[ccp4bb] Postdoctoral fellowships in structural biology at Heidelberg University Biochemistry Center
*Postdoctoral Fellowships: Structural Biology / Molecular machines in protein targeting and ribosome biogenesis* The lab of Prof. Irmi Sinning at the Biochemistry Center (BZH) of Heidelberg University seeks to recruit 4 outstanding postdoctoral researchers for a period of 2--5 years. BZH is a leading research center with a collaborative and interdisciplinary atmosphere. Prof. Sinning is an investigator of the Cluster of Excellence:Cellnetworks (http://www.cellnetworks.uni-hd.de/) and studies molecular machines involved in cellular key processes using structural biology (http://www.uni-heidelberg.de/zentral/bzh/). We are interested in co- or post-translational protein targeting pathways, membrane protein biogenesis and ribosome associated enzymes and chaperones. Together with the team of Prof. Ed Hurt (http://www.uni-heidelberg.de/zentral/bzh/) we currently develop a strong interest in the structural framework of ribosome biogenesis. The successful candidates will be encouraged to pursue these projects well integrated into a highly collaborative team of researchers. Depending on previous experience and scientific interest, the projects will involve a combination of advanced techniques in structural biology (mainly X-ray crystallography, SAXS), biochemistry, biophysics (ITC, SLS, fluorescence spectroscopy, HX-MS), and/or molecular cell biology. We routinely exploit the genome of a thermophilic fungus for structural studies; for protein crystallization we are equipped with a crystallization platform (http://xtals.bzh.uni-heidelberg.de/) and we have steady access to major European synchrotron high brilliance X-ray sources. Qualifications and experience: We are seeking skilled and passionate biochemists/structural biologists who are enthusiastic to venture into this area of research. You will join an energetic team in the resourceful environment of BZH, surrounded by world-class scientists from a wide variety of fields. Applicants should hold a Ph.D. in structural biology, biochemistry, biophysics, or in a closely related area. Research experience in structural biology, biochemistry, biophysics or molecular cell biology is desired. Good knowledge of standard cloning techniques, protein expression and purification is required, and experience in structural biology on macromolecular complexes is a plus. The ability to work independently as well as in a team, good communication skills, and excellent knowledge of English are also essential. Documents: Please submit a pdf file with the following information: a cover letter describing your motivation and experience, a scientific CV including a list of your publications, and the names and contact information of 3 referees. Address for application: irmi.sinn...@bzh.uni-heidelberg.de Contract: 2 -- 5 years (extension possible); starting date: as soon as possible. Applications will be considered until the positions are filled. Best regards, Irmi Sinning Prof. Dr. Irmi Sinning Dept. of Structural Biology Heidelberg University Biochemistry Center INF 328 69120 Heidelberg, Germany
[ccp4bb] 2nd Annual CLS Mx Data Collection School
The Canadian Macromolecular Crystallography Facility (CMCF) is excited to announce an intensive 5-day hands-on synchrotron data collection school at the Canadian Light Source (CLS) in Saskatoon. The School will take place June 5 - 9, 2012. Participants will attend a series of lectures and be actively engaged in macromolecular crystallography (Mx) data collection at CMCF beamlines. Completing the school will be an essential step to making use of the crystallography beamlines remotely and will better equip participants to effectively collect diffraction data on-site. Additionally, this year’s special topic will be an in-depth look at using COOT during structure solution and modeling with invited speaker Dr. Trevor Moraes. Participants should have a basic grounding in crystallography prior to attending the course. Application deadline is April 13, 2012. Please visit the CMCF website for more information and application form, at http://cmcf.lightsource.ca/school
[ccp4bb] synchrotron X-ray picture
Hi all, I have a vague memory of having a picture in someone's presentation once, showing a smoking hot X-ray beam emerging from the beam pipe in a hutch at a synchrotron. I think the picture might have been a double exposure, with a long exposure that captured air ionization superimposed on a normal photo (or some similar contrivance). In any case, can anyone point to or provide such a picture? I'm preparing a seminar, and I want to lend artistic verisimilitude to an otherwise bald and unconvincing narrative... Thanks in advance, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
[ccp4bb] Soaking Kinase Crystals with ATP analogues
Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland.
