Re: [ccp4bb] sticky crystals

Most obvious (maybe you did it already but that is not clear from your email), why not try seeding? - J. - Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It’s as if t

Re: [ccp4bb] sticky crystals

Hi All One more method that I heard about but never tried is to put the plate on a sonication bath and to let for a short sonication pulse. That should vibrate some liquid under your crystal. Raz -- Raz Zarivach, Ph.D. Department of Life

Re: [ccp4bb] sticky crystals

Hi Savvas, You can collect data on your crystal still in the drop, on our beamline (FIP-BM30A at the ESRF) if you are interested. Provided space group is not P1 We do that routinely. Let me know if you are interested. JL Savvas Savvides wrote: Dear colleagues, we have b

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

To go along with the EDTA comment, did you check the pH of your binding solution? Sometimes lysing E. coli causes the pH to drop below the pKa of the histidines. On Tue, Jan 27, 2009 at 8:10 PM, James Stroud wrote: > You don't have EDTA left over from your protease inhibitor, do you? Some > com

Re: [ccp4bb] sticky crystals

Hi, I had a similar story like yours.Then I added a drop of 10ul simulated mother liquot which contains much higher concentrations of all components in the normal mother liquot. Sometimes, the crystals attached to the plastic would float to the surface. If not, take another 10ul, but blew it

Re: [ccp4bb] sticky crystals

Suggestions so far have been good ones. However, the MiTeGen microtools kit: http://mitegen.com/products/microtools/microtools_kit1.shtml comes with a "MicroSaw", which is a 10-micron thick kapton saw that is intended for this purpose. That is, you don't pry the crystal off the surface, but rat

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

You don't have EDTA left over from your protease inhibitor, do you? Some commercial cocktails include it. You may have to read the fine print. James On Jan 27, 2009, at 1:00 PM, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protei

Re: [ccp4bb] sticky crystals

If you have good and bad crystals in the same drop, I've had success pushing a crummy crystal into a good crystal and having it release that way. Additionally, once I realized this was going to be a long term problem, I started coating the sitting drop depressions with a thin layer of vacuum g

Re: [ccp4bb] sticky crystals

Hi, I had this exact problem before. The method below worked for me: Take a sturdy needle (like one of the microneedles from a Hampton kit or a very thin syringe needle) and (while observing the whole thing under a scope) stick the needle into the plastic a bit away from the crystal. Push h

Re: [ccp4bb] sticky crystals

Put a small piece of dry ice on the opposite side of the plastic from the crystal. Perhaps the difference in thermal expansive coefficient will let the crystal(s) break away. Don't overdo it though. This is a trick that Gary Gilliland taught me. Jim On Wed, 28 Jan 2009, Savvas Savvides wro

[ccp4bb] sticky crystals

Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, lo

[ccp4bb] pseudo translation

Hi all,here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed tha

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

You can also try TALON cobalt affinity resin from Clontech ( http://www.clontech.com/products/detail.asp?product_id=10588&tabno=2) or the high yield PrepEase resin from USB ( http://www.usbweb.com/category.asp?special=&cat=234&id=78806). On Tue, Jan 27, 2009 at 4:34 PM, Tim Gruene wrote: > Hi Fr

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

Hi Fred, I used to worked with a protein which would not bind to the Qiagen NiNTA (really *NOT*) but beautifully to IDA resin (e.g. Amersham Pharmacia). This was under non-denaturing conditions, though, and I don't know whether this applies to the denatured state. It may be worth a try. Tim

[ccp4bb] [OFF TOPIC] his-tag doesn't bind

Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is

Re: [ccp4bb] Nvidia 3D

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0901&L=CCP4BB&T=0&F=&S =&P=192056 > -Original Message- > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > Chris Waddling > Sent:

[ccp4bb] Nvidia 3D

Has anyone taken the plunge and tried this setup with Coot or PyMol? Seems interesting: http://www.nvidia.com/object/GeForce_3D_Vision_Main.html Chris -- Dr. Christopher A. Waddling, Ph.D. University of California at San Francisco MC 2140 S126C 600 16th St., San Francisco, CA 94158-2517 (415)

Re: [ccp4bb] Problem during refinement

Eleanor Dodson wrote: ... Many of these are unlikely, and you may well correct them when rebuilding. However the pdb file output by COOT will retain the CISPEP records.. You need to check and edit these yourself.. For the record, I believe that this is no longer the case. Coot will only write

[ccp4bb] Job opening at MedImmune, Cambridge, UK

Job details for Research Scientist/Senior Research Scientist in Structural Biology/Bioinformatics, MedImmune, Cambridge, UK MedImmune Cambridge, with its distinctive science and culture, is central to AstraZeneca's plans to establish a major international presence in the research and development o

Re: [ccp4bb] fix atomic positions in REFMAC5

There is an option to do harmonic restraints: external harmonic chain [ch] residue [res] insertion [ins] atom [n] [altcode [a]] [sigma [value]] or external harmonic residues from [residue_number] [chain_name] to [residue_number] [chain_name] sigma [value] sigma 0.1 For example: exter

[ccp4bb] fix atomic positions in REFMAC5

Is there a possibility in refmac5 to fix atomic positions of some part of the molecule and to refine the other part? I couldn't find a proper keyword in keywords list:(

Re: [ccp4bb] brute force molecular replacement

That may be so, but I'm still grappling with the visual of a younger Ian on all fours under the table -- or whatever it was, I deleted the email but the visual lingers, morphing disturbingly). phx. Nicholas M Glykos wrote: Dear Jacob, Why is it called "queen of spades?" As it ha

[ccp4bb] Postdoctoral position in Protein Crystallography, Manchester UK

Posted on behalf of Lydia Tabernero (so replies to me will get ignored ;-) POSTDOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY Applications are invited for a postdoctoral position to work on the structure determination of regulatory protein complexes

Re: [ccp4bb] ccp4-6.1, imosflm, phaser

Hi Andreas, Have you a project set up in CCP4i? Make sure that your CCP4i project directory is set before you try to start Imosflm. When run through CCP4i, Imosflm uses this directory to write it's output files. You can set the project directory by clicking on the "Directories&ProjectsDir" button

Re: [ccp4bb] brute force molecular replacement

Dear Jacob, > Why is it called "queen of spades?" As it happened, I was reading Alexander Pushkin's "Queen of Spades" while writing Qs. The book touches upon the subject of how close you can get to win everything and still lose it all. I thought that this was relevant for a stochastic search,