[ccp4bb] Position for an experienced PhD-level structural biologist with sales and commercial experience
Dear All, I'd like to call your attention to a position announcement for a PhD level structural biologist to join a new biotechnology company called HarkerBIO. I am posting this announcement for any members of the CCP4 board that may be looking for positions but please note that I am not involved with the company. All enquiries should be directed to the i...@harkerbio.com email address. Thanks, Eddie --- HarkerBIO Accelerating Drug Discovery with Structural Biology 700 Ellicott Street, Buffalo, NY 14203 www.harkerbio.com HarkerBIO is a newly established biotechnology company backed by the world-renowned Hauptman-Woodward Medical Research Institute, namesake of Nobel Laureate Dr. Herbert Hauptman. Expanding on six decades of scientific research in structural biology and a proven high throughput protein crystallization technology, HarkerBIO provides unique drug discovery and development services to the pharmaceutical and biotechnology industries. Using three-dimensional structures of drug target molecules, HarkerBIO improves the early stages of drug discovery by creating visual scaffolds upon which new drugs can be built. HarkerBIO structurally guides the optimization of therapeutic candidates. By partnering with pharmaceutical and biotechnology companies, HarkerBIO is discovering and designing the next generation of drug therapies. HarkerBIO is located on the Buffalo Niagara Medical Campus, a consortium of the region's premier health care, life sciences research, and medical education institutions, all located on 120 acres in downtown Buffalo, New York. Buffalo offers cosmopolitan opportunities in an easily navigable city with a reasonable cost of living. Now with client revenues, an early and expanding customer base and business processes established, HarkerBIO is seeking an experienced PhD-level structural biologist with sales and commercial experience to drive HarkerBIO’s revenues to near-term profitability. Required Qualifications • PhD in structural biology, x-ray crystallography or structure-based drug design. • Knowledge of and access to key research contacts within pharmaceutical and biotechnology markets globally relevant to structure-based approaches. • Prior experience working in a CRO or services provider. • Experience working with and in the pharmaceutical and biotechnology industry. • Demonstrated experience in a sales, business development or commercial role within the life sciences industry, with particular emphasis working with pharmaceutical and biotechnology researchers in structural biology and related fields. • Strategic and international sales experience. • Proven capability to further build a pipeline of business, and actively manage to meet revenue milestones. • Experience with forecasting and account management. • Senior or manager-level experience in leading the following business functions: o Technical sales o Marketing o Business Development o Commercialization • The ability to work as a collaborative team member, and share responsibilities in a fast-paced and entrepreneurial environment. • Must be willing and able to travel both domestically and internationally. Compensation The selected candidate will be offered a compensation package with a base salary, incentive rewards tied to performance. A full fringe package will also include health benefits and a 401K plan. Some remote work may be an option with the ideal candidate and circumstances. To Apply Please send a CV and cover letter to: i...@harkerbio.com --- Edward Snell Ph.D. CEO Hauptman-Woodward Medical Research Institute Assistant Prof. Department of Structural Biology, SUNY Buffalo 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Heisenberg was probably here!
[ccp4bb] P3212--1's in Space Group Names?
Dear Crystallographers, I don't understand what the 1's are doing in space group names like P3212 or P3112--can someone fill me in? Not easy to google this one. JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] crystal anisotropy measurement?
