduling of work, specimen preparation, microscopic analysis, digital image processing), and (v) complete paperwork for accounting and billing. The MIC promotes research and research education covering the field of basic and advanced electron microscopy and serving clients of all race, gender and ethnicity. This opening is available immediately and interviews will begin June1, 2010 until the position is filled. Qualified candidates should forward their cover letter, CV and three letters of recommendation to Dr. Andreas Holzenburg, Director, Microscopy Imaging Center at hol...@mic.tamu.edu. Texas AM is an Equal Opportunity Employer.
Re: [ccp4bb] thread protein sequence using pdb coordinates that are not avaliable on RCSB?
Kelly, This can be done with the Swiss-PDBviewer itself. Download the viewer if you don't have it. load your pdb ( File Open PDB File... ) load the sequence you want to thread (fasta format) ( SwissModel Load Raw Sequence from Amino Acids...) then thread with ( Fit Fit Raw Sequence) Good Luck, -bob On Wed, May 12, 2010 at 12:10 PM, Kelly Daughtry kddau...@bu.edu wrote: Hello all, I would like to thread one protein sequence onto a structure I recently solved, and has not been submitted to the pdb. I found the swiss-model website, which is an excellent tool for inputing a template sequence and target sequence (which is what I want to do), but it only allows you to select a PDB ID, and not to upload your own pdb file. Does anyone know where I can do this online? I'm thisclose to just changing each amino acid in coot (with the amount of searching I've done, it's about equal time!). Thanks in advance, Kelly Daughtry *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 ***
Re: [ccp4bb] thread protein sequence using pdb coordinates that are not avaliable on RCSB?
Kelly, You can do it with swiss-model, just have to use the alignment mode. I will ask for a pdb or RCSB code. Also, in the past I have found Jigsaw 3D model server to be a comparable tool. Scott On Wed, May 12, 2010 at 10:10 AM, Kelly Daughtry kddau...@bu.edu wrote: Hello all, I would like to thread one protein sequence onto a structure I recently solved, and has not been submitted to the pdb. I found the swiss-model website, which is an excellent tool for inputing a template sequence and target sequence (which is what I want to do), but it only allows you to select a PDB ID, and not to upload your own pdb file. Does anyone know where I can do this online? I'm thisclose to just changing each amino acid in coot (with the amount of searching I've done, it's about equal time!). Thanks in advance, Kelly Daughtry *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry Biochemistry University of Denver
[ccp4bb] Positions to be filled in Lübeck
The following positions are available at the Institute of Biochemistry, University of Lübeck, Germany: PhD students and postdoctoral fellows in (structural) virology and synthetic organic chemistry 1. Postdoctoral fellow in crystallography/structural virology (initially limited to 2 years; extension up to 6 years and ”habilitation“ possible). Salary: E13 TV-L (full-time employment). Good knowledge of recombinant protein production and macromolecular crystallography is required. The successful candidate is expected to participate in the biochemistry teaching programmes of the institute in the “Medicine“ and “Molecular Life Science“ curricula. Start of work: as soon as possible. Reference number: 624/10 2. Postdoctoral fellow in virology/molecular biology/biochemistry (initially limited to 2 years; extension up to 6 years and ”habilitation“ possible). Salary: E13 TV-L (full-time employment). Knowledge of virology, in particular in the construction and use of viral replicons, as well as in recombinant production of viral proteins is required. The successful candidate is expected to participate in the biochemistry teaching programmes of the institute in the “Medicine“ and “Molecular Life Science“ curricula. Start of work: as soon as possible. Reference number: 625/10 3. 2 Postdoctoral fellows in synthetic organic chemistry (initially limited to 2 years; extension up to 4 years possible). Salary: E13 TV-L (full-time employment). Substantiated knowledge of synthesis of heterocycles and modified peptides is expected. Experience in structure-based drug design is an advantage. Start of work: 01. 10. 2010. Reference number: 626/10 4. PhD student in structural virology and drug design (initially limited to 3 years). Salary: E13 TV-L (part-time employment: 50%). Tasks include the recombinant production and X-ray structure analysis of viral proteins and the design of inhibitors. The successful candidate is expected to participate in the biochemistry teaching programmes of the institute in the “Medicine“ and “Molecular Life Science“ curricula. Start of work: as soon as possible. Reference number: 627/10 5. 2 PhD students in synthetic organic chemistry (initially limited to max. 3.5 years). Objective is the synthesis of antiviral inhibitors according to design based on crystal structures of target proteins. Salary: E13 TV-L (part time em-ployment: 50%). Start of work: 01. 10. 