[ccp4bb] Artic Express: problem with chaperone copurification

We have had good luck with making a protein soluble using the Artic Express bacterial cell line BUT we can't get rid of the chaperone that copurifies. We have tried adding ATP, MgCl2 and potassium to lysate and extensive washes and this releases a bit of the chaperone. Has anyone solved this

Re: [ccp4bb] off topic: good peak on gel filtration

In our lab we do DLS on the SEC fractions and only set up those that are monodisperse :-) On Mon, Jul 1, 2013 at 7:37 PM, El Arnaout, Toufic elarnao...@biochem.wustl.edu wrote: Hello Peter, In addition to the great comments/details, please check the following points I have now in mind..

[ccp4bb] 96 well DLS plates

We have found funding for a 96 well DLS from Wyatt. I was wondering what peoples experiences with this instrument was. Is there a better instrument out there? Does it matter where you buy the 96 well plates from? Many thanks, Gloria

[ccp4bb] structural search for homologs in pdb?

We have a protein sequence that probably contains OB folds. What is the best way to search for the top structural homologs to this sequence in the pdb? G

[ccp4bb] Structural transmission of signal across a membrane

Hello ccp4ers, I am helping a colleague develop a grant and have a vague recollection of structures of transmembrane protein receptors that signal across the membrane. Can anyone send me specific examples? Many thanks, G

[ccp4bb] limited proteolysis / ms

We are trying to apply this approach for the first time to define a crystallizable domain of our protein of interest. Can anyone send a protocol that includes exactly how to do the mass spectrometry measurement? Our core lab here doesn't know and I'm just not that gifted. Happy October, G

Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?

Are you talking about the Fig 3.1 in their book, Laboratory-build oscillation camera (Arndt, Champness, Phizackerley and Wonacott, 1973) On Thu, Oct 31, 2013 at 2:28 AM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote: one in their book, i am sure. eleanor On 30 Oct 2013, at 16:05, Gerard

Re: [ccp4bb] A question on protein microheterogenity for crystalization

We had this happen with RPA14/32 heterodimer, we kept the two peaks separate, and then grew crystals from each but in different space groups and diffraction resolution. See Habel, J. E., Ohren, J. F. and Borgstahl, G. E. O. Dynamic light scattering analysis of full-length, human RPA14/32 dimer:

[ccp4bb] in vitro Tyr phosphorylation

Does anyone know of a good way to phosphorylate the Tyr on a protein for structural studies? Is there a generic kinase that can be coexpressed or purified for phosphorylation? The pCMF Amber codon system is very expensive and Glu really doesn't mimic pTyr all that well. Any ideas/help would be

Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate

You can increase the formate concentration and it will be a cryoprotectant on its own. Test increasing amounts until you find the concentrationthat freezed clear as glass and the transfer your crystal to this. We did this with sodium formate at 7 M quite easily. On Tue, Dec 17, 2013 at 2:59

Re: [ccp4bb] in vitro Tyr phosphorylation

specific. If you don't get a better lead I can send you the sequence and a reference. Gloria Borgstahl wrote: Does anyone know of a good way to phosphorylate the Tyr on a protein for structural studies? Is there a generic kinase that can be coexpressed or purified for phosphorylation

Re: [ccp4bb] Google Gets it Right

Made my day Her biography was inspirational to me! On Mon, May 12, 2014 at 9:52 AM, lbetts0508 . laurie.betts0...@gmail.comwrote: I should have said, ALL crystallographers rejoice!! Laurie On Mon, May 12, 2014 at 10:01 AM, lbetts0508 . laurie.betts0...@gmail.com wrote: Oh

Re: [ccp4bb] PDB passes 100,000 structure milestone

I vote for Z's idea On Wed, May 14, 2014 at 12:32 PM, Zachary Wood z...@bmb.uga.edu wrote: Hello All, Instead of placing the additional burden of policing on the good people at the PDB, perhaps the entry page for each structure could contain a comments section. Then the community could

Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?