Dear Cosmo, you may calculate effective resolution of your data set along different directions using the program EFRESOL (article in press in J.Appl.Cryst., the method is described by Urzhumtseva et al., 2013, Acta Cryst D69, 1921-1934). You can download the program from http://www-ibmc.u-strasbg.fr/arn/Site_UPR9002/Sites.html or I can send it to you off list. Good luck ! Sacha Urzhumtsev De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Cosmo Z Buffalo [cbuff...@ucsd.edu] Envoyé : mardi 17 février 2015 19:55 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] crystal anisotropy measurement? Hi all, I have a anisotropic crystal with cell dimensions of 78, 78 345. Is there a method for quantitatively measuring the the anisotropy of my diffraction data and a way to compare my data to that of similarly anisotropic crystals? -Cosmo
[ccp4bb] P212121 with strong Patterson peak at 0.5, 0.5, 0.5
We have data at ~3.2 A for a crystal that looked clearly like primitive orthorhombic while indexing in HKL2000 (or Mosflm). Based on the systematic absences I called it P212121 during scaling. However, we are having some unexpected issues with finding a Mol. Rep. solution using a model that is present as is in the crystal (along with regions that are absent in the model). There seems to be a clear non-origin Patterson peak at ~0.5, ~0.5, ~0.5. In agreement with this, when I use Molrep to find two molecules of the model in the asymmetric unit, the two solutions are derived from the same rotation function but with translation solutions displaced exactly 0.5, 0.5, 0.5 fractional coordinates from each other. However, the maps after refinement don't look impressive at all. I looked at h+k+l = 2n+1 reflections. Visual inspection suggests that several of these are weaker than the corresponding h+k+l = 2n reflections, but there are certainly exceptions. I have not done a systematic calculation of what the average I and sigma are for h+k+l = 2n+1 and h+k+l = 2n are. Does this smell like a wrong space group assignment? Twinning? Or am I missing something very trivial here? Or is this a pseudo-translation we have to live with? Any kind of advice will be very helpful. I am happy to provide more information if you tell me what you need. Thanks a ton in advance. Best, JK P.S. Matthew's coefficient indicates that there could easily exist up to 6 monomers in the asymmetric unit (4 monomers if 70% of the crystal is solvent).
Re: [ccp4bb] P212121 with strong Patterson peak at 0.5, 0.5, 0.5
Hi JK, we recently worked on a crystal with similar symmetry and I would recommend you to read the two papers listed below. If you visually do see the weak reflections, the space group is primitive orthorhombic (and not body-centered orthorhombic). The strong Patterson peak at ~0.5, ~0.5, ~0.5 results from pseudo-translational non-crystallographic symmetry (tNCS), as you correctly pointed out. As a consequence the intensity distribution is different compared to a regular crystal without tNCS, which affects molecular replacement and also coordinate refinement. The tNCS could obscure the determination whether your crystallographic two-fold symmetry axes are screw axis or not, thus your space group could in principle also be P22121, P21212, P21221, P2122, P2212, P2221 or P222. This is nicely described in the first reference. In light of the tNCS, you may also want to give Phaser a try for molecular replacement, I would try all eight primitive orthorhombic space groups (keyword: SGALTERNATIVE SELECT ALL). Twinning could in theory be possible (e.g. a P2 or P21 twin with the unique beta cell angle very close to 90 degree [could be any of the three angles]), but difficult to detect together with the tNCS. Best, Simon J. A. Sundlov, A. M. Gulick, Structure determination of the functional domain interaction of a chimeric nonribosomal peptide synthetase from a challenging crystal with noncrystallographic translational symmetry. Acta Crystallogr D Biol Crystallogr 69, 1482-1492 (2013)10.1107/S0907444913009372). R. J. Read, P. D. Adams, A. J. McCoy, Intensity statistics in the presence of translational noncrystallographic symmetry. Acta Crystallogr D Biol Crystallogr 69, 176-183 (2013); published online EpubFeb (10.1107/S0907444912045374). On Tue, Feb 17, 2015 at 7:19 PM, Jayakrishnan Nandakumar User sscna...@gmail.com wrote: We have data at ~3.2 A for a crystal that looked clearly like primitive orthorhombic while indexing in HKL2000 (or Mosflm). Based on the systematic absences I called it P212121 during scaling. However, we are having some unexpected issues with finding a Mol. Rep. solution using a model that is present as is in the crystal (along with regions that are absent in the model). There seems to be a clear non-origin Patterson peak at ~0.5, ~0.5, ~0.5. In agreement with this, when I use Molrep to find two molecules of the model in the asymmetric unit, the two solutions are derived from the same rotation function but with translation solutions displaced exactly 0.5, 0.5, 0.5 fractional coordinates from each other. However, the maps after refinement don't look impressive at all. I looked at h+k+l = 2n+1 reflections. Visual inspection suggests that several of these are weaker than the corresponding h+k+l = 2n reflections, but there are certainly exceptions. I have not done a systematic calculation of what the average I and sigma are for h+k+l = 2n+1 and h+k+l = 2n are. Does this smell like a wrong space group assignment? Twinning? Or am I missing something very trivial here? Or is this a pseudo-translation we have to live with? Any kind of advice will be very helpful. I am happy to provide more information if you tell me what you need. Thanks a ton in advance. Best, JK P.S. Matthew's coefficient indicates that there could easily exist up to 6 monomers in the asymmetric unit (4 monomers if 70% of the crystal is solvent).
