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

[John R Helliwell]

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&t=395


Best wishes,

John Helliwell and Brian McMahon

[ccp4bb] modifying the ligand

[Adriana Sene]

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

Re: [ccp4bb] modifying the ligand

[Johannes Cramer]

Hi Adriana,

when I am at home, I do the following. Use zinc structure search (
http://zinc.docking.org/search/structure) to generate a smiles code (or any
other website that can do this), then take that code to phenix elbow and
generate the ligand including cif.
Other softwares that can do this off the top of my head are chemoffice and
schrödinger.

I hope this helps,
Cheers,
Johannes

2017-03-29 14:36 GMT+02:00 Adriana Sene :

> 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] Postdoctoral Associate opportunity

[Estrada, D. Fernando]

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 details on the position can be found here: 
https://www.ubjobs.buffalo.edu/postings/6006, or by searching job posting 
#R171.

Cheers,
DF Estrada

D. Fernando 
Estrada,
 PhD
Assistant Professor
Department of Biochemistry
Jacobs School of Medicine and Biomedical Sciences
University at Buffalo
Buffalo, NY 14214-3000
716-829-2767 (Office)
dfest...@buffalo.edu

[ccp4bb] CCP4 wiki is down

[Mark]

Anybody notice that the CCP4 wiki is down?

[ccp4bb] protein precipitation reg

[Akilandeswari Gopalan]

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

[ccp4bb] Large number of outliers in the dataset

[Juliana Ferreira de Oliveira]

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 around 30% and it 
doesn´t decrease (in fact if I continue refining it starts to increase).
The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:

[cid:image001.jpg@01D2A87A.D704ACD0]

So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify about 
outliers anymore. I could refine the MR solution very well, the final Rwork is 
0.2427 and Rfree = 0.2730 and validation on Phenix results in a good structure.
I run Zanuda to confirm the space group and it says that the space group 
assignment seems to be correct.
Do you think that I can improve my structure and solve it at 2.3 Å or better? 
Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify the 
resolution cut, right? What should I say?
Thank you for your help!
Regards,
Juliana

Summary data:
OverallInnerShell  OuterShell
Low resolution limit  51.51  51.51  
 2.42
High resolution limit  2.30 7.27
2.30
Rmerge   0.147   0.054  
 0.487
Rmerge in top intensity bin0.080   -
  -
Rmeas (within I+/I-)  0.155   0.057 
  0.516
Rmeas (all I+ & I-)0.155   0.057
   0.516
Rpim (within I+/I-)0.048   0.017
   0.164
Rpim (all I+ & I-)  0.048   0.017   
0.164
Fractional partial bias-0.006 -0.003
 0.146
Total number of observations83988 2907
11885
Total number unique  8145307
  1167
Mean((I)/sd(I))   9.3   23.9
 2.1
Mn(I) half-set correlation CC(1/2)0.991   0.998   
0.779
Completeness 99.9 99.5  
   100.0
Multiplicity10.3 9.5
   10.2

Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
Space group: P212121
Average mosaicity: 1.90


Juliana Ferreira de Oliveira
Brazilian Laboratory of Biosciences - LNBio
Brazilian Center for Research in Energy and Materials - CNPEM
Campinas-SP, Brazil

Re: [ccp4bb] protein precipitation reg

[Keller, Jacob]

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 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

Re: [ccp4bb] protein precipitation reg

[Roger Rowlett]

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

Re: [ccp4bb] modifying the ligand

[Gianluca Santoni]

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 find here 
http://www.daylight.com/dayhtml/doc/theory/theory.smiles.html a good 
starting point to learn SMILES


Cheers
Gian

On 03/29/2017 02:53 PM, Johannes Cramer wrote:

Hi Adriana,

when I am at home, I do the following. Use zinc structure search 
(http://zinc.docking.org/search/structure) to generate a smiles code 
(or any other website that can do this), then take that code to phenix 
elbow and generate the ligand including cif.
Other softwares that can do this off the top of my head are chemoffice 
and schrödinger.


I hope this helps,
Cheers,
Johannes

2017-03-29 14:36 GMT+02:00 Adriana Sene >:


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

Re: [ccp4bb] protein precipitation reg

[Keller, Jacob]

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

Re: [ccp4bb] protein precipitation reg

[Bonsor, Daniel]

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: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Wednesday, March 29, 2017 11:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: 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 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

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

[Phil Evans]

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 course be higher if 
you include more weak data, but minimising R is _not_ the aim of refinement. 
You should keep all the data

I don’t know what xtriage means by “large number of outliers”: perhaps someone 
else can explain

