Re: [ccp4bb] Cysteine-S-Iodine interaction

[CRAIG A BINGMAN]

I’m late to the party here, but there are (at least) 59 instances of a C-S-I 
fragment in the CCDC.

Flipping through the first third of the list, GIGBED 
(triphenylmethyl-iodo-sulfane) seems somewhat close. The S-I bond is 2.41Å and 
the C-S-I bond angle is 105.4 degrees. There may be better ones deeper in the 
list, but that’s someone else’s project to find.

(R.Minkwitz, H.Preut, J.Sawatzki, Zeitschrift fur Naturforschung,B:Chemical 
Sciences, 1988, 43, 399)

From: CCP4 bulletin board  on behalf of Paul Emsley 

Date: Friday, March 18, 2022 at 3:05 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Cysteine-S-Iodine interaction
On 17/03/2022 17:37, Mohd Syed Ahangar wrote:
>
>
> I have been doing some protein crystal soaking with some covalently
> binding fragments and in one structure I have got an extra density on
> Cysteine but that density doesn't match with the expected fragment.
> The fragment was in the form of iodide salt. when I fit the Iodine in
> the density, it fitted fairly well than any other possible chemical
> entity. From the density map it looks like something is covalently
> bound to Cysteine.
> Now my question is, can a sulphur atom of Cysteine have such an
> interaction with Iodine.


No, they are both electronegative, C-S-I is not a thing.


> The distance between S and Iodine is 2.76A in this case as shown in
> the attached figure.
> I would be grateful if someone can shed some light on this.
>

Your map (this figure) is a textbook example of a covalently linked atom
incorrectly refined with a non-bonded contact.


Paul.



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Re: [ccp4bb] TWIN?

[CRAIG A BINGMAN]

It’s difficult to tell exactly what is happening from your description and the 
attached image. Both your description and the image are OK. It’s just that 
there are a lot of ways for things to go off the rails.

Are the molecules stacked end-to-end? In that case, you may have an 
incommensurate structure where the molecule is disordered around a 
pseudo-continuous helical axis running through the crystal.

But from this view, it looks like at least two bases are swung out to the left. 
I’d omit the interaction region, refine what you can model well as well as you 
can, and see if the maps for this region clear up. Keep in mind that your model 
might be out of register, so look at the shapes of the bases carefully to see 
if you need adjust that.

On Jun 8, 2021, at 10:14 AM, maps.fa...@gmail.com 
wrote:

Hello everyone,

I am working with an RNA-only structure, data are at 3 Angstroms, and at first, 
I thought was in the C2 space group (with 6 molecules in the AU).

I can't finish building! it, because the structure seems to get in the way of 
its neighbor symmetrically! See the attached picture, please! The only 4 
residues that the structure is missing "have to go there", where one structure 
meets the other one. The R factors for this spacegroup are around 0.3.

However, Phenix Xtriage suggests the symmetry may be higher. So I reindex in 
P622, do MR with Phaser (trying all possible space groups in that point group), 
and it finds a unique solution in space group P 65 2 2 with TFZ of 50 and LLG 
>3000. Of course, the problem persists (one molecule sort of interfering with 
the neighbor), not only that but also the refinement R-factors are 
substantially higher, 0.37.

I have to say that the refinement maps look better when I am working with the 
C2 spacegroup. I can't understand, though, what is happening at that 
"supposedly interface" between the two molecules. Has anybody experienced 
anything like this in the past? Can it be a twin? Is it something else? and... 
how to fix it?

Thank you very much in advance.

Best wishes,

Almudena



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Re: [ccp4bb] Covalent Cysteine Aduct

[CRAIG A BINGMAN]

My guess would be that the chains were prepared under reducing conditions, 
mixed, and then allowed to oxidize. The surface adduct could easily form during 
this process.

On Mar 27, 2020, at 4:38 PM, Cowan, Richard H. (Dr.) 
mailto:rc...@leicester.ac.uk>> wrote:

Hi,

The Fab constructs have a c-terminal cysteines on both the heavy and light 
chains, which should form a disulphide. Adding reducing agent to the 
purification of the protein would potentially reduce this disulphide, possible 
causing issues the stability and heterogeneity? At least that's my 
understanding?

Thanks,

Dr Richard Cowan
Research Associate

HWLSB 1/05
Department of Biochemistry
University of Leicester
Lancaster Road
Leicester, LE1 9HN, U.K.