[ccp4bb] close contacts
Dear all, Is it possible to remove all the close contacts from the PDB structure in coot. Please suggest -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
Re: [ccp4bb] P3212--1's in Space Group Names?
Hi Jacob If you look at SGs P312 and P321in ITC-A you'll see that they are quite different in terms of the arrangement of the a.u.s relative to the symmetry axes. So essentially the 1's are there to distinguish these. The extra 1 or 2 after the 3 of course signifies a screw axis. Note that for a rhombohedral cell SGs R312 and R321 would be identical so there is only R32. Note that the original SG names in ITC without intervening spaces were carefully designed to be unambiguous. The extra spacing that was added by the PDB is redundant (or maybe they thought that it improves readability). Hope this helps. -- Ian On Wednesday, 18 February 2015, Keller, Jacob kell...@janelia.hhmi.org wrote: Dear Crystallographers, I don't understand what the 1's are doing in space group names like P3212 or P3112--can someone fill me in? Not easy to google this one. JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] how to reduce protein solubility
Thank you, everyone! Francesca From: Enrico Stura [est...@cea.fr] Sent: Tuesday, February 17, 2015 3:00 AM To: CCP4BB@jiscmail.ac.uk; Mattiroli,Francesca Subject: Re: [ccp4bb] how to reduce protein solubility Francesca, The most common failure is to have an excessive amount of salt (salting in/ salting out), glycerol or other solubilizing ingredient in your protein solution. I would suggest that you change the pH and reduce the salt in your protein solution, by microdialysis if you do not have much protein, and screen again. If share with ccp4bb the exact formulation of your protein solution you might get more suggestions. Enrico. On Tue, 17 Feb 2015 05:23:05 +0100, Mattiroli,Francesca francesca.mattir...@colostate.edu wrote: Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] OT: PhD position available
Dear colleagues I have an MRC-funded PhD position currently available in my lab to study proteins involved in sporulation of the human pathogen Clostridium difficile (details below). I would appreciate if you can pass the information to potential students and/or advertise it in your institutions. Many thanks Paula http://www.ncl.ac.uk/postgraduate/funding/search/list/cb115 Project details: Clostridium difficile (Cdiff) is one of the major causes of hospital acquired infections, a serious health concern that puts a significant economic burden on healthcare systems. Cdiff is resistant to most antibiotics and causes infection when the normal population of gut bacteria is disturbed by these drugs. Recent outbreaks show greater antibiotic resistance, more serious disease and higher risk of repeating infections. Clostridia forms dormant cells - spores - which are responsible for transmission and recurrent disease and can survive in the environment due to their resistance to common disinfectants, high temperatures and radiation. Our current research focuses on proteins involved in the early stages of sporulation and aim to elucidate the mechanisms and proteins involved in the engulfment of the smaller daughter cell (forespore) by the larger mother cell. In this project, we are particularly interested in the peptidoglycan (PG) remodelling activity of proteins involved in early stages of engulfment, as well as their potential interaction/interdependence with an essential mother cell to forespore communication channel. The project will involve a combination of structural biology and biophysical analysis, coupled with in vivo localisation, functional studies and peptidoglycan activity assays. The student will benefit from exceptional training in diverse disciplines: molecular and cell biology, protein purification, structure determination and PG biology to provide new understanding into Cdiff sporulation that would open new therapeutic avenues. Applicant details: The project involves a multi-disciplinary approach and students interested in bacterial infections and the challenges faced in understanding and fighting these pathogens are particularly encouraged to apply. The successful candidate would be a keen, motivated student, with an interest in microbiology and/or structural biology and an inquisitive, curious