[ccp4bb] MAX-lab beamtime Deadline March 14th 2008
___ CALL FOR PROPOSALS for beam time at MAX-lab, Lund, Sweden. Researchers using synchrotron radiation are invited to submit proposals for beam time for experiments to be carried out during the time period July 2008 to June 2009. The available facilities include beamlines for MX and SAXS, see below. Information on how to prepare and submit proposals as well as descriptions of the other experimental facilities available at MAX-lab can be found at http://www.maxlab.lu.se It is also possible to apply for Fast Access MX beam time at any time though it is recommended to submit normal proposals if possible. *** The proposal deadline is March 14th, 2008 *** ___ MACROMOLECULAR CRYSTALLOGRAPHY Beam time for macromolecular crystallography is available at: * Beamline I911-2: Fixed-wavelength (1.04 Å): 165mm marccd, mardtb, marccs sample changer * Beamline I911-3: Tunable 0.7-2.0 Å, suitable for MAD/SAD: 225mm marmosaic, kappa goniostat, semi-automatic crystal centering, Roentec fluorescence detector * Beamline I911-5: Fixed-wavelength (0.91 Å): 165mm marccd, mardtb All beamlines are equipped with user-friendly data collection software. For more information, see the beamline web site: http://www.maxlab.lu.se/beamlines/bli911 ___ SAXS Beam time is also available at the SAXS station at beamline I711. This recently developed low background set-up offers the possibility to measure in a q range down to 0.007 Å-1 depending on the chosen sample to detector distance. The set-up is further described at: http://www.maxlab.lu.se/beamlines/bli711 ___ ACCESSIBILITY AND SUPPORT MAX-lab is situated in Lund, 50 km or 30 minutes by car from Copenhagen airport (with less than 2 hour flights from e.g. London, Paris, Strasbourg, Munich, Budapest, Helsinki,...). Trains leave the airport every 20 to 40 minutes (44 minutes to Lund). Users from EU countries and associated states can apply for financial support through the project "Integrating Activity on Synchrotron and Free Electron Laser Science (IA-SFS)" within the Sixth Framework Programme (more information can be found in the beam time application instructions). ___ -- ___ Thomas Ursby, PhD Beamline manager, Cassiopeia (Beamline I911) MAX-lab, Lund University, P.O.B. 118, S-221 00 Lund, Sweden [EMAIL PROTECTED] http://www.maxlab.lu.se Phone +46-(0)733 439551, Fax +46-(0)46 2224710 Cassiopeia: MAD and Fixed-Wavelength Stations for Macromolecular Crystallography __ http://www.maxlab.lu.se/beamlines/bli911/ __
Re: [ccp4bb] anomalous signal of Mn and Ca ions
On Thursday 28 February 2008 19:54, Sun Tang wrote: > Dear All, > > In my structures, I want to assign Mn or Ca ions for some densities. > But when I did not have anomalous density in CCP4i. [snip] > I collected the data at the wavelength of 1 A. Mn has only about 1e of anomalous scattering (f") power at 1A. Ca has essentially 0. So you should not expect to see any peaks in your map. > Do I need to adjust the wavelength to maximize the anomalous signal from Mn > or Ca? Yes. To distinguish between them you would need to select an X-ray energy between their respective K-absorption edges. You can use the X-ray Anomalous Scattering server to help you: http://skuld.bmsc.washington.edu/scatter/AS_form.html This will tell you that you would need an X-ray energy less than the Mn K-edge at 6.5390 keV (1.8961 Angstrom) http://skuld.bmsc.washington.edu/scatter/AS_periodic.html > I am not sure whether I was correct. The following was what I did: > > I processed the data with HKL2000 and select anomalous signal in scaling. > In CCP4i, I selected "Run FFT-Creat Map" in the "Map& Mask Utilities". I > select "O format to cover asymmetric unit" and "Plot section on Z axis from 0 > to 1 in steps on 10". All others were by default values. I display in ono10. > > I collected the data at the wavelength of 1 A. Do I need to adjust the > wavelength to maximize the anomalous signal from Mn or Ca? > > Any ideas and suggestions are greatly appreciated! > > Sun Tang > -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[ccp4bb] anomalous signal of Mn and Ca ions
Dear All, In my structures, I want to assign Mn or Ca ions for some densities. But when I did not have anomalous density in CCP4i. I am not sure whether I was correct. The following was what I did: I processed the data with HKL2000 and select anomalous signal in scaling. In CCP4i, I selected "Run FFT-Creat Map" in the "Map& Mask Utilities". I select "O format to cover asymmetric unit" and "Plot section on Z axis from 0 to 1 in steps on 10". All others were by default values. I display in ono10. I collected the data at the wavelength of 1 A. Do I need to adjust the wavelength to maximize the anomalous signal from Mn or Ca? Any ideas and suggestions are greatly appreciated! Sun Tang - Never miss a thing. Make Yahoo your homepage.