Re: [ccp4bb] synchrotron X-ray picture
Hi Pat - I, too, have a memory of such a picture. This isn't quite the one I was thinking of, but it should hopefully serve the purpose: http://www.nsls.bnl.gov/about/imagelibrary/images/hr/Synchrotron_Light2_hires.tif (there are a couple more from the parent page: http://www.nsls.bnl.gov/about/imagelibrary/ ) - Matt On 2/1/12 1:43 PM, Patrick Loll wrote: Hi all, I have a vague memory of having a picture in someone's presentation once, showing a smoking hot X-ray beam emerging from the beam pipe in a hutch at a synchrotron. I think the picture might have been a double exposure, with a long exposure that captured air ionization superimposed on a normal photo (or some similar contrivance). In any case, can anyone point to or provide such a picture? I'm preparing a seminar, and I want to lend artistic verisimilitude to an otherwise bald and unconvincing narrative... Thanks in advance, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
On Wed, Feb 1, 2012 at 11:17 AM, Dianfan Li l...@tcd.ie wrote: I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. This isn't too surprising; most kinases undergo global conformational changes (domain closure) when binding ATP. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? I'd recommend trying ATP-gammaS - it could also be a substrate, but it's worth a look. (Is there any reason to believe that AMP-PNP is a substrate?) I've noticed that the various analogues have been known to result in different conformations in the crystal structure, so it may be a good idea to try more than one anyway. -Nat
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Dianfan Some kinases have such conformation in non-activated apo form that the ATP binding site is partially obstructed. Soaking an ATP analog may then have 3 outcomes: 1) successfully open up the binding site without damage to the crystal, 2) fail to open up the active site and the compound cannot diffuse to the active site, or 3) induce conformational changes which lead to serious disorder in the crystals (which then loose their diffraction) or even crack. Hence my question: is the ATP binding site unoccluded in the apo structure? If you're in situation #3 then soaks at low concentrations may get you to #1 as a more gentle diffusion may be better accommodated by a crystal. Or you may stay in #3, or you may have lowered the concentration so much that the crystals don't crack and you're end up in situation #2. Still a worthwhile experiment. If the ATP binding site is unoccluded then another possibility would be that the kinase-ATP analog may be more soluble than the apo kinase, in which case increasing the precipitant concentration in your soaking buffer may help. Good luck Thierry -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dianfan Li Sent: Wednesday, February 01, 2012 2:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland. 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 Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
On Feb 1, 2012, at 12:17 PM, Dianfan Li wrote: I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. 15 minutes is a long time. Scoop crystals during that time period. Do the cracked crystals diffract? Do you see the analogue? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
I assume that cocrystallization has failed? What you are experiencing is likely the effect of conformational transition caused by ligand. You can try very slow adition (even microdialysis) or if your ligand is fairly insoluble then you can just add a tiny solid particle of inhibitor to your drop and hope it dissolves slowly and saturates the crystal slowly. Hope it helps :-) Artem On Feb 1, 2012 1:31 PM, Dianfan Li l...@tcd.ie wrote: Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Hi, First, it's very much a crystallographic question. Second, the success or failure in soaking in ligands/cofactors depends quite often also to the crystal packing. Some packing forms (and the spacegroups that go with it) will tolerate the soaking even if it's accompanied with a conformational change, whereas others won't (like the spacegroup you currently have). So you could try, among the other good suggestions that you were given, more crystallization conditions, perhaps you might get lucky and come across crystals in a different spacegroup which will behave more nicely vis-a-vis soaking. Good luck, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Dianfan Li [l...@tcd.ie] Sent: Wednesday, February 01, 2012 9:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland.
[ccp4bb] Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Hi , To add to the previous comments, crystallization of GTP or ATP (or their analogues) with their kinase/ A- or G-tpases can depend on a lot of factors that were mentioned (such as packing). A simple common problem is that ATP solutions should be carefully buffered prior to their use for soaking, people tend to forget about this. A 100 mM ATP solution is pH 3 (probably not good for your protein and it has 3 acidic groups) For some classes of ATP binding proteins, acidic pH have also been shown to lower chances of successful soaking or co-crystallization. The crystallization condition is also important. High concentrations of sulphates or phosphates tend to complicate things . Same thing for high concentrations of di or tri carboxylic acids (such as citrate, tartrate or malonate). Sulfates tend to occupy the beta phosphate binding sites and at high concentrations they can outcompete an analogue. For first hand experience, I would not assume that all analogues behave the same. Especially between AMPPNP, ATPgammaS and AMPPCP (or their Guanine counterparts). the Cp analogues in our hands tend to have lower affinities. You can always try ADP AlF4 combination or ADP BeF3, if you are not afraid of beryllium . Hope this helps Good luck -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Dear Dianfan, In some cases the ATP-lid of the kinase is blocking the active site in the crystal form. In those cases the only option is to try co-crystallisation. Besides ATP and the homologs you mention you can also try ADP that as you will see in the PDB has been heavily used for kinases. Best of luck Sofia Sent via BlackBerry® from Vodafone -Original Message- From: Dianfan Li l...@tcd.ie Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Wed, 1 Feb 2012 19:17:03 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Dianfan Li l...@tcd.ie Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues Dear all, Sorry about a non-crystallographic question here. I am working on a kinase and would like to get an ATP analogue into the crystals. When soaked with AMP-PCP, the kinase crystals crack in about 15 min at 4 C. I could try other analogues like AMP-PNP etc, but those would probably behavour in a same way as AMP-PCP. Is it a good idea of trying quick soaks at high concentrations of AMP-PCP? Co-crystallization is another option I have but AMP-PCP is a substrate of the kinase (with low rate). What are other ways of getting ATP analogues into a crystal? Thanks for suggestions, Dianfan Dianfan Li, PhD College of Biochemistry and Immunology Trinity College Dublin Dublin, Ireland.
Re: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Maybe someone has suggested this already... If so, I am re-enforcing it. If the cracking is coming from actual molecular movement induced by binding (and not other reason like differing ionic strength in your soaking conditions) you could try setting up some co-crystallization and (hopefully) grow some enzyme-substrate complex crystals... hth yuri
[ccp4bb] On sodium malonate
Dear All, For the 3.4 M sodium malonate of Hampton Research, will you please tell me how the pH was regulated to 6.0, 7.0 and 8.0 separately? Cheers, Dialing
Re: [ccp4bb] On sodium malonate
Dialing, Malonic acid is dissolved in water and then pH adjusted to the desired value with NaOH. Caution: dissolving malonic acid is highly exothermic. Do it slowly, in a hood. Doug From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dialing Pretty Sent: Wednesday, February 01, 2012 7:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] On sodium malonate Dear All, For the 3.4 M sodium malonate of Hampton Research, will you please tell me how the pH was regulated to 6.0, 7.0 and 8.0 separately? Cheers, Dialing
