[ccp4bb] post doc position at NCMM in Olso, Structural Biology of membrane proteins
Postdoctoral Research Fellow - (Membrane Proteins Structural Biology) Position as Postdoctoral Research Fellow - in membrane proteins structural biology Ref.no.: 2010/ 8649 Available at the Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo. A post-doctoral position for an initial two years with possibility of extension is available in the laboratory of Dr. Preben Morth. The Postdoctoral position is funded by an NCMM grant and is available from the 1st of October. The Focus of the research will include membrane protein involved with osmoregualtion and anion transport in bacteria and higher eukaryotes. Projects focus on bicarbonate transport through the plasma membrane and the intra cellular complexes involved with their tight control. NCMM, was established in 2007 as a partner institution of the European Molecular Biology Laboratory (www.embl.org). NCMM is located in brand new laboratories in the University of Oslo and National Hospital Biomedical Campus in the Oslo Research Park and Preclinical Medicine buildings. A new Structural Biology laboratory at NCMM, headed by Dr. J. Preben Morth has been established, with focus in membrane proteins. This new laboratory is well equipped for a modern approach to structural biology, it include recombinant expression in bacterial and eukaryotic systems, new protein purification equipment and incubators. There is excellent synchrotron access to ESRF through the Norwegian bag proposal. The University of Oslo has an in-house mass spectroscopy department for fast protein identification, facilities for electrophysiology and visualization equipment with electron microscopy and confocal microscopy. Applicants must have a PhD, and should have extensive experience with Vector design and cloning in E. coli and baculovirus or yeast expression systems, protein purification and characterization. Experience with membrane proteins and protein crystallography will be an advantage but not necessary. A good command of English is required. Oslo is a vibrant city in the south of Norway, with ready access to skiing in the winter and hiking in the summer. The Oslo bay and most of the coastline offer excellent scuba diving opportunities. For more information, please see the NCMM home page: http://www.ncmm.uio.no Please also refer to the regulations pertaining to the conditions of employment for post-doctoral fellowship positions: http://www.uio.no/admhb/reglhb/personal/tilsettingvitenskapelig/regulationstermcondition.xml Salary: Postdoctoral Research Fellow (SKO, 1352), pay grade: 57 – 64 (NOK 448 200– 510 000 depending on experience and qualifications) The application must include: Application letter including a statement of interest, summarizing the applicant's scientific work and interests and describing how she/he fits the description of the person we seek CV (summarizing education, positions, pedagogical experience, administrative experience and other qualifying activity), including a list of published and unpublished works Copies of educational certificates, transcript of records, letters of recommendation A complete list of publications and up to 5 academic works that the applicant wishes to be considered by the evaluation committee Names and contact details of 2-3 references (name, relation to candidate, e-mail and telephone number) Extended Applicant Form, to be filled in by the applicant http://www.uio.no/om/jobb-ved-uio/ledige-stillinger/2010/vitenskapelige/sokerskjema_GBR.rtf Closing date for applications: 15. August, 2010 The application should be sent electronically as a single pdf-file to recruitm...@ncmm.uio.no labelled with Ref.no.: 2010/8649 The University of Oslo has an agreement for all employees, aiming to secure rights to research results a.o. The University of Oslo has a goal of recruiting more women in academic positions. Women are encouraged to apply. In accordance with the University of Oslo’s equal opportunities policy, we invite applications from all interested individuals regardless of gender or ethnicity. Contacts: Requests for application information should be directed to CAO Elin Kaurstad E-mail: e.k.kaurs...@ncmm.uio.no Requests for further information and inquiries regarding the post can be directed to Group Leader J.Preben Morth, PhD. E-mail: j...@mb.au.dk Please see the following URL for further details and guidelines for appointment to research fellowships at the University of Oslo: http://www.matnat.uio.no/internt/administrasjonen/personal/Forskrifter-phd-postdoc-eng.pdf The University of Oslo (UiO) wishes to achieve a more equal gender distribution of scientific staff. Female candidates are encouraged to apply. UiO has an agreement for all employees, aiming to secure rights to research results a.o. Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds
Re: [ccp4bb] Phenix Autobuild
use fasta my_amazing_protein THISMESSAGESHOULDHAVEBEENSENTTOTHEPHENIXBB ;-) Preben On 03/08/2010, at 14.19, Md. Munan Shaik wrote: Hi everybody, I have a problem with Phenix autobuild. I want to input sequence file together with the pdb and mtz that I got from molecular replacement. But I have no idea which types of sequence files its required. if anybody please help me out. thanks === Md. Munan Shaik Department of Chemical Biology University of Padova via G. Colombo 03 Padova 35131, Italy Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/en/j...@mb.au.dk
Re: [ccp4bb] R factor R free struck
hi remember to reindex your data to P21212 in case you used Phaser to search all alternative orthorhombic SG's and it found P22121 Preben On 03/10/2010, at 04.56, Jack Russel wrote: Hi all, I have collected a data at 2.9 Å and the solved the structure using phaser . the space group comes to be P2 21 21. There are 4 molecules in Assymetric unit and an octamer is generated according to the symmetry. But after repeated rounds of rigid body refinement with REFMAC5 and model building with coot the R factor had been struck at 40% and R free at 50%. So my first question is whether my solution after phaser is correct. And if it is how can i lower the R factor and Rfree. The second question is it possible to have such a large difference between R factor and R free. Thanks in advance
Re: [ccp4bb] R factor R free struck
the reindexing have no effect on the R-factor, I did indeed mean to remember to transform the co-ordiantes according to the indexing :-) On 04/10/2010, at 11.27, Ian Tickle wrote: On Sun, Oct 3, 2010 at 6:34 PM, J. Preben Morth pr...@bioxray.au.dk wrote: hi remember to reindex your data to P21212 in case you used Phaser to search all alternative orthorhombic SG's and it found P22121 Preben Hi, re-indexing the data without also remembering to transform the co-ordinates (using the matrix transpose of the inverse of the re-indexing operator) would almost certainly cause the R factors to increase! But even assuming you did that, why should re-indexing/transformation have any effect at all on the R factors? Cheers -- Ian Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/en/j...@mb.au.dk