Re: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column?
Eric, I second Chun's reply. As the Volumetric flowrate (ml/min) is equal to the linear flowrate times the area of the column, and the linear flowrate is fixed for a particular resin (Sigma is terrible at providing this value for their resins) you basically just need to increase the diameter of you column to increase the allowable volumetric flowrate. I typically find that a 2 cm ID column gives a pretty good flowrate with agarose resins, so much so that I usually use a peristaltic pump to slow the rate of loading so I have time to drink my morning coffee. As for washing and elution I usually carry these out at the same flowrate as loading (including GST resins). Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452 Eric Dollins wrote: Dear protein purifiers, Off topic question: Is there a general rule for how fast you can load, wash and elute from affinity columns, e.g. glutathione agarose? The product insert from Sigma says load under gravity flow. For the volume of cell lysate I have, gravity loading would take an excruciatingly long time. I want to hook up a peristaltic pump to speed things along, but don't really have a feel for just how fast one can load a column in general (I realize this is also dependent on the construct, the buffer, etc). What about the subsequently wash or elution? Thanks for help Eric
Re: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column?
Dear Eric, There are several essential variables that govern protein chromatography (whether affinity, ion exchange, sizing, or other). It would be silly of me to reproduce a protein chromatography handbook here, so instead I would just list some practical pointers: If your column is e.g. 10-15 ml of any HF agarose (High-flow, crosslinked) packed into say an XK16 column (16 mm i.d.) then you can expect flow rates anywhere between 9-14 ml/min with a low viscosity buffer. Naturally, things tend to slow down if you have viscous buffers or suspended microparticulates. Practically speaking, clarified E. coli lysate can typically be loaded on such a column at 7-10 ml/min whereas clarified insect cell lysate tends to require 5-8 ml/min flow. This of course assumes that the back pressure is monitored and kept within reasonable limits (most manufacturers give you considerably lower pressure limits than both the column and the resin can actually withstand). Things to watch out for include exponential clogging - if your flow rate is slightly too high, the resin begins to compress, causing further increase in pressure, which in turn compresses the resin even more and so on until either the upper limit on pressure is triggered or the column is crushed/exploded. Obviously, larger i.d. allows for higher flow rate - for instance, 50 ml HF agarose packed into an XK26 column can usually sustain 20+ ml/min flow, likewise 150 ml of the same resin packed into an XK50 can do 40+ ml/min. Conversely, the larger the column (assuming the same design and materials) the less pressure it can withstand (e.g. ~1.8 MPa for XK16, ~1.2 MPa for XK26, and only ~0.6 MPa for XK50). For resins made of polystyrene (such as e.g. MonoQ/S or Q15/S15, etc.) crushing is less of an issue (the beads tend to be 'springy') whereas for size exclusion columns made of e.g. Superdex or Sephacryl crushing can be quite a problem. For somewhat exotic resins such as MonoBeads, ToyoPearl, etc. the maximum allowable pressure can exceed that of the vessel they're in (which is why stainless steel columns are sometimes employed with these resins). If you have to deal with particularly nasty lysates, don't forget that batch-binding can be an easy and practical (but somewhat messy) solution. In some cases I even had to resort to washing the resin on a Buchner funnel with a glass wool filter. Anything to get the protein. Good luck. Happy purifying! Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eric Dollins Sent: Thursday, February 28, 2008 5:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column? Dear protein purifiers, Off topic question: Is there a general rule for how fast you can load, wash and elute from affinity columns, e.g. glutathione agarose? The product insert from Sigma says load under gravity flow. For the volume of cell lysate I have, gravity loading would take an excruciatingly long time. I want to hook up a peristaltic pump to speed things along, but don't really have a feel for just how fast one can load a column in general (I realize this is also dependent on the construct, the buffer, etc). What about the subsequently wash or elution? Thanks for help Eric -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED]
Re: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column?