Phone +44 (0) 116 229 7077


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Paul Emsley 
mailto:pems...@mrc-lmb.cam.ac.uk>>
Sent: 27 March 2020 21:33
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Covalent Cysteine Aduct


On 27/03/2020 21:06, Cowan, Richard H. (Dr.) wrote:


  although [BME] seems unlikely, since the crystallized protein is a Fab.


I don't follow.



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Re: [ccp4bb] Ligand identification

[CRAIG A BINGMAN]

My only comment is that you should consider not only what is in reservoir, but 
also anything present in your sample buffer.

On Jul 18, 2019, at 3:02 PM, Nicola Evans 
<251ca3b4615e-dmarc-requ...@jiscmail.ac.uk>
 wrote:

(well conditions: 0.2 M Magnesium chloride hexahydrate, 0.1 M BIS-TRIS pH 5.5, 
25% PEG 3,350). As this is an the crystal contact region the molecule isn't 
likely to be biologically significant. The data have been solved to 1.9Å. I 
have attached a screenshot of the mysterious density.




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Re: [ccp4bb] beryllium chloride

[CRAIG A BINGMAN]

Bob,

Yes, Be salts are wildly toxic. They are also useful in structural studies on 
enzymes that perform phosphoryl transfers, almost always as beryllium fluoride 
complexes. The PDB contains about 200 such structures.

Craig

On Apr 1, 2019, at 9:37 PM, Sweet, Robert 
<27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk>
 wrote:

With all respect, this conversation make my skin crawl a little. I've been 
taught that beryllium salts are EXTREMELY toxic.  Please study this: 
https://pubchem.ncbi.nlm.nih.gov/compound/beryllium_chloride




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Re: [ccp4bb] beryllium chloride

[CRAIG A BINGMAN]

You can purchase beryllium oxide through a number of vendors. After careful 
neutralization with an appropriate amount of HCl, you should have BeCl2 in 
solution. I usually use pH paper to check the pH in such small volume 
operations.

> On Apr 1, 2019, at 4:07 PM, Alexandra Deaconescu 
>  wrote:
> 
> Hello,
> 
> Is anyone aware of a company that sells Beryllium chloride in the US? Sigma 
> does not carry it any longer, and a quick Google search failed to reveal 
> alternatives.
> 
> Thank you very much,
> 
> Alexandra
> 
> 
> -- 
> Alexandra Deaconescu, B.E., Ph.D.
> Assistant Professor
> Brown University
> 
> Office: (401) 863-3215
> Wet Lab: (401) 863-6729
> Computational Lab: (401) 863-7031
> 
> For Mail:
> Laboratories of Molecular Medicine
> 70 Ship St. GE-4
> Providence, RI 02903
> 
> For Courier:
> Laboratories of Molecular Medicine
> Brown University
> 70 Ship St., Chestnut St. Loading Dock
> Providence, RI 02903
> 
> Website: www.deaconesculab.com
> 
> Admin
> Ms. Christina Fournier
> Email: christina_fournier[at]brown.edu
> Mailing Address:
> Box G-E, Brown University,
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> 
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Re: [ccp4bb] RNA pdb molecular weight

[CRAIG A BINGMAN]

I’m guessing you need to be closer than ~320 atomic mass units per 
ribonucleobase? (Which neglects modified bases and counter ions and assumes 25% 
each of ACGU?)

On Nov 16, 2018, at 10:19 AM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:

Hi,

I’m not an RNA person. Can anyone suggest a method to calculate the mass of a 
RNA PDB?  I’d like the protons to also be considered in the calculation. Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
85 Saint Nicholas Terrace, CDI 2.318
New York, NY 10031
http://www.khayatlab.org/
212-650-6070




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Re: [ccp4bb] To post to ccp4bb...

[CRAIG A BINGMAN]

A phospholipid headgroup might be rotationally disordered and washed out, 
especially if it isn’t exactly the correct binding partner.  It does look very 
much like lipid density to me. Attempts to model a diacyl glycerol and PEGs 
should be revealing.

On Jul 2, 2018, at 10:36 AM, Edward A. Berry 
mailto:ber...@upstate.edu>> wrote:

Is it a membrane protein?
Looks like a phospholipid without the phosphate. diglyceride maybe?
But I see not much blue density. Presumably this fills out if you contour
at a lower but still reasonable level?