2010. Reference number: 628/10 The Institute of Biochemistry investigates the molecular basis of infection by RNA-viruses with the objective to design and develop new antiviral drugs (see PLoS Pathogens 5, e1000428 (2009); Protein Science 18, 6 (2009); J. Mol. Biol. 383, 1081 (2008); Chem. Biol. 15, 597 (2008)). The work involves recombinant production, crystallisation and X-ray structure analysis of the proteins as well as design and chemical synthesis of inhibitors. All instrumentation necessary for this research is implemented in the institute, including direct access to synchrotron radiation at DESY. Please consult our webpages (www.biochem.uni-luebeck.de) for further information. The salary scale is regulated by the collective labor agreement for the public service of the federal states (TV-L), if the requested qualifications are satisfied. The final pay-scale allocation is subject to change. Working hours are according to § 6 TV-L. The University seeks to increase the number of women in those areas where they are underrepresented and therefore explicitly encourages women to apply. Applications from disabled individuals are especially welcome. Further information can be obtained from the Director of the Institute of Biochemistry, Prof. Dr. Rolf Hilgenfeld, tel. +49-451-500-4060; hilgenf...@biochem.uni-luebeck.de. Applicants interested in one of these positions are invited to send their complete resumé, including the names of two possible referees, before Mai 31, 2010, indicating the respective reference number, to Universität zu Lübeck – Dezernat Personal – Ratzeburger Allee 160, 23538 Lübeck, Germany -- Dr. Jeroen R. Mesters Gruppenleiter Strukturelle Neurobiologie und Kristallogenese Institut für Biochemie, Universität zu Lübeck Zentrum für Medizinische Struktur- und Zellbiologie Ratzeburger Allee 160, D-23538 Lübeck Tel: +49-451-5004065, Fax: +49-451-5004068 Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.selfish-brain.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
[ccp4bb] noncovalent interactions
Hi, Which free online servers are best for calculation of non covalent interactions present in several pdb structures? Thanks for any insights provided by the community members... Cheers GM
Re: [ccp4bb] Ion exchange protein lost
Like Juergen suggested, your protein is most likely stuck to the column, either because it never was really was refolded or because it doesn't like to be at pH 4.5. Do you really need such a low pH to have it bind to source 15S. Check the pI of the protein and perhaps some different pH buffer or try an anion exchange column if possible. Ursula On 5/11/10 7:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
Re: [ccp4bb] Ion exchange protein lost
Human gcsf has a pI ~ 6. As Ursula suggests you might be better off with anion exchange. There are protocols that show you can both refold and purify onto such column type. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabetat medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 12/05/2010 19:17, Ursula Schulze-Gahmen wrote: Like Juergen suggested, your protein is most likely stuck to the column, either because it never was really was refolded or because it doesn't like to be at pH 4.5. Do you really need such a low pH to have it bind to source 15S. Check the pI of the protein and perhaps some different pH buffer or try an anion exchange column if possible. Ursula On 5/11/10 7:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
Re: [ccp4bb] thread protein sequence using pdb coordinates that are not avaliable on RCSB?
Thanks for all the suggestions. I was able to use swiss-pdb viewer to accomplish my goal within 2 minutes of installing! Very useful software in general! I didn't realize all of the tools present. Thank you everyone! Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Wed, May 12, 2010 at 12:46 PM, Scott Pegan scott.d.pe...@gmail.comwrote: Kelly, You can do it with swiss-model, just have to use the alignment mode. I will ask for a pdb or RCSB code. Also, in the past I have found Jigsaw 3D model server to be a comparable tool. Scott On Wed, May 12, 2010 at 10:10 AM, Kelly Daughtry kddau...@bu.edu wrote: Hello all, I would like to thread one protein sequence onto a structure I recently solved, and has not been submitted to the pdb. I found the swiss-model website, which is an excellent tool for inputing a template sequence and target sequence (which is what I want to do), but it only allows you to select a PDB ID, and not to upload your own pdb file. Does anyone know where I can do this online? I'm thisclose to just changing each amino acid in coot (with the amount of searching I've done, it's about equal time!). Thanks in advance, Kelly Daughtry *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry Biochemistry University of Denver
Re: [ccp4bb] Micro-g Crystal Growth and the literature