approach to research. The successful candidate must have an undergraduate degree awarded at an upper second or higher (or equivalent) in Biochemistry, Chemistry, Molecular Biology or related subject (for 4 year programme) or a Masters degree (for 3 year programme). Equivalent experience will also be considered. Studentship details: The studentship will cover a tax-free stipend of approximately £14,057 per year, research costs and tuition fees, and is available to UK students and to EU citizens who have been in the UK for the three years prior to the academic year 2015-16. Fees only studentships (no stipend) are available to EU citizens who have been a resident of the EU but not UK. For further details, contact paula.salg...@ncl.ac.uk For application, please use application portal www.ncl.ac/uk/postgraduate/applyhttp://www.ncl.ac/uk/postgraduate/apply === Dr Paula S. Salgado Lecturer in Macromolecular Crystallography Institute for Cell and Molecular Biosciences Faculty of Medical Sciences 3rd Floor Cookson Building Newcastle University Newcastle upon Tyne, NE2 4HH, UK Tel: +44 (0)191 208 7432 Fax: +44 (0)191 208 7424 Email: paula.salg...@ncl.ac.ukmailto:paula.salg...@ncl.ac.uk
[ccp4bb] 20th Anniversary Structural Biology Symposium - Registration is OPEN
20th ANNIVERSARY SCSB Structural Biology Symposium You and your colleagues are cordially invited to join us for the20th Anniversary Structural Biology Symposium to be held at Levin Hall on the University of Texas Medical Branch Campus on beautiful Galveston Island on Saturday, May 2nd, 2015. A. Keith Dunker, Indiana Univ. School of Med. Lewis Kay, University of Toronto Junji Iwahara, Univ. Texas Medical Branch George Rose, Johns Hopkins University Susan S. Taylor, Univ. California, San Diego Robert D. Wells, Texas AM Health Science Center http://www.scsb.utmb.edu/symposium Registration is open. -- Yours sincerely, Mark A. White, Ph.D. Associate Professor of Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics Macromolecular X-ray Laboratory, Basic Science Building, Room 6.630 University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Cell. (281) 734-3614 Fax. (409) 747-1404 mailto://mawh...@utmb.edu http://xray.utmb.edu QQ: In seeking wisdom thou art wise; in imagining that thou hast attained it - thou art a fool. - Lord Chesterfield
[ccp4bb] Computational Crystallography Newsletter - Volume 6, Number 1
I am pleased to announce the publication of the latest issue of the Computational Crystallography Newsletter: http://www.phenix-online.org/newsletter/ A listing of the articles and short communications is given below. Please note that the newsletter accepts articles of a general nature of interest to all crystallographers. Please send any articles to me at nwmoria...@lbl.gov noting that there is a Word Template on the website to streamline production. January 2015 Articles Improved Probabilistic Estimates of Biocrystal Solvent Content Rapid evaluation of non-bonded overlaps in atomic models Short Communications Plan a SAD experiment, scale SAD data, and analyze your anomalous signal Validation of carbohydrate structures in CCP4 6.5 Disulfide bond restraints Fitting Tip #9: Avoiding excess cis peptides at low resolution or high B FAQ - Tips for coordinated metal refinement Cheers Nigel --- Nigel W. Moriarty Building 64R0246B, Physical Biosciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : nwmoria...@lbl.gov Fax : 510-486-5909 Web : CCI.LBL.gov
Re: [ccp4bb] Refinement with phase/FOM and HL coefficients
Potentially there is more information in the 4 parameters HLA HLB HLC HLD which can describe a bi-modal distribution of phase probabilities( that would be generated by experimental phasing) than can be carried by the 2 parameter PHI FOM which will describe a uni-modal distribution. However if HLC=HLD=0.0 as will be so if the phase estimates are derived from a structure factor calculation the two options are equivalent . On 17 February 2015 at 09:14, Mohamed Noor mohamed.n...@staffmail.ul.ie wrote: Dear all In a few places (Refmac and Phenix, maybe there are also others), there is an option to use either phase/FOM or HL for refinement and DM. Is there any difference between these two? The dataset in question is a Fe SAD dataset. Thanks. Mohamed
[ccp4bb] crystal anisotropy measurement?