Phil



> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira 
>  wrote:
> 
> 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 around 30% and it 
> doesn´t decrease (in fact if I continue refining it starts to increase).
> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>  
> 
>  
> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify 
> about outliers anymore. I could refine the MR solution very well, the final 
> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a good 
> structure.
> I run Zanuda to confirm the space group and it says that the space group 
> assignment seems to be correct.
> Do you think that I can improve my structure and solve it at 2.3 Å or better? 
> Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify the 
> resolution cut, right? What should I say?
> Thank you for your help!
> Regards,
> Juliana
>  
> Summary data:
> OverallInnerShell  OuterShell
> Low resolution limit  51.51  51.51
>2.42
> High resolution limit  2.30 7.27  
>   2.30
> Rmerge   0.147   
> 0.054   0.487
> Rmerge in top intensity bin0.080   -  
> -
> Rmeas (within I+/I-)  0.155   0.057   
> 0.516
> Rmeas (all I+ & I-)0.155   0.057  
>  0.516
> Rpim (within I+/I-)0.048   0.017  
>  0.164
> Rpim (all I+ & I-)  0.048   0.017 
>   0.164
> Fractional partial bias-0.006 -0.003  
>0.146
> Total number of observations83988 2907
> 11885
> Total number unique  8145307  
> 1167
> Mean((I)/sd(I))   9.3   23.9  
>2.1
> Mn(I) half-set correlation CC(1/2)0.991   0.998   
> 0.779
> Completeness 99.9 99.5
>  100.0
> Multiplicity10.3 9.5  
>  10.2
>  
> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
> Space group: P212121
> Average mosaicity: 1.90
>  
>  
> Juliana Ferreira de Oliveira
> Brazilian Laboratory of Biosciences - LNBio
> Brazilian Center for Research in Energy and Materials - CNPEM
> Campinas-SP, Brazil

Re: [ccp4bb] protein precipitation reg

[David Briggs]

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
>
>
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs

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

[Mark J van Raaij]

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 well-ordered aa from the input model, refine at different 
resolutions and compare the difference maps for that aa. Or calculate omit maps 
at different resolutions and compare those.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

> On 29 Mar 2017, at 17:44, Phil Evans  wrote:
> 
> 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 course be higher 
> if you include more weak data, but minimising R is _not_ the aim of 
> refinement. You should keep all the data
> 
> I don’t know what xtriage means by “large number of outliers”: perhaps 
> someone else can explain
> 
> Phil
> 
> 
> 
>> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira 
>>  wrote:
>> 
>> 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 around 30% 
>> and it doesn´t decrease (in fact if I continue refining it starts to 
>> increase).
>> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>> 
>> 
>> 
>> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify 
>> about outliers anymore. I could refine the MR solution very well, the final 
>> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a 
>> good structure.
>> I run Zanuda to confirm the space group and it says that the space group 
>> assignment seems to be correct.
>> Do you think that I can improve my structure and solve it at 2.3 Å or 
>> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify 
>> the resolution cut, right? What should I say?
>> Thank you for your help!
>> Regards,
>> Juliana
>> 
>> Summary data:
>> OverallInnerShell  OuterShell
>> Low resolution limit  51.51  51.51   
>> 2.42
>> High resolution limit  2.30 7.27 
>>2.30
>> Rmerge   0.147   
>> 0.054   0.487
>> Rmerge in top intensity bin0.080   - 
>>  -
>> Rmeas (within I+/I-)  0.155   0.057  
>>  0.516
>> Rmeas (all I+ & I-)0.155   0.057 
>>   0.516
>> Rpim (within I+/I-)0.048   0.017 
>>   0.164
>> Rpim (all I+ & I-)  0.048   0.017
>>0.164
>> Fractional partial bias-0.006 -0.003 
>> 0.146
>> Total number of observations83988 2907   
>>  11885
>> Total number unique  8145307 
>>  1167
>> Mean((I)/sd(I))   9.3   23.9 
>> 2.1
>> Mn(I) half-set correlation CC(1/2)0.991   0.998  
>>  0.779
>> Completeness 99.9 99.5   
>>   100.0
>> Multiplicity10.3 9.5 
>>   10.2
>> 
>> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
>> Space group: P212121
>> Average mosaicity: 1.90
>> 
>> 
>> Juliana Ferreira de Oliveira
>> Brazilian Laboratory of Biosciences - LNBio
>> Brazilian Center for Research in Energy and Materials - CNPEM
>> Campinas-SP, Brazil

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

[Nicholas Larsen]

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
2.6 A cutoff.  You are right that some reviewers might raise objections if
the R-factors are too high.

Good luck,
Nick

On Wed, Mar 29, 2017 at 11:56 AM, Mark J van Raaij 
wrote:

> 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 well-ordered aa from the input model, refine at
> different resolutions and compare the difference maps for that aa. Or
> calculate omit maps at different resolutions and compare those.
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
>
> On 29 Mar 2017, at 17:44, Phil Evans  wrote:
>
> 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 course
> be higher if you include more weak data, but minimising R is _not_ the aim
> of refinement. You should keep all the data
>
> I don’t know what xtriage means by “large number of outliers”: perhaps
> someone else can explain
>
> Phil
>
>
>
> On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira <
> juliana.olive...@lnbio.cnpem.br> wrote:
>
> 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
> around 30% and it doesn´t decrease (in fact if I continue refining it
> starts to increase).
> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>
> 
>
> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify
> about outliers anymore. I could refine the MR solution very well, the final
> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a
> good structure.
> I run Zanuda to confirm the space group and it says that the space group
> assignment seems to be correct.
> Do you think that I can improve my structure and solve it at 2.3 Å or
> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to
> justify the resolution cut, right? What should I say?
> Thank you for your help!
> Regards,
> Juliana
>
> Summary data:
> OverallInnerShell  OuterShell
> Low resolution limit  51.51  51.51
>   2.42
> High resolution limit  2.30 7.27
>2.30
> Rmerge   0.147
>   0.054   0.487
> Rmerge in top intensity bin0.080   -
>  -
> Rmeas (within I+/I-)  0.155   0.057
>   0.516
> Rmeas (all I+ & I-)0.155   0.057
>   0.516
> Rpim (within I+/I-)0.048   0.017
>   0.164
> Rpim (all I+ & I-)  0.048   0.017
>   0.164
> Fractional partial bias-0.006 -0.003
> 0.146
> Total number of observations83988 2907
>11885
> Total number unique  8145307
>  1167
> Mean((I)/sd(I))   9.3
>   23.9 2.1
> Mn(I) half-set correlation CC(1/2)0.991   0.998
>   0.779
> Completeness 99.9
> 99.5 100.0
> Multiplicity10.3
> 9.5   10.2
>
> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
> Space group: P212121
> Average mosaicity: 1.90
>
>
> Juliana Ferreira de Oliveira
> Brazilian Laboratory of Biosciences - LNBio
> Brazilian Center for Research in Energy and Materials - CNPEM
> Campinas-SP, Brazil
>
>
>