(I noticed these words of wisdom did not make it to the thread. Hereby belatedly corrected.) The relevant TLI here is ROI: for the money to put one crystallization plate in space, you can employ enough postdocs probably to solve all membrane proteins in the human genome terrestrially 3 times over. (Of course, biologists would also need to get savvy enough to sucker governments into thinking it's a good idea for national defence.) Quite apart from the fact that crystallization and data collection are only two of many steps in a totally non-linear process, and that the cheapest (...?) way to get good data from sub-optimal crystals that actually matter is (a) to go to a state-of-the-art synchrotron and (b) go back to the lab for a few weeks. But maybe the quickest way to find your answer is to locate your local crystallographer (preferably with pharma experience and/or with a few recent grant rejections) and invite him out for a beer. The question is big and the real answer is check out a text book (cue Bernard...), certainly too big for a bulletin board reply read by people few of whom I believe take the reported improvements of space crystals seriously -- by seriously I mean sure-I'll-swipe-my-own-credit-card-if-NASA-won't seriously. (Ah, that was fun...! :-) phx. On 10/05/2010 04:49, Jack Reynolds wrote: TITLE: Extracting trends from two decades of microgravity macromolecular crystallization history (2005) (Judge, Snell and van der Woerd). Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown . . . in the reduced acceleration environnments of an orbiting spacecraft. TITLE: Macromolecular Crystallization in Microgravity Generated by a Superconducting Magnet (2006) (Wakayama, Yin, Harata, Kiyoshi, Fujiwara and Tanimoto). About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. TITLE: The crystallization of biological macromolecules under microgravity: a way to more accurate three-dimensional structures? (2002) (Lorber). The crystallization of proteins . . . in a microgravity environment can produce crystals having lesser defects than crystals prepared under normal gravity on earth. Such microgravity-grown crystals can diffract X-rays to a higher resolution and have a lower mosaic spread. TITLE: Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination. (2004) (Han, Cang, Zhou, Wang, Bi, Colelesage, Delbaere, Nahoum, Shi, Zhou, Zhue and Lin) . . . careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution, and a lower average temperature factor than the best ground-based control crystal. TITLE: JAXA-GCF project - High-quality protein crystals grown under microgravity environment for better understand of protein structure. (2006). (Sato, Tanaka, Inaka, Shinozaki, Yamanaka, Takahashi, Yamanaka, Hirota, Sugiyama, Kato, Saito, Sano, Motohara, Nakamura, Kobayashi, and Yoshitomi.) JAXA has developed technologies for growing, in microgravity, high-quality protein crystals, which may diffract up to atomic resolution, for a better understanding of 3-dimensional rpotein structures through X-ray diffraction experiments. TITLE: A Comparison between Simulations and Experiments for Microgravity Crystal Growth in Gradient Magnetic Fields. (2008). (Poodt, et al.). Microgravity protein crystal growth is expected to lead to an improvement of protein crystal quality, compared to crystals grown under normal gravity, due to the suppression of buoyancy driven convection. This is highly relevant, because for protein structure determination by X-ray diffraction, protein crystallization is often the quality limiting step. TITLE: Macromolecular crystallization in microgravity. (2005) (Snell and Helliwell). Density difference fluid flows and sedimentation of growing crystals are greatly reduced when crystallization takes place in a reduced gravity environment. TITLE: Comparison of space- and ground-grown Bi2Se.21Te2.79 thermoelectric crystals. (2010). (Zhou, et al.) The compositions of the space crystal grown along growth direction were more homogeneous than that of the ground crystal grown. The crystallization of space crystal grown was obviously improved. That's just a handful of quotes from a few of the sources I have accumulated over the last few
Re: [ccp4bb] noncovalent interactions
You could try LPC CSU Server: http://bip.weizmann.ac.il/oca-bin/lpccsu -Gyan On Wed, May 12, 2010 at 1:02 PM, gauri misra kamga...@gmail.com wrote: Hi, Which free online servers are best for calculation of non covalent interactions present in several pdb structures? Thanks for any insights provided by the community members... Cheers GM
Re: [ccp4bb] software to represent raw reflections in hkl zones
Tillmann,
I've added a jiffy script to synthesize pseudo-precession photos from a
rotation dataset, to the latest PHENIX package. We build this package
nightly, so any PHENIX bundle with a version number greater than
dev-402 will workhowever I see that dev-402 is not yet on the
public Web site (http://www.phenix-online.org) so I'll attach the python
script to this email for standalone use.
Here's what you'll need:
1) Make sure any recent PHENIX is installed and on path.
2) Make sure Mosflm is on path as ipmosflm
3) Index using two frames in the dataset:
labelit.index /data/project/dataset1_1_E1_###.img 1 90
4) Generate the precession photo:
labelit.python precession_photo.py bravais_choice=1 \ #id number for
the bravais setting as listed by labelit.index
pixel_width=600 resolution_outer=3.0 intensity_full_scale=1000 \
plot_section=H,K,0 image_range=1,90 pdf_output.file=HKL.pdf
5) These command line options should be self explanatory, but --help
gives a fuller description.
Nick Sauter
Tillmann Heinisch wrote:
Hi,
I have problems solving the structure of a protein crystal which seems to be
disordered. In order to investigate the disorder it would be useful to have a
precision photograph that shows reflections only in the [0kl] plane. Does
anyone know software that can transform raw data to give intensity distribution
in distinct zones of hkl?