Hi all, I have a anisotropic crystal with cell dimensions of 78, 78 345. Is there a method for quantitatively measuring the the anisotropy of my diffraction data and a way to compare my data to that of similarly anisotropic crystals? -Cosmo
Re: [ccp4bb] Cis-peptide bond checking
Dear all, The version of Refmac that I currently use through ccp4i has If following features are found in coordinate file then make restraints to maintain them: activated by default for cis-peptides. I usually deactivate it during refinement, but this means that if one does nothing and moving atoms around creates a cis-bond, which is not so difficult to do with drag and refine at low resolution, the cis-bond is likely to be maintained. This probably helps them to creep in. Wouldn't it be a more sensible default to not create restraints for them by default? How many are genuine non-proline cis peptides? 0.x%? P.S: guilty as any, I just found one such error in one of my structures. Cheers, Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dkmailto:josea.cuesta.se...@carlsberglab.dk From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan Croll Sent: Monday, February 16, 2015 9:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cis-peptide bond checking Dear Wouter, That does sound like a useful tool indeed - finding the proverbial needle in a haystack! That's the challenge with such a rare event: rather like a true Ramachandran outlier, when they do occur they're usually a sign of an important motif in your protein that should be remarked upon. To others making the same point: yes, I'm well aware of the existence of true cis peptides, and both re-calculate the background rate in high-res structures and briefly discuss their nature in my paper (my personal favourite example is tissue transglutaminase (2q3z) which contains two - one of which is induced by the formation of a vicinal disulfide bond. It's believed that reduction of the disulfide switches the backbone back to trans to activate the enzyme). But I'm currently unaware of any protein that contains more than 3-4 cis bonds that stand up under scrutiny, while there are many models out there with tens of, or up to a few hundred. For examples of erroneous assignment at high res look at 3ncq, 2gec or 2j82. It's not such a problem at high resolution, but at lower resolutions I'm more concerned about why the cis bonds have crept into the model. Are they simple innocuous oversights (as pointed out by Robbie Joosten, most - but certainly not all - appear in poorly-defined density), or have they come about due to accidentally force-fitting a loop that is fundamentally wrong (e.g. due to an adjacent strand being out of register)? In most cases it's of course the former, but what worries me is the example of a structure I found (since corrected by the authors) that had 86 cis bonds (1.4%), yet only 0.4% Ramachandran and RSRZ outliers. In a good structure one would expect an erroneous cis bond to introduce an outlier in some other metric - but it seems equally possible that in a bad structure it could bring an outlier back into a favoured region. Hope this clarifies my point. Cheers, Tristan From: wouter.t...@radboudumc.nlmailto:wouter.t...@radboudumc.nl wouter.t...@radboudumc.nlmailto:wouter.t...@radboudumc.nl Sent: Monday, 16 February 2015 9:55 PM To: Tristan Croll; CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cis-peptide bond checking Dear Tristan, Thank you for your post of earlier today regarding the problem of cis and trans peptide planes in the PDB. We also realised this problem a while ago and an article describing this problem and a solution is presently under review at Acta Cryst. D. After analysis of the PDB we can state with 95% certainty that ~4600 trans - cis flips in ~2800 entries (and ~70K peptide-plane flips) are needed in the PDB. Around a third of the trans - cis corrections concern non-prolines. We hope to be able to deal with the problem of cis - trans corrections later. In the tradition of our group, the software to detect these flips is already available at swift.cmbi.ru.nl. Hopefully, the referees of our article consider this topic just as important as you and I do :-). Kind regards, Wouter Touw and Gert Vriend On 02/16/2015 10:58 AM, Tristan Croll wrote: Dear all, My apologies for the spam-like nature of my post, but I would like to draw your attention to an important issue (outlined in an upcoming short communication to Acta D, which will appear at doi:10.1107/S1399004715000826 once it's online). At present, neither the structural quality checks in commonly-used crystallography packages nor those run on deposition of a structure to the PDB are flagging the presence of non-proline cis peptide bonds. This has led to the presence of many erroneous cis bonds creeping into the PDB - primarily in low-resolution structures as one would expect, but I have identified clearly erroneous examples in
[ccp4bb] Zero or non-zero FOM DELFWT coefficients?