-- 
[This e-mail message may contain privileged, confidential and/or 
proprietary information of H3 Biomedicine. If you believe that it has been 
sent to you in error, please contact the sender immediately and delete the 
m

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

[Nicolas FOOS]

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 of 
Xtriage about outlier is due to this high mosaicity. What is the 
diagnostic of Xtriage in terms of possible twinning? I am also wondering 
about a pseudo translation.

Maybe try to re-processed your data in this direction.

Hope to help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 29/03/2017 17:56, Mark J van Raaij wrote:
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 well-ordered aa from the input model, refine at 
different resolutions and compare the difference maps for that aa. Or 
calculate omit maps at different resolutions and compare those.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij 



On 29 Mar 2017, at 17:44, Phil Evans > wrote:


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 course be higher if you include more 
weak data, but minimising R is _not_ the aim of refinement. You 
should keep all the data


I don’t know what xtriage means by “large number of outliers”: 
perhaps someone else can explain


Phil



On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira 
> wrote:


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 around 30% and it doesn´t decrease (in fact 
if I continue refining it starts to increase).

The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:



So I decided to cut the data at 2.6A and Xtriage analysis doesn’t 
notify about outliers anymore. I could refine the MR solution very 
well, the final Rwork is 0.2427 and Rfree = 0.2730 and validation on 
Phenix results in a good structure.
I run Zanuda to confirm the space group and it says that the space 
group assignment seems to be correct.
Do you think that I can improve my structure and solve it at 2.3 Å 
or better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need 
to justify the resolution cut, right? What should I say?

Thank you for your help!
Regards,
Juliana

Summary data:
OverallInnerShell  OuterShell
Low resolution limit  51.51 
 51.51   2.42
High resolution limit  2.30 
7.272.30
Rmerge   0.147 
  0.054   0.487
Rmerge in top intensity bin0.080   - 
 -
Rmeas (within I+/I-)  0.155 
  0.057   0.516
Rmeas (all I+ & I-)0.155 
  0.057   0.516
Rpim (within I+/I-)0.048 
  0.017   0.164
Rpim (all I+ & I-)  0.048 
  0.017   0.164
Fractional partial bias-0.006 
-0.003 0.146
Total number of observations83988 2907 
   11885
Total number unique  8145307 
 1167
Mean((I)/sd(I))   9.3 
  23.9 2.1
Mn(I) half-set correlation CC(1/2)0.991   0.998 
  0.779
Completeness 99.9 
99.5 100.0
Multiplicity10.3 
9.5   10.2


Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
Space group: P212121
Average mosaicity: 1.90


Juliana Ferreira de Oliveira
Brazilian Laboratory of Biosciences - LNBio
Brazilian Center for Research in Energy and Materials - CNPEM
Campinas-SP, Brazil

Re: [ccp4bb] protein precipitation reg

[Roger Rowlett]

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 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 11:53 AM, David Briggs wrote:

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

--
--

David Briggs PhD
https://about.me/david_briggs

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

[Robbie Joosten]

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 find a 
decent cut-off. The procedure is a bit of work, but you can use the PDB-REDO 
server (pdb-redo.eu) if you want a ready-made solution.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: Nicolas FOOS
Verzonden: woensdag 29 maart 2017 18:19
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: 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 of Xtriage about outlier is due to this 
high mosaicity. What is the diagnostic of Xtriage in terms of possible 
twinning? I am also wondering about a pseudo translation.
Maybe try to re-processed your data in this direction.

Hope to help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19


On 29/03/2017 17:56, Mark J van Raaij wrote:
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 well-ordered aa from the input model, refine at different 
resolutions and compare the difference maps for that aa. Or calculate omit maps 
at different resolutions and compare those.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij

On 29 Mar 2017, at 17:44, Phil Evans 
mailto:p...@mrc-lmb.cam.ac.uk>> wrote:

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 course be higher if 
you include more weak data, but minimising R is _not_ the aim of refinement. 
You should keep all the data

I don’t know what xtriage means by “large number of outliers”: perhaps someone 
else can explain

Phil



On 29 Mar 2017, at 14:54, Juliana Ferreira de Oliveira 
mailto:juliana.olive...@lnbio.cnpem.br>> wrote:

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 around 30% and it 
doesn´t decrease (in fact if I continue refining it starts to increase).
The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:



So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify about 
outliers anymore. I could refine the MR solution very well, the final Rwork is 
0.2427 and Rfree = 0.2730 and validation on Phenix results in a good structure.
I run Zanuda to confirm the space group and it says that the space group 
assignment seems to be correct.
Do you think that I can improve my structure and solve it at 2.3 Å or better? 
Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify the 
resolution cut, right? What should I say?
Thank you for your help!
Regards,
Juliana

Summary data:
OverallInnerShell  OuterShell
Low resolution limit  51.51  51.51  
 2.42
High resolution limit  2.30 7.27
2.30
Rmerge   0.147   0.054  
 0.487
Rmerge in top intensity bin0.080   -
  -
Rmeas (within I+/I-)  0.155   0.057 
  0.516
Rmeas (all I+ & I-)0.155   0.057
   0.516
Rpim (within I+/I-)0.048   0.017
   0.164
Rpim (all I+ & I-)  0.048   0.017   
0.164
Fractional partial bias-0.006 -0.003
 0.146
Total number of observations83988 2907
11885
Total number unique  8145  

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

[Jeffrey, Philip D.]