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
For this particular project, we mounted two native crystals from the crystal drop before adding the Ta6Br12(2+). The Ta6Br12(2+) was added as powder and left o/n. Since various amounts of Ta6Br12(2+) was added this way we never knew what the concentration was, many crystals were ruined his way, I suspect that the final concentration of Ta6Br12(2+) in the drops with useful crystals had a [Ta6Br12(2+)] below 1mM. The structure was initially solved by SIRAS using a native and derivative dataset collected on a crystal originating from the same drop. This derivative was collected at the peak. Most of the phasing power came from the isomorphous signal. However later as Poul mentioned, the data we got the best result from, was a dataset collected at an energy above the absorption edge of Ta and used both the anomalous and the isomorphous signal to get the best phase information. Preben On 04/10/2010, at 21.48, Jacob Keller wrote: - Original Message - From: Poul Nissen p...@mb.au.dk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, October 04, 2010 2:21 PM Subject: Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?) [1] the signal from Ta6Br12 is enormous and one will typically focus on low resolution (below 7 Å) so radiation sensitivity can be handled by a fairly low dose data collection We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth JP et al. 2007) and found that although we got the strongest anom. diff. Fourier peaks from a data set collected on the Ta peak, we got far better SAD phases from a data set collected on the high-energy remote wavelength. This I think is also often observed for SeMet. Interesting phenomenon--has it been documented, I wonder? I wonder which datasets were collected first? If the peak was collected first, as usual I think, and one assumes an exponential decay of the resonant signal as a function of radiation dose, it makes sense that the resonant signal would be more constant after the first data set, where the decay curve would have flattened out a bit. This would also be true for two consecutive data sets collected at high energy. Also, I think the decay function itself is steeper at the peak wavelength, leading to a less-internally-consistent data set at the peak. Does this argument hold water? Jacob Keller J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no, j...@mb.au.dk Tel: +47 2284 0600 http://www.ncmm.uio.no/research/ncmm-embl-group-leaders/
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin I would also like to comment. I our recent structure determination of the sodium pump (3.5 A) (see morth JP et al 2007) we did not have experimental phasing to more than 6 A for the Ta6Br12 clusters and 7 A for the Pt sites. Both with extensive multicrystal averaging and phase combination it was possible to trace the structure. The data was what you could call lousy, but in the end I was able to identify the 3 Rubidium ions present in the data set based on the their anomalous scattering power. Not detectable in the reflection statistics of the native data set (see Schack VR et al 2008). So even though your selenium sites will not help the phasing initially, they will still guide the model building extensively (see Hunte C et al 2005 Nature, 3.5 A res structure with Se) with your improved model phases the selenium site positions will improve and at a later stage they will be valuable, when combined as additional phase information. The initial maps always look really bad, your 3 years of heavy atom derivatisation might not have been in vain, you can still use the initial Se phases to try to locate more heavy atoms sites (Fredslund F et al 2006 jmb) good luck Preben On 11/05/2009, at 05.24, Engin Ozkan wrote: Wow, I got quite a number of responses. Thanks everyone. Let's elaborate. Petr, I don't know my anomalous signal, because I haven't yet done the experiment. If I had, I would have definitely talked about chi2 values for I+/I- merged and unmerged, Anomalous Patterson maps, or other measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great paper to read on that, I suggest every grad student to present it in their Crystallography Journal Club). I was merely pondering today, but I plan to do the experiment very soon (crystals are waiting for synchrotron time). About your example, 2.9 A diffracting crystals (with 4 A anomalous signal) are in the doable range, as you suggested. The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Engin On 5/10/09 2:50 PM, Leiman Petr wrote: Dear Engin Ozkan, You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity? This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28 which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution. Sincerely, Petr --- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Engin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, whereI/ sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick- Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them.
Re: [ccp4bb] Phasing at Low Resolution
Dear Martin You can either add the cluster as a powder, just dip the tip of a needle in the powder and gently approach the drop with the needle, the Ta6Br12 crystals will spray itself onto the drop. Then you just wait for the crystal to turn green, I usually marked the position where the powder had hit the drop, then mounted crystals lying with various distance from the powder site. This was how we derivatized the Na,K ATPase crystals (pH 7, 14 % PEG2kmme), with the H-ATPase crystal it was a bit more tricky, the cluster would not dissolve that readily, and one way to get around this was to dissolve the Ta6Br12 in water (approx 5 mM) then use a speed vac to increase the concentration even further. The highly concentrated Ta6Br12 will form a small round green ball, that you can break with a needle and transfer a tiny droplet to your crystal drop. The same approach was used with orange Pt ( Pt(II)- terpyridine chloride) best of luck Preben On 14/05/2009, at 20.29, Martin Jinek wrote: Dear all Now that tantalum clusters have been mentioned I have recently had issues with the solubility of the Ta6Br12 cluster (obtained from Jena Bioscience) in PEG-based crystallisation conditions at pH 8.0-8.5. Has anybody also experienced this? Is there a trick to getting the compound to dissolve? Best regards, Martin Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/da/j...@mb