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Re: [ccp4bb] help identifying unknown density

[CRAIG A BINGMAN]

You may have crystallized an enzyme that uses a phosphoramidate intermediate.

https://en.wikipedia.org/wiki/Phosphoramidate


On Oct 9, 2017, at 10:40 AM, Stephen Cusack 
> wrote:

Dear All,

   I am refining the crystal structure of an E. coli expressed protein at 2.65 
A resolution. The crystals grew in

0.1 M MES pH6, 0.7 M sodium formate pH 6. Ni-NTA was used in the purification.

There are four molecules in the asymmetric unit. A particular histidine in each 
molecular has clear, strong extra density which looks like a metal bonded to an 
imidazole nitrogen (distance approx 2 A) with three other co-ordinating atoms 
in a triangular planar arrangement (see attached screen shot for unbiased extra 
density, atoms are only put in the density for illustrative purposes).

Has anyone seen anything like this ? Any suggestions ?

thanks very much

Stephen

--

**
Dr. Stephen Cusack, FRS
Head of Grenoble Outstation of the European Molecular Biology Laboratory (EMBL)
Group leader in structural biology of protein-RNA complexes and viral proteins
**

Email: cus...@embl.fr
Website: http://www.embl.fr
Tel: (33) 4 76 20 7238Secretary (33) 4 76 20 7123
Fax:(33) 4 76 20 7199
Postal address:   EMBL Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 
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des Martyrs, 38000 Grenoble, France
**

Re: [ccp4bb] A question about Cys and fluoro benzene ring

[CRAIG A BINGMAN]

As others have suggested, it looks very much like a substitution at the 
fluorine position.  I might add that high resolution electrospray mass spec 
should be very useful in this case, because I suspect that this adduct will  
survive sample preparation, and it would provide strong and orthogonal 
experimental evidence that a chemical reaction had occurred at that position, 
specifically that fluorine had been lost during the reaction. To make your life 
easier, I’d definitely run an unreacted protein sample as a control (subjecting 
it to the full experimental procedure for creating the reaction.) The mass 
resolution of electrospray is amazing, but in many cases there are 
complications to the spectrum from associated ions. You definitely want the 
control spectrum for the unreacted protein to be from exactly the same solution 
conditions as your experimental sample.

> On Aug 23, 2017, at 10:01 AM, Cheng Zhang  wrote:
> 
> Hi everyone,
> 
> We recently got a structure of a receptor bound to a ligand. The ligand has a 
> fluoro methyl benzene ring moiety, which is close to a Cys residue in the 
> receptor. The density for the ligand and the Cys seems to suggest a covalent 
> bond. However, I don't know if a covalent bond is chemically possible. Also, 
> I believe Cys is rarely involved in cation-pi interactions? Any suggestions 
> for placing the Cys and the fluoro methyl benzene ring?
> 
> Thanks! 
> 
> Cheng
> 
> 
> 
> -- 
> -
> Cheng Zhang

Re: [ccp4bb] Unknown positive electron density

[CRAIG A BINGMAN]

Betty,

I think that f’’ for Ba at this wavelength is around 3.5 electrons, and f'' for 
As is expected to be about 3 electrons. Is there a nearby crystallographic 
symmetry axis?

Craig

On Aug 21, 2017, at 5:05 PM, Betty Chu 
<chube...@umd.edu<mailto:chube...@umd.edu>> wrote:

Hi Craig,

The data collection wavelength was 0.92 Angstroms. Since we observe anomalous 
signal for Ba at this wavelength, we would expect greater anomalous signal if 
As were present. There is a possibility for weak anomalous signal in this 
positive density, but the weak anomalous signal only shows up if I try to model 
a Ba in the density. Without modelling anything, there is no anomalous signal.

This is what the map looks like after one round of refinement with the Ba in 
the density. But since there are waters that are 1.6 Angstroms, 1.9 Angstroms, 
and 2.2 Angstroms away from the Ba, which is smaller than the coordination 
distance between Ba and water, we are skeptical of the Ba being there.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/O4lI2iKvRsQUoRo

Thank you,
Betty

On Mon, Aug 21, 2017 at 5:21 PM, CRAIG A BINGMAN 
<cabing...@wisc.edu<mailto:cabing...@wisc.edu>> wrote:
What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
<chube...@umd.edu<mailto:chube...@umd.edu>> wrote:

Hi Shailesh,

When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?

Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
<shailes...@gmail.com<mailto:shailes...@gmail.com>> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
<chube...@umd.edu<mailto:chube...@umd.edu>> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
<pradeeppal...@gmail.com<mailto:pradeeppal...@gmail.com>> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
<chube...@umd.edu<mailto:chube...@umd.edu>> wrote:
Dear ccp4bb,

I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.

Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,

Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] Unknown positive electron density

[CRAIG A BINGMAN]

What is the data collection wavelength/energy? Would you expect significant 
anomalous diffraction from As at this wavelength?

On Aug 21, 2017, at 11:37 AM, Betty Chu 
> wrote:

Hi Shailesh,

When I modelled in the Barium ion with octahedrally coordinated waters and ran 
the refinement, the distances from the barium to some of the waters ended up 
being too close (<2.2 Angstroms). Also, the positive electron density is 
connected. If the density indicated barium with coordinated waters, would that 
mean there are multiple ones present in the positive density?

Here are more views of the connected positive density.

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/j5CgZIJe6NKgUlZ

https://cbsostorage.chem.umd.edu/owncloud/index.php/s/YbiNFbT8AW1iHUQ

On Mon, Aug 21, 2017 at 12:00 PM, Shailesh Tripathi 
> wrote:
Looks like Ba2+. Since it exist with coordination number 6 or above check what 
geometry water is following there (trigonal bipiramidal or so on). Water might 
also be shared by symmetry related Ba cation.



Shailesh Kumar Tripathi,
Phone: 9686289668


On Mon, Aug 21, 2017 at 9:17 PM, Betty Chu 
> wrote:
Yes, I have. The cacodylate ion does not fit well into the density.

On Mon, Aug 21, 2017 at 11:44 AM, Pradeep Pallan 
> wrote:
Did you try modelling in a cacodylate ion (CH3)2AsO2-?

On Mon, Aug 21, 2017 at 10:19 AM, Betty Chu 
> wrote:
Dear ccp4bb,

I am refining a 1.40 Angstrom data set for a DNA oligonucleotide. While the 
model for the DNA fits very well into the density, there is a patch of positive 
electron density in the solvent space that we are having trouble with.

The screenshot can be viewed through this link:
https://cbsostorage.chem.umd.edu/owncloud/index.php/s/J5cKnOpCC4vb1VC

In the screenshot, the yellow color is the anomalous map and a barium ion is 
fitted into density near the positive green electron density.

The oligonucleotide was purchased from IDT. The crystallization condition is 
15% MPD, 120 mM BaCl2, and 30 mM NaCaC pH 6.4. I have tried modelling Ba2+ with 
coordinated waters, MPD, and sodium cacodylate into the electron density, but 
none of those fit well.

Any suggestions regarding the identity of this electron density is much 
appreciated. Thank you!

Sincerely,

Betty Chu
Paukstelis Research Group
Department of Chemistry and Biochemistry
University of Maryland, College Park



--

---
Pradeep Pallan

Re: [ccp4bb] large number in ASU

[CRAIG A BINGMAN]

Congratulations!

I think you are now looking for additional crystallographic and 
non-crystallographic symmetry, because finding 40 particles in arbitrary 
positions and orientations is going to be brutal.

I wouldn’t take the cell and point group assignment from XDS at face value.  
Rather I think you should put XDS_ASCII.HKL through phenix.xtriage and 
POINTLESS in the CCP4 suite of programs.  Both can be invoked from the command 
line.

pointless HKLIN XDS_ASCII.HKL
phenix.xtriage XDS_ASCII.HKL

That handles the crystallographic symmetry part.  Let us know how it turns out.

It is also possible that there is extensive NCS, and if the space group 
assignment holds, I’d ask again about that.  As Jacob Keller’s response 
suggests, most people are going to suggest that you lock down crystallographic 
symmetry before looking at NCS.

Craig



On Apr 26, 2017, at 2:34 PM, Jademilson Santos 
> wrote:

Greetings all,

I am having trouble with a data set and would like to know if somebody can 
help. I'm working with a protein of approximately 50 kDa, which I have 
successfully crystallized. The crystals diffracted at a resolution of 3,65 
angstroms and upon initial processing using XDS i obtained the following 
information:

space group: P21
ISA = 33.3
cell unit: a=285.2, b=135.9, c= 287.5, α=90, β=117.5, γ=90

Matthews coefficient indicates that there are 40 molecules in the asymmetric 
unit

I am currently running the program Phaser (Phenix) to determine the phase via 
molecular replacement with a model that has 49% homology and query coverage of 
94% and the program is taking extremly long to finish. In this case in which 
there is an extremly high number of molecules in the asymmetric unit, is this 
actually possible? Does someone know how to work with these values and is there 
a specific strategy which i must follow?

