Re: [ccp4bb] [EXTERNAL] [ccp4bb] Viewing samples under liquid nitrogen

Clear Pyrex beaker/dish/bowl filled with liquid nitrogen on the microscope stand. Place pin on a wand and hold at an angle in the bath. Probably best to do when the safety-officer is not around. Can you mount on a home source with the cryo-stream on? Best, Dan From: CCP4 bulletin board on

Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

Hopefully, someone will be able to share 293 Met- media. But if not, could you not use DMEM media with FBS, then deplete with DMEM with no glutamine, no methionine, no cysteine (thermos fisher 21013024) but supplemented with glutamax and cystine? The following paper describes it in the large

Re: [ccp4bb] ITC question -dimer vs monomer

You may want to look at the following paper concerning Il8, dimerization, binding and ITC. http://m.jbc.org/content/279/35/36175.full Get Outlook for Android From: CCP4 bulletin board on behalf of Bernhard Rupp Sent: Friday, October 4,

Re: [ccp4bb] Secrets of ZYMIT

Could you take a solution of it, buffer exchange/size exclusion and then perform MS fingerprinting to identify the protease if you don't have any luck with the companies? Best Dan Get Outlook for Android From: CCP4 bulletin board on

Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

Would it be possible to add a public annotations section to the PDB, to allow us to potentially flag/warn whoever downloads that particular structure, there could be something wrong with it, such as wrong space group, no/poor density fitting for ligand. Something similar to PubPeer maybe?

Re: [ccp4bb] Fo-Fc density close to cysteine residue

Have you mass-speced the protein before crystallization to make sure it wasn’t derivatized during expression and/or purification, or compared the mass spec of the crystals verses purified protein? Any fancy reagents or other reductants used during purification? What about S-Acetyl-cysteine

Re: [ccp4bb] W. Friedrich's thesis title

According to the German Wikipedia page it is correct. https://de.m.wikipedia.org/wiki/Walter_Friedrich_(Biophysiker) Maybe it wasn't deposited in the library until 1912. Dan Get Outlook for Android From: CCP4 bulletin board on behalf of

Re: [ccp4bb] A Max Perutz question

https://www.nature.com/articles/449144a Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zachary A.

Re: [ccp4bb] off topic: Membrane protein "into" soluble protein

In theory yes, you can fuse the termini to restrain the protein. You could use T4 lysozyme and insert in the loop which they use for crystallizing GPCRs (many references, including https://www.ncbi.nlm.nih.gov/pubmed/25450769). The potential problems are that you have no idea where the termini

Re: [ccp4bb] KCl in SDS-PAGE workarounds?

SDS is soluble, whereas the potassium salt of dodecyl sulphate is insoluble. You could try 18-Crown-6 ether to chelate the potassium, though I don't know if the crown ether would then affect the gel. Dan Daniel A. Bonsor PhD Institute of Human Virology, University of Maryland at Baltimore

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

I have had some luck with adding ~5-10ul of 60%-70% dilution of the reservoir to the drop. The larger volume of the drop appears to slow down the whizzing of the crystals and allows you to get a few crystals. Though it still occurs. You could also cool the area down or move into the cold room

Re: [ccp4bb] efresol download?

Is this not it? http://www-ibmc.u-strasbg.fr/spip-arn/rubrique257?lang=en At the bottom of the page. Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 From: CCP4 bulletin

Re: [ccp4bb] off-topic: negative thermal shift upon ligand binding

What are you using to dissolve the compounds? If DMSO or other organics, you maybe witnessing unfolding due to the organics. Have you done a control of just the solution with the protein. Some compounds may drastically change the pH of the buffer your protein is in. You maybe observing a

[ccp4bb] Arcimboldo, ShelxE, Windows

Dear All, Does Arcimboldo actually run on WIndows as I can see it is installed and so is ShelxE, but I have a warning in the GUI saying "ShelxE is not found and option to run on 'this machine' is disabled. Is this a bug, do I need alter something so get it to recognize ShelxE or it does not

Re: [ccp4bb] Se-Met and Se-Cys double labelling

To perform double labelling on your protein in an NON auxotrophic strain may be difficult. For the seleomet side, you can shut down the biosynthesis of Met by the addition of lysine, phenylalanine, threonine, isoleucine and valine; various protocols exist online. However to shutdown

Re: [ccp4bb] Problems with an exonuclease

Several further notes after contemplation, lunch and a slow day. If the protein is His-tagged, you could stick the unfolded protein to Ni-Resin and extensively wash with 8M Urea to remove DNA/Triton-X-100, elute, and then refold. This may be easier than multiple sonications/centrifugations. Or

Re: [ccp4bb] Problems with an exonuclease

It will either be two things. DNA or residual Triton-X-100. When you say, cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the pellet and then centrifuged again? If the latter, try sonication. I wash my IBs at least 4 times with the following buffers; 1. 20mM Tris, 500mM

Re: [ccp4bb] protein precipitation reg

Probably the price, HEPES is nearly 5-8 fold more expensive. PBS is fine, unless you have to add divalents. TRIS is your (cheap) friend! Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel:

Re: [ccp4bb] off topic: Alternatives to GE HiTrap columns

You could try Goldbio.com. I have not used the His-columns from them, but have used the Protein A columns and they work as well as the Protein A from GE. Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland

Re: [ccp4bb] Fwd: [ccp4bb] Remittance advice - Invitation to edit

The Nigerian Prince wants your money. Get Outlook for Android From: CCP4 bulletin board on behalf of Anindito Sen Sent: Sunday, December 11, 2016 12:45:16 PM To: CCP4BB@JISCMAIL.AC.UK

Re: [ccp4bb] Off-topic: Expression of human single-pass membrane protein

How are you targeting the protein to the membrane and to which one? Which strain of E coli are you using? BL21(De3) may not be the best. Especially if the gene is not codon optimized. You could try Rosetta cells. Furthermore, you may get better expression with the Walker Strains (C41 and C43)

[ccp4bb] Reindexing of lattice

Hi, I am trying to reindex a P212121 lattice to change the axis order from 32.62, 64.89, 103.16 to 64.89, 103.16, 32.62. Looking at the REINDEXING manual I can't find how to reindex the lattice. Does anyone know how I should do this and an example of the execute script that I should use?

Re: [ccp4bb] Interesting DNA contamination

Several different approaches may help you to separate DNA from protein including; 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. 2) Use a low concentration of

Re: [ccp4bb] Off topic - Mercury Lamp for Akta

As per usual the CCP4BB community has offered several different on the board and directly to me. Companies suggested include; Sonntek (www.sonntek.comhttp://www.sonntek.com) -- $695. Kinesis (www.hplclampsandspares.kinesis.co.ukhttp://www.hplclampsandspares.kinesis.co.uk or

[ccp4bb] Off topic - Mercury Lamp for Akta

The mercury lamp (28-4042-25) offered by GE Healthcare for Akta systems come with the housing and is priced at $1345. We have plenty of sparing housing units and are interested in just the lamp. Does anyone here have any cheaper alternatives/suggestions than buying the lamp and housing? Thanks