Juliana,

I think if you compare otherwise equivalent refinement runs in phenix.refine 
with
refinement.input.xray_data.outliers_rejection=True   (the default)
and
refinement.input.xray_data.outliers_rejection=False
then this will tell you if there's any meaningful difference in the refinement 
statistics (or map).  This doesn't mean you have to use phenix.refine on your 
structure beyond that point, just that this is likely the cleanest comparison 
that I can think of.

Phil Jeffrey
Princeton

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Juliana Ferreira 
de Oliveira [juliana.olive...@lnbio.cnpem.br]
Sent: Wednesday, March 29, 2017 9:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [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 around 30% and it 
doesn´t decrease (in fact if I continue refining it starts to increase).
The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:

[cid:image001.jpg@01D2A87A.D704ACD0]

So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify about 
outliers anymore. I could refine the MR solution very well, the final Rwork is 
0.2427 and Rfree = 0.2730 and validation on Phenix results in a good structure.
I run Zanuda to confirm the space group and it says that the space group 
assignment seems to be correct.
Do you think that I can improve my structure and solve it at 2.3 Å or better? 
Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to justify the 
resolution cut, right? What should I say?
Thank you for your help!
Regards,
Juliana

Summary data:
OverallInnerShell  OuterShell
Low resolution limit  51.51  51.51  
 2.42
High resolution limit  2.30 7.27
2.30
Rmerge   0.147   0.054  
 0.487
Rmerge in top intensity bin0.080   -
  -
Rmeas (within I+/I-)  0.155   0.057 
  0.516
Rmeas (all I+ & I-)0.155   0.057
   0.516
Rpim (within I+/I-)0.048   0.017
   0.164
Rpim (all I+ & I-)  0.048   0.017   
0.164
Fractional partial bias-0.006 -0.003
 0.146
Total number of observations83988 2907
11885
Total number unique  8145307
  1167
Mean((I)/sd(I))   9.3   23.9
 2.1
Mn(I) half-set correlation CC(1/2)0.991   0.998   
0.779
Completeness 99.9 99.5  
   100.0
Multiplicity10.3 9.5
   10.2

Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
Space group: P212121
Average mosaicity: 1.90


Juliana Ferreira de Oliveira
Brazilian Laboratory of Biosciences - LNBio
Brazilian Center for Research in Energy and Materials - CNPEM
Campinas-SP, Brazil

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

[Eleanor Dodson]

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 (therefore a much better structure ] and
reduce the noise.

There are many reasons for data problems, and AIMLESS gives plots and
output  to help you identify how to sort these out. One common one is
radiation damage - you can usually see this in the batch scaling - and you
may get better data by omitting the last images.

Then Ice rings are a constant problem - these are reported usually in the
data processing..

Eleanor


On 29 March 2017 at 17:58, Jeffrey, Philip D. 
wrote:

> Juliana,
>
> I think if you compare otherwise equivalent refinement runs in
> phenix.refine with
> refinement.input.xray_data.outliers_rejection=True   (the default)
> and
> refinement.input.xray_data.outliers_rejection=False
> then this will tell you if there's any meaningful difference in the
> refinement statistics (or map).  This doesn't mean you have to use
> phenix.refine on your structure beyond that point, just that this is likely
> the cleanest comparison that I can think of.
>
> Phil Jeffrey
> Princeton
> --
> *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Juliana
> Ferreira de Oliveira [juliana.olive...@lnbio.cnpem.br]
> *Sent:* Wednesday, March 29, 2017 9:54 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [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
> around 30% and it doesn´t decrease (in fact if I continue refining it
> starts to increase).
>
> The Wilson plot graph is not fitting very well between 2.3 and 2.6 Å:
>
>
>
>
>
> So I decided to cut the data at 2.6A and Xtriage analysis doesn’t notify
> about outliers anymore. I could refine the MR solution very well, the final
> Rwork is 0.2427 and Rfree = 0.2730 and validation on Phenix results in a
> good structure.
>
> I run Zanuda to confirm the space group and it says that the space group
> assignment seems to be correct.
>
> Do you think that I can improve my structure and solve it at 2.3 Å or
> better? Or I can finish it with 2.6 Å? To publish at 2.6 Å I need to
> justify the resolution cut, right? What should I say?
>
> Thank you for your help!
>
> Regards,
>
> Juliana
>
>
>
> Summary data:
>
> OverallInnerShell  OuterShell
>
> Low resolution limit  51.51  51.51
>   2.42
>
> High resolution limit  2.30
> 7.272.30
>
> Rmerge   0.147
> 0.054   0.487
>
> Rmerge in top intensity bin0.080
> -  -
>
> Rmeas (within I+/I-)  0.155   0.057
>   0.516
>
> Rmeas (all I+ & I-)0.155   0.057
>   0.516
>
> Rpim (within I+/I-)0.048   0.017
>   0.164
>
> Rpim (all I+ & I-)  0.048   0.017
>   0.164
>
> Fractional partial bias-0.006 -0.003
> 0.146
>
> Total number of observations83988 2907
>11885
>
> Total number unique  8145307
>  1167
>
> Mean((I)/sd(I))   9.3
> 23.9 2.1
>
> Mn(I) half-set correlation CC(1/2)0.991   0.998
>   0.779
>
> Completeness 99.9 99.5
> 100.0
>
> Multiplicity10.3
> 9.5   10.2
>
>
>
> Average unit cell: 37.57 51.51 88.75 90.00 90.00 90.00
>
> Space group: P212121
>
> Average mosaicity: 1.90
>
>
>
>
>
> Juliana Ferreira de Oliveira
>
> Brazilian Laboratory of Biosciences - LNBio
>
> Brazilian Center for Research in Energy and Materials - CNPEM
>
> Campinas-SP, Brazil
>
>
>