Regards

Jademilson Celestino dos Santos

Laboratory of Applied Structural Biology
Department of Microbiology
Institute of Biomedical Sciences
University of São Paulo- USP

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

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 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-.
> 
> JPK

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

> 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

No, I have not specifically noted that before.

Additionally, why would you use a positively charged buffer on a weak cation 
exchange resin?  The Ni affinity resins, in addition to their noteworthy 
affinity for various metals, are also weak cation exchange resins.  Binding a 
positively charged buffer to a negatively charged column material can cause pH 
effects that you probably weren’t expecting.

Tris may have some uses.  But using it in a mobile phase with Ni affinity 
columns or as a final sample buffer aren’t the best cases for choosing Tris.  I 
understand it is widely used in aquaculture applications where they are 
treating tens of thousands of gallons of water, and price of the buffer 
substance is an actual consideration.

Craig

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[CRAIG A BINGMAN]

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 strong competitive inhibitor in your enzyme assays, 
especially as above.
Tris has an absolutely miserably bad change in pKa vs. temperature.  It is 
larger than -0.03 pKa/dT(C).  It can be a catastrophically bad choice for 
flash-freezing protein aliquots.

If you taken the time and incurred the expense of preparing a macromolecular 
sample for crystallization studies, and you are worried about the price 
difference between Tris and HEPES, in my opinion you are absolutely worried 
about the wrong things.

Why are people substantially concerned about the buffering capacity of a buffer 
for final sample preparation?  You have a purified protein, presumably without 
substrate present.  Exactly what do you think is generating or absorbing 
hydrogen ions in that solution?  Oxidation of reducing agent should be about 
the only thing that is taxing the buffer.  From the example below, oxidation of 
5 mM BME will put some pressure on the buffer, but unfortunately Tris 
accelerates the oxidation of BME relative, to, say, HEPES. And surely you 
aren’t just letting the protein sit and oxidize in the refrigerator? Oh you 
might be since when you tried to snap freeze it in Tris, it turned into cooked 
egg white because the pH went to over 10 before it vitrified.  
(http://www.sciencedirect.com/science/article/pii/S0031942200801429)

Isn’t the whole point to use a small amount of buffer so you can easily push 
the pH around in crystallization screens? (At which point the sample is usually 
in 100+ mM buffer.)

On Mar 29, 2017, at 2:03 PM, Hughes, Jon 
> 
wrote:

...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] 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 
[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 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 7.5 has very 
little buffer capacity (about 15% of the total concentration in the acid 
direction). We typically use Tris-Cl pH 8.0, which is closer to the Tris pKa 
and has good buffer capacity for both acid and base. For pH 7.5 we would 
typically use HEPES as the storage buffer.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 3/29/2017 9:38 AM, Akilandeswari Gopalan wrote:
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 dialysis, the 
proteins precipitate. Kindly suggest some solutions to avoid aggregation. pI of 
one protein is 9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with 
20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.

Thank you
Regards
Akila

--
Akilandeswari G

--


Fehler! Es wurde kein Dateiname angegeben.




David Briggs PhD
Fehler! Es wurde kein Dateiname 
angegeben.about.me/david_briggs

Re: [ccp4bb] No improvement in R-factor after Refmac.

[CRAIG A BINGMAN]

You really need to approach such situations with caution.  Examination of the 
relatively small number of axial reflections probably show that there might be 
twofold screw axes in all three directions.  But a non-crystallographic 
microscopic translation of nearly 0.5 in the direction of a crystallographic 
axis will give the same pattern of strong and weak reflections as a 
crystallographic twofold screw axis.  If I were you, I would be very sure to 
try molecular replacement in all possible orthorhombic space groups.  Several 
programs, including Phaser, will organize that exhaustive search across all 
eight possibilities for you.

On Mar 17, 2017, at 11:56 PM, Polisetty Satya Dev 
> wrote:

Hi,

We checked all possible space groups of orthorhombic crystal system using Scala 
and Pointless but the statistics show that P212121 is the possible space group.

Thank You,
Satya Dev

On Fri, Mar 17, 2017 at 8:03 PM, Teplyakov, Alexey [JRDUS] 
> wrote:
Check the space group. It may be orthorhombic with a pure rotational axis (e.g. 
P21212) or even monoclinic.