Hi Eric, Check the max pressure of the resin. Some tell you the flow-rate in cm/hr also. You can calculate that to ml/min with your column dimension. Most resins can stand 5 ml/min flow-rate in an ID2.5cm column. However, most people will recommend a slower flow-rate for glutathione agarose, claiming slow on rate. That's a myth. In our hands, glutathione resins binds as fast as Ni resins. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 [EMAIL PROTECTED] www.accelagen.com -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Eric Dollins Sent: Thursday, February 28, 2008 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column? Dear protein purifiers, Off topic question: Is there a general rule for how fast you can load, wash and elute from affinity columns, e.g. glutathione agarose? The product insert from Sigma says load under gravity flow. For the volume of cell lysate I have, gravity loading would take an excruciatingly long time. I want to hook up a peristaltic pump to speed things along, but don't really have a feel for just how fast one can load a column in general (I realize this is also dependent on the construct, the buffer, etc). What about the subsequently wash or elution? Thanks for help Eric -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED]
[ccp4bb] O-glycosylation in ccp4/refmac
Hello, I am trying to model an O-glycosylation modification at a threonine, but this seems to be problematic when I try to refine the model in refmac. The run fails, and the log says that a new ligand was found (the sugar). It offers a lib.cif file to add to the library and try the refinement again. I looked at this file and it looked fine (i.e. it wrote out the correct bond that is made between the sugar and threonine). However, when I tried to refine again with the lib.cif file, the run failed again and gave the same message. Thanks for any advice!
[ccp4bb] Off topic: General rule for maximum flow rate for affinity column?
Dear protein purifiers, Off topic question: Is there a general rule for how fast you can load, wash and elute from affinity columns, e.g. glutathione agarose? The product insert from Sigma says load under gravity flow. For the volume of cell lysate I have, gravity loading would take an excruciatingly long time. I want to hook up a peristaltic pump to speed things along, but don't really have a feel for just how fast one can load a column in general (I realize this is also dependent on the construct, the buffer, etc). What about the subsequently wash or elution? Thanks for help Eric -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED]
Re: [ccp4bb] Glu+Arg solutions for solubility
I have not tried to make 1M solutions of the indicated AA, but i have had good luck of using what they suggest in that specific paper. By using 50mM of each (Glu and Arg) I was able to increase the solubility of a 44kDa protein (a Serpin) from ~1mg/ml to above 17mg/ml. So in my case it really worked. This was for the purpose of doing liquid state NMR on that protein. I does help to solubilize the two aa in 1:1 ratio as the two combined improve their individual solubility. Jan Jensen On Thu, February 28, 2008 4:33 pm, Paul Paukstelis wrote: > posting for a colleague: > > In Golovanov et al. 2004 they indicate it is possible to make a 1M > solution of free amino acids (not amino acid salts) Glu+Arg in the > context of generating solutions for keeping proteins soluble. Has anyone > been able to do this? > >
[ccp4bb] Glu+Arg solutions for solubility
posting for a colleague: In Golovanov et al. 2004 they indicate it is possible to make a 1M solution of free amino acids (not amino acid salts) Glu+Arg in the context of generating solutions for keeping proteins soluble. Has anyone been able to do this?
[ccp4bb] ELRIG crystallography meeting
announcement: ELRIG Protein Crystallography meeting Hinxton Hall, near Cambridge, UK 1st April 2008 The European Laboratory Robotics Interest Group (ELRIG) is holding a free one day meeting on the 1st April 2008 at Hinxton Hall, Cambridge, UK. These meetings bring together vendors and scientists interested in robotic crystallisation and and a strong list of speakers has been assembled this year. Please see http://www.elrig.org/ for details. jan Jan Löwe Laboratory of Molecular Biology Medical Research Council Hills Road Cambridge CB2 0QH UK email: [EMAIL PROTECTED] phone: +44 (0)1223 252969 fax : +44 (0)1223 213556 WWW: http://www2.mrc-lmb.cam.ac.uk/groups/JYL/index.html