Many Thanks for your help,
Tillmann
def usage():
return Python script to generate a synthetic photograph.
Requirements: Phenix must be installed and on path.
Mosflm must be installed and ipmosflm on path.
Usage:
1. First index the dataset using labelit.index. Typical syntax to index two
images is
labelit.index /home/data/project/dataset1_1_E1_###.img 1 90
2. Then
labelit.precession_photo command line arguments
Example:
labelit.precession_photo bravais_choice=1 \\
pixel_width=600 \\
resolution_outer=3.0 \\
intensity_full_scale=1000 \\
plot_section=H,K,0 \\
image_range=1,90 \\
pdf_output.file=HKL.pdf
3. Command line arguments explained:
labelit.precession_photo --help
Known limitations:
a) Since the source images are rotation photographs, there is an inherent
approximation in assuming that the data was obtained at the central
rotation angle (0.5 degree in a 0.0 to 1.0 rotation).
b) Real precession photographs are obtained with a finite-width annulus;
however the approximation here is one of an infinitesmal-width opening.
c) This is a naive implementation that reads all of the data into memory
first before calculating the image. More efficient algorithms will be
implemented later.
Author: Nick Sauter, LBNL; May 8, 2010.
Released under the CCTBX license; see cctbx.sf.net.
import math,pickle,os
import libtbx.phil
precession_master_phil = libtbx.phil.parse(
labelit_precession {
pixel_width = 1500
.type = int
.help = Width of pixel grid on which to calculate the synthesized image;
must be sufficiently large to sample the Bragg spots
resolution_outer = 4.0
.type = float
.help = The high-resolution limit (Angstroms) for the synthesized image
bravais_choice = None
.type = int
.help = Crystal setting (integer ID#), as enumerated in labelit.index
output, chosen for the synthesized section; print a list with
labelit.stats_index
plot_section = None
.type = str
.help = Miller indices of the section to be synthesized, comma separated,
no spaces, such as H,0,L
image_range = None
.type = ints(size=2)
.help = Range of data frames (inclusive) to be used for synthesized image,
i.e., min frame no.,max frame no., no spaces
intensity_full_scale = 255
.type = int
.help = Image intensity to be used as full scale (black)
pdf_output {
file = None
.type = str
.help = File name for pdf output
}
}
)
from scitbx import matrix
from scitbx.array_family import flex
from cctbx.crystal_orientation import crystal_orientation
from cctbx.uctbx import unit_cell
from spotfinder.diffraction.imagefiles import
Spotspickle_argument_module,image_files
from labelit.dptbx import AutoIndexEngine, Parameters
from labelit.command_line.imagefiles import QuickImage
from annlib_ext import AnnAdaptor
def get_precession_parameters(sources=[]):
parameters = precession_master_phil.fetch(sources=sources)
#validation not done here
return parameters
def ai_factory(inputpd):
subpd = inputpd[best_integration]
ai =
AutoIndexEngine(inputpd[endstation],inputpd[recommended_grid_sampling])
P = Parameters(xbeam=float(inputpd[xbeam]),ybeam=float(inputpd[ybeam]),
distance=float(inputpd[distance]),twotheta=float(inputpd[twotheta]))
ai.setBase(P)
ai.setWavelength(float(inputpd['wavelength']))
ai.setMaxcell(float(inputpd['ref_maxcel']))
ai.setDeltaphi(float(inputpd['deltaphi'])*math.pi/180.)
Re: [ccp4bb] General user proposal deadline May 15, 2010
Dear All, Just a reminder that May 15, 2010 is the deadline for the July-August 2010 Rapid Access Proposal cycle for PX beamtime at the ALS. The BCSB beamlines (5.0.1,5.0.2, 5.0.3, 8.2.1 8.2.2) can all be operated remotely and are all equipped with ADSC Q315(R) detectors. Beamlines 8.2.2, 8.2.1 and 5.0.2 are tuneable stations whereas 5.0.1 and 5.0.3 are fixed-wavelength beamlines. Control software for the beamlines features an interface allowing ultra high-throughput screening, automated annotation of diffraction quality and on the fly indexing and collection strategy feedback. The automounters in use on sector 5 support the unipuck and the ALS puck (both commercially available at http://www.crystalpositioningsystems.com). Beamlines 8.2.1 and 8.2.2. support in addition to the uni- and ALS puck the Rigaku system (http://www.rigaku.com/) To submit a proposal, please go to the ALS website link listed below: http://alsusweb.lbl.gov/4DCGI/WEB_GetForm/PXProposalEntry.shtml/Initialize Thank you, Stacey Ortega 510-495-2450