From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Polisetty Satya Dev
Sent: Friday, March 17, 2017 9:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] No improvement in R-factor after Refmac.

Dear all,
I solved a structure at 2.0 A resolution with R-work and R-free values 0.25 and 
0.32 respectively and I am stuck at Refmac step where there is no further 
reduction in R-factor.

The above stated values were obtained after several rounds of manual refinement 
followed by refmac. There are also areas where electron density is missing 
around peptide backbone in one of the monomer in ASU.

Can anyone please tell me how can I improve the electron density and R-factor.

The structure solution was obtained using Phaser MR and here are the data 
statistics:

Average unit cell: 81.95, 100.40, 156.96,   90.00, 90.00, 90.00,
Space group: P212121,
Completeness 99.5,
Multiplicity 6.4,
Four monomers per ASU.
Solvent content: 47%.

Thank you everyone,
Satya Dev,
JNCASR.

Re: [ccp4bb] Crystallization with no precipitant

[CRAIG A BINGMAN]

On Mar 1, 2017, at 8:19 PM, Shaun Lott 
> wrote:

They diffracted, but weren't much use to us in the end, as they were hard to 
reproduce or optimise. Hopefully you have more luck than we did!

This sometimes happens with extremely soluble proteins.  If these are the only 
crystals you get, I might suggesting driving the protein concentration as high 
as possible (even to over 100 mg/mL in some cases) and then re-screening at 
substantially higher protein concentration than in the first pass.

Re: [ccp4bb] Crystallization with no precipitant

[CRAIG A BINGMAN]

> Could it be possible that the robot never dispensed the reservoir solution 
> and the protein just crystallized against the reservoir solution through 
> simple concentration? I have just set up a couple of drops just against 
> reservoir solution. If this is the case, has anyone else experienced this and 
> had luck with this and got diffraction?

Yes.  Some proteins will crystallize in centrifugal concentrators. 

Re: [ccp4bb] Crystalization in low PH

[Craig A. Bingman]

I'm not convinced that you need a conventional buffer at pH 2 or 3.  At pH 2, 
the hydrogen ion concentration is 10 mM.  If you want to use something else, 
the second pKa for sulfuric acid is around 2.  The first pKa for phosphoric 
acid is slightly higher than 2.  Lactic acid has a pKa close to 3.  Formic acid 
has a pKa just under 4.  Most of these numbers were in an appendix in the first 
chemistry text you ever used.  wink  These numbers imply pretty strongly that 
most crystallization screens emphasizing common salts will require determined 
modification to hit these low pH values, because many stabilizing anions in the 
Hoffmeister series will be partially or completely protonated at these pH 
values.  PEG and organic screens will require a smaller hammer to retrofit.

On Nov 6, 2011, at 11:19 PM, 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 compare the differences between 
 the crystals in regular PH and low PH.
 
 I was wondering how people set up the boxes in low PH, as usual buffers are 
 mostly less acidic.
 
 Regards
 
 Sam

Re: [ccp4bb] yellow crystals

[Craig A. Bingman]

In another thread, you indicated that there were no identifiable cofactor 
binding sites in your protein, so we are down to less common situations.  Some 
proteins are spontaneously decorated with pyridoxal on surface lysine residues. 
 In some cases, this has absolutely nothing to do with the enzymatic activity 
of the protein.  

On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote:

 Hi -- has anyone had crystals that are colored in regular (unpolarized) 
 light?  Mine are yellow, and I'm not aware of anything in the buffer 
 conditions that might cause this.  I read online that glutaraldehyde can turn 
 protein crystals a golden color, but as far as I know there isn't any of that 
 in the well.  Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8.  
 Any explanations?
 
 Thanks!
 Caitlyn
 
 
 Caitlyn C. Yeykal
 Mrksich Group/Adams Group
 Dept. of Biochemistry, University of Chicago
 929 E. 57th St., Rm 547B
 Chicago, IL  60615
 cait...@uchicago.edu

Re: [ccp4bb] atomic scattering factors in REFMAC

[Craig A. Bingman]

That is correct.  We saw this in every selenomethionyl protein structure that 
was determined at CESG.  There are two reasons for the negative density defects 
at Se atoms.   As you note, the default scattering factors for Se are incorrect 
for these experiments, as f' is large in Se SAD experiments.  Additionally, the 
fractional incorporation of Se is often less than 100%.  For our autoinduction 
media, it was fairly consistently around 90 mole percent Se.  Your mileage will 
vary on the last effect.  In addition to these two effects, which are 
particular to SeMet around the Se K edge, both regular methionine and 
selenomethionine have somewhat disordered sidechain conformations more often 
than you might guess (without anomalous diffraction data poking you in the eye 
and showing definitively that the Se is in more than one place.)

On Oct 31, 2011, at 10:57 AM, Ivan Shabalin wrote:

 I noticed that if I refine a structure containing SeMet, then Se atoms 
 usually have big negative (red) peeks of difference map and high B-factors. 
 As I understand from the diffraction theory and from some discussions at 
 CCP4bb, that may result because in REFMAC the atomic scattering factors are 
 internally coded for copper radiation (CuKa).

Re: [ccp4bb] raw data deposition

[Craig A. Bingman]

I strongly suspect that it is much more cost effective to have the PDB archive 
a unit of data than it is to have it archived at the lab or department level.  
So I suspect that more money will be available for doing science if we turn 
over archival responsibilities for image data to the kind folks at the PDB, who 
really know what they are doing and capture efficiencies of scale that are out 
of reach for most labs.  

On Oct 27, 2011, at 3:47 PM, Adrian Goldman wrote:

 2) I agree with Susan.  In a time of limited funding, is this the most 
 important use of money?

Re: [ccp4bb] IUCr committees, depositing images

[Craig A. Bingman]

On Oct 16, 2011, at 4:18 PM, Bosch, Juergen wrote:

 The rest is comparable to a collection of stamps, although with the benefit 
 as BR mentioned of adding an additional hurdle/layer to falsifying structures.

Not if you are interested in scattering that falls between reciprocal lattice 
maxima, or if you want to preserve the possibility of applying future data 
reduction packages.  

Re: [ccp4bb] IUCr committees, depositing images

[Craig A. Bingman]

I'm simultaneously embarrassed to have not read the thread to completion before 
replying, and also totally pumped that I would answer a question the same way 
as Bernhard Rupp!

On Oct 16, 2011, at 7:34 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote:

 Ø  Not if you are interested in scattering that falls between reciprocal 
 lattice maxima, or if you want to preserve the possibility of applying future 
 data reduction packages.  
  
 Yep, this is exactly what I expressed in my original statement:
  
 “diffuse solvent contributions, commensurate and incommensurate 
 superstructures, split reflections,  and similar stuff that presently just 
 gets indexed away”
  
 BR

Re: [ccp4bb] should the final model be refined against full datset

[Craig A. Bingman]

Recent experience indicates that the PDB is checking these statistics very 
closely for new depositions.  The checks made by the PDB are intended to 
prevent accidents and oversights made by honest people from creeping into the 
database.  Getting away with something seems to imply some intention to 
deceive, and that is much more difficult to detect.

On Oct 14, 2011, at 3:09 PM, Robbie Joosten wrote:

 The deposited R-free sets in the PDB are quite frequently 'unfree' or the 
 wrong set was deposited (checking this is one of the recommendations in the 
 VTF report in Structure). So at the moment you would probably get away with 
 depositing an unfree R-free set ;)
 

Re: [ccp4bb] should the final model be refined against full datset

[Craig A. Bingman]

We have obligations that extend beyond simply presenting a best model.  

In an ideal world, the PDB would accept two coordinate sets and two sets of 
statistics, one for the last step where the cross-validation set was valid, and 
a final model refined against all the data.  Until there is a clear way to do 
that, and an unambiguous presentation of them to the public, IMO, the gains won 
by refinement against all the data are outweighed by the Confusion that it can 
cause when presenting model and associated statistics to the public.


On Oct 14, 2011, at 3:32 PM, Jan Dohnalek wrote:

 Regarding refinement against all reflections: the main goal of our work is to 
 provide the best possible representation of the experimental data in the form 
 of the structure model. Once the structure building and refinement process is 
 finished keeping the Rfree set separate does not make sense any more. Its 
 role finishes once the last set of changes have been done to the model and 
 verified ...
 
 J. Dohnalek

Re: [ccp4bb] is codon optimization worth it?

[Craig A. Bingman]

Rosetta strains carry a plasmid that supplies several tRNAs that match codons 
that are rare in E. coli.  This is quite different than being explicitly 
optimized to express mammalian proteins.  We (the Center for Eukaryotic 
Structural Genomics, a PSI-1 and PSI-2 center) used strains with the same tRNA 
plasmid (either pRARE or pRARE2) to express proteins from yeast, Arabidopsis, 
some thermophilic eukaryotes, mouse, frog, human and zebrafish proteins.  It 
seemed to work just fine for all of them.
 
On Sep 30, 2011, at 9:55 AM, Ed Pozharski wrote:

 On Fri, 2011-09-30 at 10:49 -0400, Patrick Loll wrote:
 Has anyone encountered a case in which a construct with the native
 sequence expressed poorly (or not at all?) in Rosetta(DE3), but the
 corresponding construct with a codon-optimized sequence expressed
 well? (The gene in question is from cerevesiae)
 
 
 Wait, isn't Rosetta optimized for mammalian genes, not yeast (but maybe
 codon bias in yeast matches that)?
 
 A sideways suggestion would be to express in yeast. Extra bonus - it
 smells like beer :)
 
 -- 
 Hurry up before we all come back to our senses!
   Julian, King of Lemurs

Re: [ccp4bb] Database of Known Medically-Relevant Mutations

[Craig A. Bingman]

I agree with Ethan that this is a niche that OMIM fills with some 
(considerable) degree of success.

http://www.omim.org/

Uniprot is also covering this in more detail, both in individual protein 
records, and in aggregate, e.g.

http://www.uniprot.org/docs/humpvar


On Sep 27, 2011, at 1:52 PM, Jacob Keller wrote:

 Dear Crystallographers,
 
 does anyone have a favorite site which lays out in a clear way all of
 the known medically-relevant or phenotype-changing mutations of a
 given protein and their phenotypes with references? Some of the
 existing sites are very busy graphically and somewhat difficult to
 interpret, making what would seem to be a simple task take much
 longer...
 
 Jacob Keller
 
 -- 
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 cel: 773.608.9185
 email: j-kell...@northwestern.edu
 ***

Re: [ccp4bb] Aging PEGs

[Craig A. Bingman]

Commercial polyethylene glycol is contaminated with polymers that have aldehyde 
groups at the ends.  Other impurities include some amount of internal epoxide 
linkages.  The aldehyde groups can be additionally oxidized to carboxylic 
acids.  I would assume that oxidation to terminal carboxylic acids explains the 
change in PEG pH vs. time.  The pH will also change as the PEG solution comes 
to equilibrium with atmospheric carbon dioxide.  The aldehyde content increases 
with time after the PEG is dissolved in water and stored under normal 
atmospheric gas.  These PEGs can react with protein amine groups.  

On Aug 24, 2011, at 2:18 PM, Jacob Keller wrote:

 A while ago I measured the pH's of old and new PEGs and found them
 very different, and internally attributed all old vs new PEG issues
 to pH. Upon reflection, this seems too simplistic. Are there other
 known mechanisms of crystallization capacities of PEGs of various
 ages?
 
 Jacob Keller
 
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 cel: 773.608.9185
 email: j-kell...@northwestern.edu
 ***

Re: [ccp4bb] anomalous scatterer

[Craig A. Bingman]

This is from Ethan's very useful site, which should be known to everyone doing protein work at synchrotron sources:http://skuld.bmsc.washington.edu/scatter/http://skuld.bmsc.washington.edu/scatter/AS_form.htmlThe usual caveats apply... these calculations, especially for f", are often not especially accurate very near the peak. Chemistry causes small shifts in edge energies compared to the elemental state, and f" is often larger than the calculated value. Still, you can see that your experiment was conducted close to and above (energy) the Fe K-edge, Accordingly Fe is expected to make nontrivial dispersive and anomalous contributions at this energy.On Aug 9, 2011, at 11:38 AM, Huiming Li wrote:Hi All, I am working on a Tb binding protein on which I collected anomalous data at Tb edge of 1.648 A. Each protein is designed to bind one Tb. There are two copies of the protein in an ASU. I have two questions. First, I am only able to see one copy of the protein with Tb bound, and no density on the other copy. Isn't this a bit surprising? Second, there is one additional peak on each monomer at the site where Fe is known to bind, and Fe has an edge of 1.739A. At 1.648A, f' and f'' of Fe is only about 1/5 of Tb. Is it possible Fe also shows some anomalous signal at Tb edge?Thank you,Huiming Li, Ph.D.Immune Disease InstituteChildren's Hospital BostonHarvard Medical SchoolBoston, MA 02115