Re: [ccp4bb] modifying the ligand

[Paul Emsley]

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 be used to do that.




Let's imagine that you have access to Coot... The details of how to go 
about this depend on the nature of your input. Let's also imagine that 
you are starting from a SMILES string, then:


Calculate -> Ligand Builder -> File -> Input from SMILES -> 
CC(=O)Nc1ccc(O)cc1 -> OK


.. click, click, edit (in a hopefully intuitive manner) [1]

Apply (wait as Coot runs Acedrg for you)... -> molecule appears (and an 
mmCIF dictionary is written to the file system (which can be used as 
import to Coot/Lidia (just try that with your "paid-for" software)).


You can use File -> Save As... to save in various output formats, press 
the SMILES icon to get a 'cut&pasteable' string.


Paul.

[1] 
https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/screenshots/coot-lidia-pep.png

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

[Gianluca Santoni]

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 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 of Xtriage about outlier is due to
  this high mosaicity. What is the diagnostic of Xtriage in terms of
  possible twinning? I am also wondering about a pseudo translation.
  
  Maybe try to re-processed your data in this direction. 

Hope to help.
Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 29/03/2017 17:56, Mark J van Raaij
  wrote:


  
  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 well-ordered aa from the
  input model, refine at different resolutions and compare the
  difference maps for that aa. Or calculate omit maps at
  different resolutions and compare those.

  
Mark J van Raaij
  Dpto de Estructura de Macromoleculas
  Centro Nacional de Biotecnologia - CSIC
  calle Darwin 3
  E-28049 Madrid, Spain
  tel. (+34) 91 585 4616
  http://wwwuser.cnb.csic.es/~mjvanraaij

  
  
  

  On 29 Mar 2017, at 17:44, Phil Evans 
wrote:
  
  
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 course be higher if
  you include more weak data, but minimising R is _not_
  the aim of refinement. You should keep all the data
  
  I don’t know what xtriage means by “large number of
  outliers”: perhaps someone else can explain
  
  Phil
  
  
  
  On 29 Mar 2017, at
14:54, Juliana Ferreira de Oliveira 
wrote:

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 around 30%
and it doesn´t decrease (in fact if I continue
refining it starts to increase).
The Wilson plot graph is not fitting very well
between 2.3 and 2.6 Å:



So I decided to cut the data at 2.6A and Xtriage
analysis doesn’t notify about outliers anymore. I
could refine the MR solution very well, the final
Rwork is 0.2427 and Rfree = 0.2730 and validation on
Phenix results in a good structure.
I run Zanuda to confirm the space group and it says
that the space group assignment seems to be correct.
Do you think that I can improve my structure and
solve it at 2.3 Å or better? Or I can finish it with
2.6 Å? To publish at 2.6 Å I need to justify the
resolution cut, right? What should I say?
Thank you for your help!
Regards,
Juliana

Summary data:
Overall    InnerShell  OuterShell
Low resolution limit 

[ccp4bb] AW: [ccp4bb] protein precipitation reg

[Hughes, Jon]

...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, 
mailto:kell...@janelia.hhmi.org>> 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.
[Das Bild wurde vom Absender entfernt.]



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

Re: [ccp4bb] Coot render tool missing

[Xiao Lei]

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 edit:
set PATH=%COOT_PREFIX%\bin;%COOT_PREFIX%\libexec;%COOT_PREFIX%\
python27;%PATH%;*C:\CCP4-7\7.**0\bin*). My CCP4 installation folder is
C:\CCP4-7\7.0\bin. To locate files, I just simply use Win7 search file from
the start menu.







*2. - edit the raster3d.py file
(C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\)
from (line 44 or so):r3d_exe = find_exe("render", "PATH")tor3d_exe =
find_exe("render", "CCP4_BIN", "PATH")*

After I did this, Coot can find Raster3D PATH, but I still have an error of
"Some errors in Raster3D"  in terminal windows and fail to render.

*3. Download:*



*https://raw.githubusercontent.com/bernhardcl/coot/master/python/raster3d.py
and
replace the same file (maybe make a backup first) in
C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\*

*4. Done. Works!*


Thanks Bernhard!



On Wed, Mar 29, 2017 at 11:58 AM, Xiao Lei  wrote:

> Hello B.,
>
> Works.Thank you very much!
>
> On Wed, Mar 29, 2017 at 8:14 AM, B.Lohkamp  wrote:
>
>>
>> Hello,
>>
>> I see. Sorry. Now I remember this and I fixed this elsewhere too. Please
>> try the following. Download:
>>
>> https://raw.githubusercontent.com/bernhardcl/coot/master/pyt
>> hon/raster3d.py
>>
>> and replace the same file (maybe make a backup first) in
>> C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\
>>
>> This should work.
>>
>> Bernhard
>>
>> Hi B. ,
>>>
>>> I followed your suggestions and now Wincoot can find render.exe, but it
>>> has a warning message of "this copy of render was not built with tiff
>>> support BL Warning: some error in rester3D" .
>>>
>>> I attach the picture here. I'd appreciate if you have any idea of how to
>>> fix it.
>>>
>>> Thanks a lot.
>>>
>>> Inline image 1
>>>
>>> On Tue, Mar 28, 2017 at 12:54 AM, B.Lohkamp >> > wrote:
>>>
>>>
>>> remove the % around the ccp4 path and you are set.
>>>
>>> B
>>>
>>> Hi Bernhard,
>>>
>>> Thank you for your help. I attach my runwincoot.bat below. My
>>> render.exe
>>> path is "C:\CCP4-7\7.0\bin". What should I do to add to the
>>> PATH? I
>>> could not understand the grammar well, should I add as :
>>> set
>>> PATH=%COOT_PREFIX%\bin;%COOT_PREFIX%\libexec;%COOT_PREFIX%\p
>>> ython27;%PATH%;%C:\CCP4-7\7.0\bin%
>>>   ?
>>> 
>>> 
>>> --
>>> @set LANG=en
>>> title WinCoot
>>>
>>>
>>> set COOT_PREFIX=C:\WinCoot
>>> set COOT_GUILE_PREFIX=C:/WinCoot
>>>
>>> set COOT_HOME=%COOT_PREFIX%
>>> set COOT_BACKUP_DIR=%COOT_PREFIX%\coot-backup
>>>
>>> set COOT_SHARE=%COOT_PREFIX%\share
>>> if not exist "%CLIBD_MON%" (
>>>   echo no $CLIBD_MON found setting COOT_REFMAC_LIB_DIR
>>>   set COOT_REFMAC_LIB_DIR=%COOT_SHARE%\coot\lib
>>> )
>>> set COOT_SCHEME_DIR=%COOT_SHARE%/coot/scheme
>>> set COOT_STANDARD_RESIDUES=%COOT_SHARE%\coot\standard-residues.p
>>> db
>>> set COOT_PIXMAPS_DIR=%COOT_SHARE%\coot\pixmaps
>>> set COOT_RESOURCES_FILE=%COOT_SHARE%\coot\cootrc
>>> set COOT_DATA_DIR=%COOT_SHARE%\coot
>>> set COOT_REF_STRUCTS=%COOT_SHARE%\coot\reference-structures
>>> set COOT_PYTHON_DIR=%COOT_PREFIX%\python27\lib\site-packages\coo
>>> t
>>> set COOT_REF_SEC_STRUCTS=%COOT_SHARE%\coot\ss-reference-structur
>>> es
>>>
>>> set PYTHONHOME=%COOT_PREFIX%\python27
>>>
>>> set
>>> GUILE_LOAD_PATH=%COOT_GUILE_PREFIX%/share/guile/1.8;%COOT_GU
>>> ILE_PREFIX%/share/guile;%COOT_GUILE_PREFIX%/share/guile/gtk-
>>> 2.0;%COOT_GUILE_PREFIX%/share/guile/gui;%COOT_GUILE_PREFIX%/
>>> share/guile/www;%COOT_GUILE_PREFIX%/share/guile/site
>>>
>>> set SYMINFO=%COOT_SHARE%\coot\syminfo.lib
>>>
>>> set
>>> PATH=%COOT_PREFIX%\bin;%COOT_PREFIX%\libexec;%COOT_PREFIX%\p
>>> ython27;%PATH%
>>>
>>> start /affinity 1 coot-bin.exe %*
>>> 
>>> 
>>> ---
>>>
>>> On Mon, Mar 27, 2017 at 10:24 PM, B.Lohkamp >> 
>>> >>
>>> wrote:
>>>
>>>
>>> Hi ...,
>>>
>>> Hi Ethan,
>>>
>>> Thank you for the information. I have ccp4i installed o

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 
mailto:jon.hug...@bot3.bio.uni-giessen.de>> 
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, 
mailto:kell...@janelia.hhmi.org>> 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] [ccp4bb] protein precipitation reg

[Mark Wilson]

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

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
 wrote:

>
>
>
>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

Re: [ccp4bb] [ccp4bb] protein precipitation reg

[Chun Luo]

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 [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark 
Wilson
Sent: Wednesday, March 29, 2017 1:12 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: 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

Mark A. Wilson
Associate Professor
Department of Biochemistry/Redox Biology Center University of Nebraska
N118 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
mwilso...@unl.edu 






On 3/29/17 2:53 PM, "CCP4 bulletin board on behalf of CRAIG A BINGMAN"
 wrote:

>
>
>
>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. 

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

[ccp4bb] Proline derivatives

[Jan van Agthoven]

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

[Janet Newman]

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 that about 
1/3 of the time the protein was significantly (4C or more) stable in some other 
buffer system than the one it was in.
Ristic, M., Rosa, N., Seabrook, S.A., Newman, J., 2015. Formulation screening 
by differential scanning fluorimetry: how often does it work? Acta 
Crystallographica Section F Structural Biology Communications 71, 1359–1364. 
doi:10.1107/S2053230X15012662

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 ?

Cheers, Janet

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of CRAIG A 
BINGMAN
Sent: Thursday, 30 March 2017 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: 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

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

[Keller, Jacob]

>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]

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] Coot render tool missing

[B.Lohkamp]

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 C:\yourwincootinstallationdirectory) *

 I use Notepad to open the .bat file and do edit:
set
PATH=%COOT_PREFIX%\bin;%COOT_PREFIX%\libexec;%COOT_PREFIX%\python27;%PATH%;*C:\CCP4-7\7.**0\bin*).*
 *My
CCP4 installation folder is C:\CCP4-7\7.0\bin. To locate files, I just
simply use Win7 search file from the start menu.

*2. - edit the raster3d.py file
(C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\)
from (line 44 or so):

r3d_exe = find_exe("render", "PATH")

to

r3d_exe = find_exe("render", "CCP4_BIN", "PATH")*
*
*
After I did this, Coot can find Raster3D PATH, but I still have an error
of "Some errors in Raster3D"  in terminal windows and fail to render.

*3. Download:*

*https://raw.githubusercontent.com/bernhardcl/coot/master/python/raster3d.py


and replace the same file (maybe make a backup first) in
C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\*

*4. Done. Works!*
*
*

Thanks Bernhard!



On Wed, Mar 29, 2017 at 11:58 AM, Xiao Lei mailto:xiaolei...@gmail.com>> wrote:

Hello B.,

Works.Thank you very much!

On Wed, Mar 29, 2017 at 8:14 AM, B.Lohkamp mailto:b.lohk...@gmail.com>> wrote:


Hello,

I see. Sorry. Now I remember this and I fixed this elsewhere
too. Please try the following. Download:


https://raw.githubusercontent.com/bernhardcl/coot/master/python/raster3d.py



and replace the same file (maybe make a backup first) in
C:\yourwincootinstallationdrirectory\python27\lib\site-packages\coot\

This should work.

Bernhard

Hi B. ,

I followed your suggestions and now Wincoot can find
render.exe, but it
has a warning message of "this copy of render was not built
with tiff
support BL Warning: some error in rester3D" .

I attach the picture here. I'd appreciate if you have any
idea of how to
fix it.

Thanks a lot.

Inline image 1

On Tue, Mar 28, 2017 at 12:54 AM, B.Lohkamp
mailto:b.lohk...@gmail.com>
>>
wrote:


remove the % around the ccp4 path and you are set.

B

Hi Bernhard,

Thank you for your help. I attach my runwincoot.bat
below. My
render.exe
path is "C:\CCP4-7\7.0\bin". What should I do to add
to the PATH? I
could not understand the grammar well, should I add as :
set


PATH=%COOT_PREFIX%\bin;%COOT_PREFIX%\libexec;%COOT_PREFIX%\python27;%PATH%;%C:\CCP4-7\7.0\bin%
  ?


--
@set LANG=en
title WinCoot


set COOT_PREFIX=C:\WinCoot
set COOT_GUILE_PREFIX=C:/WinCoot

set COOT_HOME=%COOT_PREFIX%
set COOT_BACKUP_DIR=%COOT_PREFIX%\coot-backup

set COOT_SHARE=%COOT_PREFIX%\share
if not exist "%CLIBD_MON%" (
  echo no $CLIBD_MON found setting COOT_REFMAC_LIB_DIR
  set COOT_REFMAC_LIB_DIR=%COOT_SHARE%\coot\lib
)
set COOT_SCHEME_DIR=%COOT_SHARE%/coot/scheme
set
COOT_STANDARD_RESIDUES=%COOT_SHARE%\coot\standard-residues.pdb
set COOT_PIXMAPS_DIR=%COOT_SHARE%\coot\pixmaps
set COOT_RESOURCES_FILE=%COOT_SHARE%\coot\cootrc
set COOT_DATA_DIR=%COOT_SHARE%\coot
set
COOT_REF_STRUCTS=%COOT_SHARE%\coot\reference-structures
set
COOT_PYTHON_DIR=%COOT_PREFIX%\python27\lib\site-packages\coot
set
COOT_REF_SEC_STRUCTS=%COOT_SHARE%\coot\ss-reference-structures

set PYTHONHOME=%COOT_PREFIX%\python27

set


GUILE_LOAD_PATH=%COOT_GUILE_PREFIX%/share/guile/1.8;%COOT_GUILE_PREFIX%/share/guile;%COOT_GUILE_PREFIX%/share/guile/gtk-2.0;%COOT_GUILE_PREFIX%/share/guile/gui;%COOT_GUILE_PREFIX%/share/guile/www;%COOT_GUILE_PREFIX%/share/guile/site

set SYMINFO=%COOT_SHARE%\coot\syminfo.lib

set

 

Re: [ccp4bb] protein precipitation reg

[Debanu Das]

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 are you dialyzing? Is it after SEC? Could your
protein be too concentrated at that point since your yield is high
leading to some precipitation? How severe is the loss?
2) Did you try more purification before dialysis?
3) Are you removing the detergent in the buffer you are dialyzing against?
4) Can you try buffer exchange during concentration instead of dialysis?
5) Try increasing your NaCl concentration and adding 5-10% glycerol to
improve protein solubility.
6) Did you try cleaving the C-term His-tag before dialysis? Did you
try N-term His tag?
7) Do you really need to dialyze? Did you try assays or
crystallization trials with the purification buffer? You can run a SEC
column without imidazole to remove that before crystallization.
8) PSI/SBKB TargetTrack can be great resource to look at
expression/purification protocols for similar proteins:
http://sbkb.org/tt/
9) Also look up the Tb Structural Genomics resource to see if there is
anything on this target or related targets: http://www.webtb.org/

Best,
Debanu

On Wed, Mar 29, 2017 at 6:38 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
>

Re: [ccp4bb] protein precipitation reg

[Akilandeswari Gopalan]

Dear all,
I have used the following buffers for purification and dialysis. this is
fyi.