You can prevent them from falling off by also removing 5 microliter or so of mother liquor from the drop and repositioning it back over the reservoir On Wed, Jul 2, 2014 at 3:38 PM, Patrick Loll pat.l...@drexel.edu wrote: You can cut a small piece of sponge and put that into the reservoir;

Re: [ccp4bb] Disorder or poor phases?

a recent experience in our lab with molecular replacement (wt and disordered point mutant; same space group and unit cell) was solved with a combination of two methods. 1. We made omit maps in the disordered region at several lower resolutions. The region became interpretable after suffereing

[ccp4bb] SUMO(ULP-1) protease

My fellow crystallographers, We are thinking the SUMO/His vectors would be nice to have in the lab aresenal... but. The stumbling block is that the protease needed for cleavage is very expensive at crystallography scale. SUMO(ULP-1) protease costs ~$700/mg fusion protein. It would not be a

Re: [ccp4bb] Calculation of volume/size of cavity

We used VOIDOO recently and it worked well Kleywegt, G. J. Jones, T. A. (1994). Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr. D Biol. Crystallogr. 50, 178-185. On Thu, May 31, 2012 at 12:41 PM, Zhou, Tongqing (NIH/VRC) [E]

[ccp4bb] CONH2 not COOH at TER

How would I tell REFMAC to replace the C-terminal Pro in my synthetic peptide with a CONH2 not the natural CT.

[ccp4bb] retract Fwd: CONH2 not COOH at TER

-- From: Gloria Borgstahl gborgst...@gmail.com Date: Thu, Aug 23, 2012 at 12:25 PM Subject: CONH2 not COOH at TER To: ccp4bb@jiscmail.ac.uk How would I tell REFMAC to replace the C-terminal Pro in my synthetic peptide with a CONH2 not the natural CT.

Re: [ccp4bb] Professor Dame Louise Johnson

This indeed is sad news for today. I just wanted to note that Professor Johnson's early papers on time-resolved crystallography truly inspired me to continue in crystallography, influenced my decision for my first postdoctoral position and to push the limits. I still have the carefully

Re: [ccp4bb] Nobel Prizes for 3D Molecular Structure

Daniel Schectman's 2012 nobel prize for the discovery of quasicrystals is missing. On Sat, Oct 13, 2012 at 5:01 PM, Joel Sussman joel.suss...@weizmann.ac.ilwrote: Just want to make sure anyone interested in Nobel Prizes knows about this existing page:

Re: [ccp4bb] suitable buffer for CD studies

I have found it is best to test the absorption of your buffer in the wavelength range you are interested in. If you are going to do a temperature study with CD perhaps at 222 nm, then test your buffer there with your UV spec. You want to have little or no absorption. Or do the range 200-270 nm

[ccp4bb] buried surface area

Hello all, What is the easiest way, these days, to calculate the buried surface area between two subunits of a protein? Thanks, and Happy Friday,

[ccp4bb] Buried Surface Area - summary

Here was the query.What is the easiest way, these days, to calculate the buried surface area between two subunits of a protein? Here are the software and links received (thank you all):1.4+ votes for PISA - This following website is great and does a very nice job of analyzing protein interfaces

Re: [ccp4bb] small lines in diffraction pattern

. ** Gloria Borgstahl Eppley Institute for Cancer Research and Allied Diseases 987696 Nebraska Medical Center 10732A Lied Transplant Center Omaha, NE 68198-7696 http://sbl.unmc.edu Office (402) 559-8578 FAX (402) 559-3739

[ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms

of a disulfide bond on a 2-fold connecting two homodimers). So I polled the collective knowledge of the great ccp4bb group. On Wed, Dec 8, 2010 at 10:57 AM, Gloria Borgstahl gborgst...@gmail.com wrote: My fellow crystallographers, I wanted to take a poll. How many of you have ever had a protein

[ccp4bb] TEV protease

We are using TEV protease to make a big batch of protein for structural studies. We usually use thrombin, so this is our first time using this enzyme on a large scale. Boy is it expensive! Does anyone know of a bulk source for this enzyme and what ratios of use do you recommend. Imidazole

[ccp4bb] Thank you! TEV protease

I have learned alot about TEV protease and thank you all for your help Going to start making it ourselves!!!

Re: [ccp4bb] strange density

I'm voting with Roger this time. If I were you I would model a nickel in there (unless you have a better candidate) if it is right, the distances to the His should be like seen in a SOD active site. Then you can model the bonds and waters. You may need partial occupancy on the metal. Reminds me

[ccp4bb] titering baculovirus ?

Hi Guys, we are learning to work with Sf9 cells and Carol in my lab wanted me to ask you the following question. Many thanks for any help, G I need to titer a baculovirus stock in my suspension-adapted Sf9 cells. I know that these can be encouraged to attach better to tissue culture plastic if

Re: [ccp4bb] Another paper structure retracted

Dale, This is exactly the conversation I just had with my student Jason, right on! The paper we are writing just now, this is figure 1. But I always get rejected by Nature, so go figure. On Thu, Aug 11, 2011 at 1:25 PM, Dale Tronrud det...@uoxray.uoregon.eduwrote: I agree with Prof. Tomchick:

Re: [ccp4bb] IUCr committees, depositing images

I just want to jump in to state that I am ALL FOR the notion of depositing the images that go with the structure factors and the refined structure. Through the years, I have been interviewing folks about the strange satellite diffraction they saw, but ignored, used the mains that they could

Re: [ccp4bb] Crystalization in low PH

Glutaraldehyde works best at low pH On Mon, Nov 7, 2011 at 8:40 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Mon, 2011-11-07 at 05:19 +, Sam Arnosti wrote: Hi everyone I have a protein that is extraordinarily stable at PH=3.0 or even 2.0. I want to crystallize it in the  low PH and

Re: [ccp4bb] Crystallizing protein sitting in PBS

A thing we frequently forget is that phosphate can be a precipitating agent try a phosphate grid screen, just like you would with ammonium sulfate. If your protein likes PBS, it may want to crystallize with phosphate See Enrico Stura's footprint screen for example On Tue, Nov 15, 2011 at 5:25

[ccp4bb] molrep question - how get our ducks in a row?

Hello all, We are solving a superstructure of a protein complex with 2 parts. Built 6 of the first part and they are all sensibly stacked next to each other. Then we read this into molrep as the fixed model and solved for the second part. The solution was found but the 6 for the second model are

[ccp4bb] dynapro DLS cuvettes

Does any one know of a source of these cuvettes? Protein Solution doesn't exist anymore and Wyatt no longer has these.

[ccp4bb]

Be aware that some buffers are temperature sensitive and change pH, if this pH change heads toward the pI of the protein it can crash out. On Tue, Aug 19, 2014 at 6:37 PM, Prince, D Bryan dbryan.pri...@astrazeneca.com wrote: Dear Prashant, I have been working with a protein-protein

Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

Thanks Mark, this is a good tip what would you use for Rosetta cells though? Tetracyclin? On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson mwilso...@unl.edu wrote: Hi Ivan, We've had good luck with the addition of chloramphenicol ~1 hour prior to harvest, as described in: Carrió, M. M., and

[ccp4bb] Nova F- (topic off in left field)

Apparently these are not for sale anymore by EMD Millipore yet it is what is recommended for plasmid preps with pETcoco-2. Does anyone know of a source of these cells or a substitute cell line we could use? Thanks, Gloria

Re: [ccp4bb] Demonstration for 2nd graders?

I have had young ones grow lysozyme crystals in just a few minutes, using eye droppers and petri dishes. The crystals grow very fast, you can watch them grow in the microscope. Also they grow large enough you can see them by eye. Some izit dye would be fun to add (never did that). Then I let

Re: [ccp4bb] Strange Ancient Diffraction Pattern...

Ah Ha! drum roll A quasicrystal ! On Wed, Apr 1, 2015 at 7:00 AM, Julia Griese gri...@dbb.su.se wrote: This one appears to be of a similar age. It has a most puzzling, but pretty pentagonal pattern (and a backstop). Unfortunately Mosflm doesn't appear to support the image format.

Re: [ccp4bb] cryo condition

High concentration of ammonium formate is a cryosolvent On Mon, May 4, 2015 at 5:20 PM, Tristan Croll tristan.cr...@qut.edu.au wrote: What about nature's favourite cryoprotectant, trehalose? From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of

Re: [ccp4bb] [RANT] Reject Papers describing non-open source software

Did they stop supporting it due to lack of renewed funding and having to cut staff that had the knowledge? I'm pretty sure you only know part of the story. On Tue, May 12, 2015 at 11:48 AM, James Stroud xtald...@gmail.com wrote: I hereby call on the broadest community of academics and

Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle curiosity ...

Eddie Snell for President On Wed, Nov 9, 2016 at 11:02 AM, Edward Snell wrote: > As a Brexit and Trumpet affected person having a foot in both countries > ,this topic is too far off the normal discussion on CCP4 and probably > better taken up privately. CCP4 is not a

Re: [ccp4bb] Crystal with ZERO diffraction

I have learned to refrain from catapulting my crystals. Happy holidays, God bless us ... everyone (with diffracting crystals) On Wed, Dec 21, 2016 at 2:35 PM, Keller, Jacob wrote: > >In a second case we were working on a beamline on the west coast of the > US. Every

Re: [ccp4bb] Shipping crystals for RT data collection

I would recommend just mounting them in capillaries without loops, as this will travel better. Put slugs of mother liquor on either side, seal with wax and then coat wax and onto the end of capillary glass lightly with fingernail polish. We have had terrible luck with FedEx delivering damaged

Re: [ccp4bb] Granada Crystallization Boxes?

ne might > have some. > > Best wishes, Patrick > > > On 11 July 2017 at 19:04, Gloria Borgstahl <gborgst...@gmail.com> wrote: > >> I have recently found out that these are no longer being manufactured or >> sold commercially. But, as fortune has it, we have ju

[ccp4bb] Granada Crystallization Boxes?

I have recently found out that these are no longer being manufactured or sold commercially. But, as fortune has it, we have just been funded to fly some large quartz capillaries crystallization experimente up to the International Space Station for neutron crystallography. Our experimental design

Re: [ccp4bb] Granada Crystallization Boxes?

gt; Rm. ND10.214A > Dallas, TX 75390-8816 > diana.tomch...@utsouthwestern.edu > (214) 645-6383 (phone) > (214) 645-6353 (fax) > > On Jul 11, 2017, at 1:04 PM, Gloria Borgstahl <gborgst...@gmail.com> > wrote: > > I have recently found out that these are no longer being manuf

[ccp4bb] Fwd: Please register - 7th Annual SBMB Workshop - July 13 - Omaha, NE

, etc. If you can, please register to present a research poster. The associated journal club is included in the website and will be broadcasted remotely if you wish to participate from your office. Thank you for your interest, Gloria Borgstahl and Luis Marky Please share this email. To register

Re: [ccp4bb] Aperiodic protein crystals

Thanks James, Yes Charles I have an active NSF grant on this topic. We should talk, Gloria On Mon, Sep 18, 2017 at 11:05 AM, James Holton wrote: > > I believe Gloria Borgstahl's lab at UNMC has done a little bit of work on > this. > > > -James Holton > > MAD Scientist

[ccp4bb] Fwd: Granada Crystallization Boxes?

-- From: Gloria Borgstahl <gborgst...@gmail.com> Date: Tue, Jul 11, 2017 at 1:04 PM Subject: Granada Crystallization Boxes? To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@jiscmail.ac.uk> I have recently found out that these are no longer being manufactured or sold commercially. But,

Re: [ccp4bb] PDB search help

Our RPA14/32 crystals from 2007 included full length protein for both subunits (RPA14 and RPA32), but only the central OB fold of RPA32 could be modelled. The crystals included RPA32(1-270) but only 42-176 could be modelled. I remember being very frustrated by not being able to visualize the

Re: [ccp4bb] AW: Differences in a homodimer protein

I have seen this in MnSOD. We had a tetramer in the ASU and each active site had different ligands in our peroxide soak. I assumed the crystal lattice can influence these things or it is inappropriate metal incorporation. I would highly recommend doing ICP-MS on a dissolved crystal and on your

Re: [ccp4bb] AW: Re: [ccp4bb] another unknown density problem

If you mean you truncated the low resolution data to 5 angstrom, I wouldn't recommend that. On Fri, Nov 3, 2017 at 12:24 PM, Eleanor Dodson wrote: > That map does not look like a 5A map? > I guess you mean something else.. > Eleanor > > On 3 November 2017 at 11:00,

Re: [ccp4bb] "Atomic resolution"

My answer is not precise, I just try to give students something they can remember, so they don't go and model water for example at an inappropriate resolution. Of course phasing matters as well. On Thu, Jan 11, 2018 at 2:31 PM, Keller, Jacob wrote: >>I tell people it

[ccp4bb] phosphates

Doe any one know what resolution is needed to distinguish phosphate from carbon in a difference map?

Re: [ccp4bb] "Atomic resolution"

I tell people it is when your resolution is less than the bond length that connects the two atoms. On Thu, Jan 11, 2018 at 1:59 PM, Thomas Edwards wrote: > Dear Jacob, > > Ah... this old chestnut! > > Current EM people say that they are at atomic resolution because they

Re: [ccp4bb] protein quasicrystals?

I am very interested in this topic, and have found incommensurately modulated crystals and few examples of possible protein quasicrystals. Do you have any images you could share? On Tue, Feb 13, 2018 at 9:09 AM, Yu Qiu wrote: > Hi, > > > > I have been trying to crystallize a

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I am also a bit late in responding, I have a few incommensurately modulated protein crystal datasets that you would be welcome to use in your course. It would be neat for students to at least know that this type of diffraction exists. As far as I know, they can only be processed

[ccp4bb] nonenzymatic removal of His tag?