Hello All, I've noticed that the figure-of-merit (FOM) and weighted difference map coefficients (e.g. DELFWT) for the test set (FreeR_flag = 0) of reflexions in an MTZ file as produced by a certain well-known ML refinement program are all zero, while the same coefficients produced by another well-known refinement program are non-zero. I can see that there might be arguments to justify both approaches, e.g. the values for the test set might be over-estimated so having them all zero is safer, but on the other hand a non-zero value even if it's biased must still be closer to the correct value than zero is. I was wondering if anyone knows of a reference where the pros and cons of the alternative approaches are discussed? Cheers -- Ian
Re: [ccp4bb] how to reduce protein solubility
Hi Francesca, Try some zinc (1mM + ) in your protein buffer? Zinc tends to make a lot of things less soluble in my hands. Dave On Tue, 17 Feb 2015 04:34 Mattiroli,Francesca francesca.mattir...@colostate.edu wrote: Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca
Re: [ccp4bb] how to reduce protein solubility
Hi Francesca, You could try using reductive methylation, this has always lead to a loss of solubility (and protein!) in my experience. Jena bioscience and Hampton research sell kits. Best regards, Paul.. [EvotecLogoMail] Paul A. McEwan Ph.D. Principal Scientist, Structural Biology +44. (0)1235 838802 +44. (0)1235 863139 (Fax) paul.mce...@evotec.commailto:paul.mce...@evotec.com www.evotec.comhttp://www.evotec.com/ Evotec (UK) Ltd. 114 Innovation Drive Milton Park Abingdon Oxfordshire OX14 4RZ (United Kingdom) From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Briggs Sent: 17 February 2015 08:12 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to reduce protein solubility Hi Francesca, Try some zinc (1mM + ) in your protein buffer? Zinc tends to make a lot of things less soluble in my hands. Dave On Tue, 17 Feb 2015 04:34 Mattiroli,Francesca francesca.mattir...@colostate.edumailto:francesca.mattir...@colostate.edu wrote: Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom.
Re: [ccp4bb] how to reduce protein solubility
Francesca, The most common failure is to have an excessive amount of salt (salting in/ salting out), glycerol or other solubilizing ingredient in your protein solution. I would suggest that you change the pH and reduce the salt in your protein solution, by microdialysis if you do not have much protein, and screen again. If share with ccp4bb the exact formulation of your protein solution you might get more suggestions. Enrico. On Tue, 17 Feb 2015 05:23:05 +0100, Mattiroli,Francesca francesca.mattir...@colostate.edu wrote: Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] Refinement with phase/FOM and HL coefficients
Dear all In a few places (Refmac and Phenix, maybe there are also others), there is an option to use either phase/FOM or HL for refinement and DM. Is there any difference between these two? The dataset in question is a Fe SAD dataset. Thanks. Mohamed
Re: [ccp4bb] how to reduce protein solubility
Just a possibility for salvage of your already set-up drops: You can spike the reservoirs with some highly concentrated precipitant (no matter what as long as it sucks more water out of your drop). It does not solve your problem but maybe you can revive a few drops and get more information from your experiment. I use a syringe with a needle and poke through the tape into the reservoir and top it off with the high conc. precip. The tiny hole is easy to re-tape and does not hinder observation. Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mattiroli,Francesca Sent: Tuesday, February 17, 2015 5:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] how to reduce protein solubility Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca
