Re: [ccp4bb] protein precipitation reg

Hi Akila, In addition to what others have asked about the dialysis buffer, a few more comments that might help to decide next steps because the precipitation (note precipitation and aggregation are related but not synonymous) may be due to several different or related reasons: 1) At what stage

Re: [ccp4bb] Coot render tool missing

Just to clarify, 1 and 2 is obsolete when using 3. B Hi All, With Bernhard's help, I fixed this issue of render tool in Coot in Win7. I summarize what I did here: 1.* - add C:\yourccp4installationdirectory\bin to PATH in the coot startup script (runwincoot.bat in

Re: [ccp4bb] [ccp4bb] protein precipitation reg

Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an old name for sulfuric acid. > On Mar 29, 2017, at 8:40 PM, Keller, Jacob wrote: > >> And if we are going to pour scorn and vitriol on Tris, why not mention its >> large dpKa/dT of 0.03 pH

Re: [ccp4bb] [ccp4bb] protein precipitation reg

>And if we are going to pour scorn and vitriol on Tris, why not mention its >large dpKa/dT of 0.03 pH units/deg ? Hah! That's what many people are doing when they make buffers: pouring vitriol (HCl) on TRIS! I prefer to pour concentrated HEPES, and get two buffers without adding any extra Cl-.

Re: [ccp4bb] [ccp4bb] protein precipitation reg

Just to point out that whatever buffer you purify your protein into is possibly not the one that will keep your protein happiest. We had the opportunity of testing about 250 proteins in DSF against 26 different buffer / salt combinations (in triplicate, with lots of controls) and found out

[ccp4bb] Proline derivatives

Hi Everyone, Does anyone know what’s the fastest way the find all commercially available proline derivatives on carbon gamma? Thanks, Jan

Re: [ccp4bb] [ccp4bb] protein precipitation reg

> On Mar 29, 2017, at 4:15 PM, Chun Luo wrote: > > In addition to price, the prevalence of Ni purification may be another reason > for Tris popularity. Some His-tagged constructs don't bind to Ni well in > HEPES. I wonder if anyone has similar experience or comments. —Chun

Re: [ccp4bb] [ccp4bb] protein precipitation reg

In addition to price, the prevalence of Ni purification may be another reason for Tris popularity. Some His-tagged constructs don't bind to Ni well in HEPES. I wonder if anyone has similar experience or comments. --Chun -Original Message- From: CCP4 bulletin board

Re: [ccp4bb] [ccp4bb] protein precipitation reg

I heartily concur with Craig. Tris can be a dangerous buffer for many reasons, including those listed below. In addition, as a primary amine, it can complicate work with metalloproteins and has moderate nucleophilicity. There is almost always a better buffer choice than Tris. Best regards, Mark

Re: [ccp4bb] [ccp4bb] protein precipitation reg

There are almost always better choices than Tris buffer. Mo Cleland used to call it “Trash” buffer. He is no longer with us, but today I will happily carry that flag in his honor. Tris may show up in your crystal structure, especially at carbohydrate binding sites. Tris may be a surprisingly

Re: [ccp4bb] Coot render tool missing

Hi All, With Bernhard's help, I fixed this issue of render tool in Coot in Win7. I summarize what I did here: 1.* - add C:\yourccp4installationdirectory\bin to PATH in the coot startup script (runwincoot.bat in C:\yourwincootinstallationdirectory) * I use Notepad to open the .bat file and do

[ccp4bb] AW: [ccp4bb] protein precipitation reg

...it's just a wonderful tradition! there's an interesting description of the history of tris in maniatis cheers jon Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von David Briggs Gesendet: Mittwoch, 29. März 2017 17:53 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb]

Re: [ccp4bb] Large number of outliers in the dataset

In addition to what Nicolas has pointed out, is it quite suspicious to me that you have the same multiplicity in the high resolution shell as in the low resolution. On Mar 29, 2017 18:17, Nicolas FOOS wrote: Dear Juliana, all the statistics

Re: [ccp4bb] modifying the ligand

On 29/03/17 13:36, Adriana Sene wrote: Hi Hi, I am new here, I am looking for some way to modify one of my ligand. I have some cl atoms in the ligand which I wanted to remove and add other functional groups. If some one can tell me, how it can be done or which tools either free or paid can

Re: [ccp4bb] Large number of outliers in the dataset

If you have outliers - worry about why? (By the way - what is the multiplicity?} Look at the AIMLESS list of rejections/ the scale factor for different batches/ reports of ice rings/ etc There has to be a reason, and with snsible reprocessing you can probably but much better resolution data

Re: [ccp4bb] Large number of outliers in the dataset

I agree that you should try to use all the data. There is nothing wrong with solving your structure with the data you trust and then extending the resolution when your model is in an advanced state of refinement. If you worry whether your data has added value, you can use paired refinement to

Re: [ccp4bb] protein precipitation reg

Exactly. Tris is very cheap. HEPES not so much. On the other hand, zwitterionic buffers have significant advantages in terms of controlling inorganic anion or cation concentrations. ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of

Re: [ccp4bb] Large number of outliers in the dataset

Dear Juliana, all the statistics presented here looks good in terms of resolution cut (maybe I will be less sever). For me the point is about the mosaicity you report 1.90 it's high in my opinion. How looks you images? I am wondering if the indexation is really right. And maybe the complain

Re: [ccp4bb] Large number of outliers in the dataset

One thing that sometimes helps me in this situation is reprocess and refine in lower symmetry like P21. It could be you have pseudosymmetry and need to model more molecules in the AU to better reflect your data. Sometimes this can help. If that doesn't work, then you may have to stick with your

Re: [ccp4bb] Large number of outliers in the dataset

To be really convinced I think you should also compare the maps at 2.6 and 2.3 Å. If the 2.3 Å map looks better, go for it. If it doesn’t look better, perhaps you are adding noise, but the I/sigma and CC1/2 values suggest you aren’t. Perhaps try 2.5 and 2.4 Å also. And perhaps remove a

Re: [ccp4bb] protein precipitation reg

It doesn't cost as much as HEPES, iirc. On Wed, 29 Mar 2017, 16:36 Keller, Jacob, wrote: > A bit off topic, but I’ve always wondered how TRIS got so popular what > with it’s pKa of 8.3—does anyone know? > > > > JPK > > > > *From:* CCP4 bulletin board

Re: [ccp4bb] Large number of outliers in the dataset

It is not clear to me why you believe that cutting the resolution of the data would improve your model (which after all is the aim of refinement). At the edge CC(1/2) and I/sigI are perfectly respectable, and there doesn’t seem to be anything wrong with the Wilson plot. Th R-factor will of

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] protein precipitation reg

A bit off topic, but I’ve always wondered how TRIS got so popular what with it’s pKa of 8.3—does anyone know? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, March 29, 2017 11:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein

Re: [ccp4bb] modifying the ligand

Hi Adriana, I support Johannes answer, but personally, I prefer to write directly the smiles string for your ligand and then modify it. Since the code depends on the starting atom, it could be tricky to determine where to mutate if you did not write it by yourself in first place. You can

Re: [ccp4bb] protein precipitation reg

What are you dialyzing against? Your storage solution should typically be buffered away from the pI and contain at least a small amount of kosmotropic salt, e.g. NaCl. Some proteins will require additional stabilizing/solubilizing agents such as glycerol or reducing agents. FYI, Tris-Cl, pH

Re: [ccp4bb] protein precipitation reg

And what are you dialyzing it against? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Akilandeswari Gopalan Sent: Wednesday, March 29, 2017 9:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein precipitation reg Dear all, I am a PhD student doing structural

[ccp4bb] Large number of outliers in the dataset

Hello, I have one dataset at 2.3 Å (probably it can be better, I/σ = 2.1 and CC1/2 = 0.779, the summary data is below), but when I perform Xtriage analysis it says that “There are a large number of outliers in the data”. The space group is P212121. When I refine the MR solution the Rfree stops

[ccp4bb] protein precipitation reg

Dear all, I am a PhD student doing structural studies on a few proteins from Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned into pet22b with c terminal His tag. the proteins are expressing well. upon purification I am getting good yield of protein but during

[ccp4bb] CCP4 wiki is down

Anybody notice that the CCP4 wiki is down?

[ccp4bb] Postdoctoral Associate opportunity

Dear Colleagues, I'm happy to promote an opportunity for a postdoctoral associate in my lab. Our principle interest is the structural and function of vitamin D related cytochromes P450. We also interface regularly with the state of the art Hauptman-Woodward Medical Research Institute. More

[ccp4bb] modifying the ligand

Hi I am new here, I am looking for some way to modify one of my ligand. I have some cl atoms in the ligand which I wanted to remove and add other functional groups. If some one can tell me, how it can be done or which tools either free or paid can be used to do that. bet Adiana

[ccp4bb] IUCr DDDWG Workshop on "Research Data Management" will be at ACA 2017 in New Orleans

Dear Colleagues, We draw your attention to the next IUCr Diffraction Data Deposition Working Group (DDDWG) Workshop which is entitled "Research Data Management" to be held at the ACA 2017 in New Orleans. Details can be found here:- http://forums.iucr.org/viewtopic.php?f=21=395