Hello, friends in crystallography, A colleague just asked me this question. He is worried about trace protease interfering with the receptors he is studying in cell-based experiments using a 110 amino acid protein we made for him. He has been unable to make the peptide synthetically. The

Re: [ccp4bb] how many crystallographers are there?

You might want to quote how many researchers use the PDB instead. Maybe an inquiry to the PDB could give you that statistic. That would give you a big number On Wed, May 29, 2019 at 1:54 PM Scott Horowitz wrote: > Hi all, I was recently asked how many biological > crystallographers plus

Re: [ccp4bb] challenges in structural biology

Sorry to be late chiming in on this post (survived RAGBRAI). I think the challenges (crystallization, perdeuteration) and benefits of neutron crystallography (where are those protons) could be included. We are now in an era of using cryotrapping with neutrons which I think is really cutting edge

[ccp4bb] invitation for in-person or remote participation in ORNL STS workshop

mistry, and Medicine (2) Cell Signaling at Membranes (3) Macromolecular Complex Assembly and Disassembly (4) Bioinspired Materials and Technology We are looking forward to your participation! On behalf of Biology Session Chairs: Dr. Gloria Borgstahl University of Nebraska Medical Center Dr. Y

[ccp4bb] E. coli BirA biotin ligase expression/purification

Dear friends in crystallography, I know this seems unrelated, but it really isn't ... please forgive me. We are trying to use the Avitag/BirA system to specifically biotinylate target proteins in an economical manner. Are any of you purifying the BirA enzyme in your lab for biotinylation and if

[ccp4bb] protein expression in human cells

Hello CCP4-ers, I was wondering what people have found to be the best human cell line expression system for making a large quantity of purified recombinant protein. Any information and protocols would be greatly appreciated. Happy 2020, Gloria

Re: [ccp4bb] Shipping samples for neutron diffraction

Actually we don't declare anything to TSA, Jahaun just carries them in his carryon and it is fine. Easy peasy? On Wed, Feb 19, 2020 at 3:48 PM < 0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote: > > ... and don't say you're travelling with heavy water! > > Jon Cooper > > On 19 Feb 2020

[ccp4bb] insect secretion recommendations?

Hi Friends, We are secreting Spike Ecto domain into the media from insect cells for purification. As we scale up I am wondering what is recommended for collecting the media from large volumes of culture. Centrifugation? Filtration of some kind? I imagine we need to be gentle to not lyse the

Re: [ccp4bb] Vote for cryoEM

I personally don't tweet. On Tue, Mar 31, 2020 at 12:21 PM Sweet, Robert < 27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk> wrote: > Real Men (and possibly Women too) Don't Tweet. > > Bob > > > From: CCP4 bulletin board on behalf of James > Holton >

Re: [ccp4bb] disinfecting keyboards

We were looking at these, they look like fun. https://www.wetkeys.com/Soft-touch-Comfort-Hygienic-Washable-Keyboard-USB-p/kbstfc106-w.htm On Tue, May 5, 2020, 7:20 PM James Holton wrote: > All joking aside, there has been a furor of attention on UV-based > disinfection of late. Some of it

[ccp4bb] Contouring Patterson map?

What is the best way to display Harker sections... these days? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to

[ccp4bb] Contouring Patterson map?

What is the best way to display Harker sections these days? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members

Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

I was thinking the form of GFP that dimerizes. This would also make it easy to track where the protein is. On Tue, Sep 22, 2020 at 1:28 PM Diana Tomchick < diana.tomch...@utsouthwestern.edu> wrote: > Any dimeric tag should work if you add a long enough linker to satisfy > your distance

[ccp4bb] AcTEV protease

Another protein purification question: Does anyone know what is AcTEV protease that is sold by Thermofisher Scientific? Is this the same as SuperTEV? It works well but is so expensive. Thanks for any advice, Gloria To

[ccp4bb] MBP Ab for native protein

Hello friends, Can anyone recommend an antibody that works well forbbonding native MBP tag? Many thanks, G To unsubscribe from the CCP4BB list, click the following link:

[ccp4bb] TEV vs HRV3C

Hello my fellow structural biologists, I am contemplating why some choose the HRV3C protease site over TEV for their fusion proteins. Does anyone know? Can HRV3C be made easily in homelab? Does anyone have a plasmid? Thank you, G

[ccp4bb] SAXS models and data

Do we deposit these anywhere? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a