*Lysis buffer*:

25mM Tris pH 7 or 7.5 or 8

100-500mM NaCl (increase in salt concentration increased precipitation of
the protein in the column itself)

 *5mM Beta mercaptoethanol *

*0.5% Triton X 100 *

I have tried with other buffers also.

a. HEPES buffer pH7.5

b. Phosphate buffer pH 7.8

c. MOPS buffer pH 8



*Wash and Elution Buffer*:

25mM Tris pH 7 or 7.5 or 8

100-500mM NaCl

20 and 30mM Imidazole for wash

300mM for elution





*Dialysis Buffer*:

1. Tris 25mM pH 7

2. Tris 25mM pH 7.5

3. Tris 25mM pH 8

4. Tris 25mM pH 7.5, 5% glycerol

5. Tris 25mM pH 7.5, 10% glycerol

6. Tris 25mM pH 7.5, 20% glycerol

7. Tris 25mM pH7.5, 50mM NaCl

8. Tris 25mM pH7.5, 100mM NaCl

9. Tris 25mM pH7.5, 1mM MgCl2

10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu


In all these cases the protein precipitates. i have tried to do buffer
exchange also. i can see precipitate sticking on the walls of the tube
during the process.

On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das  wrote:

> 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 are you dialyzing? Is it after SEC? Could your
> protein be too concentrated at that point since your yield is high
> leading to some precipitation? How severe is the loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer you are dialyzing against?
> 4) Can you try buffer exchange during concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try assays or
> crystallization trials with the purification buffer? You can run a SEC
> column without imidazole to remove that before crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to look at
> expression/purification protocols for similar proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics resource to see if there is
> anything on this target or related targets: http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 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
> >
>



-- 
Akilandeswari G
JRF
C/O Dr. Alamelu Raja
National Institute for Research in Tuberculosis
Chetput, Chennai

Re: [ccp4bb] protein precipitation reg

[Paul Miller]

Only one of your dialysis buffers has a decent (100 mM) NaCl. Maybe you could 
try higher salt IN COMBINATION with glycerol. There's also NDSBs that stabilise 
proteins.

Also, could the high immidazole be keeping the protein happy? You could test 
this by dialysing into the same buffer with a range of immidazole 
concentrations. You could try other related natural compounds, histidine, 
histamine, as well to understand this process better.

Cheers, Paul

Paul Steven Miller (PhD)
Postdoctoral Researcher
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive
Oxford
OX3 7BN


 Original message 
>Date: Thu, 30 Mar 2017 11:32:05 +0530
>From: CCP4 bulletin board  (on behalf of Akilandeswari 
>Gopalan )
>Subject: Re: [ccp4bb] protein precipitation reg  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear all,
>   I have used the following buffers for purification
>   and dialysis. this is fyi.
>
>   Lysis buffer:
>
>   25mM Tris pH 7 or 7.5 or 8
>
>   100-500mM NaCl (increase in salt concentration
>   increased precipitation of the protein in the column
>   itself)
>
>    5mM Beta mercaptoethanol 
>
>   0.5% Triton X 100 
>
>   I have tried with other buffers also.
>
>   a. HEPES buffer pH7.5
>
>   b. Phosphate buffer pH 7.8
>
>   c. MOPS buffer pH 8
>
>    
>
>   Wash and Elution Buffer:
>
>   25mM Tris pH 7 or 7.5 or 8
>
>   100-500mM NaCl 
>
>   20 and 30mM Imidazole for wash
>
>   300mM for elution
>
>    
>
>    
>
>   Dialysis Buffer:
>
>   1. Tris 25mM pH 7
>
>   2. Tris 25mM pH 7.5
>
>   3. Tris 25mM pH 8
>
>   4. Tris 25mM pH 7.5, 5% glycerol
>
>   5. Tris 25mM pH 7.5, 10% glycerol
>
>   6. Tris 25mM pH 7.5, 20% glycerol
>
>   7. Tris 25mM pH7.5, 50mM NaCl
>
>   8. Tris 25mM pH7.5, 100mM NaCl
>
>   9. Tris 25mM pH7.5, 1mM MgCl[2
>
>   ]10.  Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM
>   L-Glu
>
>   In all these cases the protein precipitates. i have
>   tried to do buffer exchange also. i can see
>   precipitate sticking on the walls of the tube during
>   the process. 
>
>   On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das
>wrote:
>
> 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 are you dialyzing? Is it after
> SEC? Could your
> protein be too concentrated at that point since
> your yield is high
> leading to some precipitation? How severe is the
> loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer
> you are dialyzing against?
> 4) Can you try buffer exchange during
> concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and
> adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before
> dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try
> assays or
> crystallization trials with the purification
> buffer? You can run a SEC
> column without imidazole to remove that before
> crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to
> look at
> expression/purification protocols for similar
> proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics
> resource to see if there is
> anything on this target or related targets:
> http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 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
> >
>
>   --
>   Akilandeswari G
>   JRF
>   C/O Dr. Alamelu Raja
>   National Institute for Research in Tuberculosis
>   Chetput, Chennai