[ccp4bb] OT: Free Webinar - 3D Molecular Graphics, Movies, Documents and Presentations

[Andrew Orry]

Dear all,

We are holding a webinar next week (Feb 4th 9AM PST) highlighting the 
tools available in our free molecular visualization software tools-  
ICM-Browser (Desktop - Win/Mac/Linux), iMolview (iPad/Android), and 
ActiveICM (Plugin for Windows Powerpoint and Web Browsers).


There is more information on how to register here: 
http://www.molsoft.com/training.html#webinars


The webinar topics include:
- Easy 3D stereo viewing using Anaglyph Stereo ( 
http://www.molsoft.com/news.html#anaglyph3D - no expensive glasses, 
hardware or monitors required).

- How to annotate, color, and change the 3D representation of molecules.
- How to prepare publication quality molecular images.
- How to make fully interactive 3D slides including smooth and blending 
transitions.
- How to import fully interactive 3D slides into Windows PowerPoint, web 
pages and iPad/Android devices.
- Molecular movie making made easy using - movies from screenshots and 
slides.


We hope you can join us.
Thanks,

--
Andrew Orry Ph.D.
Senior Research Scientist
MolSoft LLC
--
www.molsoft.com
www.twitter.com/molsoft
https://www.facebook.com/Molsoft

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Edward A. Berry]

If the pK's are well separated, so that only one is titrating at a time ( assume
only two species exist at a time), the number of equiv of NaOH to add would be :

  1/(1+10^(pK1-pH)) + 1/(1+10^(pK2-pH)) + 1/(1+10^(pK3-pH))

where each term transitions from 0 to 1 as pH passes through it's pKa

In principle the calculated buffers should be more accurate, especially in the strong 
buffering regions where a small error in weighing out doesn't affect pH much. They depend 
on accurate pKa values, but these have probably been determined by a physical chemist with 
a hydrogen electrode (not glass electrode) in a junctionless system with temperature 
control.  They are probably more reproducible, since analytical balances don't drift as 
much or show as much temperature dependence as pH electrodes. However if you publish just 
giving pH and someone else tries to reproduce your results by adjusting a 1M stock 
solution at room temperature to the pH and then diluting it to 10 mM in the assay at 4C, 
they are likely to get a very different pH.


Also check the label on your NaOH bottle to see the water content and adjust the MW 
accordingly- NaOH is often less than 90% NaOH by weight.


Finally if the reason for using a tribasic buffer is to get similar conditions over a wide 
pH range, this is a delusion because different buffer species are present at different 
pH's.  MES^- is a lot more like MOPS^- than citrate^- is like citrate^-2, ionically 
speaking. And the ionic strength will vary greatly from one end of the range to the other, 
as you go from mostly neutral citric acid to mostly Na2- or Na3-citrate.


eab

Katherine Sippel wrote:

Alternatively you could make a stock solution of citric acid (say 1 M for 
example) and
stock solution of sodium citrate (also 1 M). Mix them in the appropriate ratio 
to ballpark
the right pH and just adjust up or down with the stock solution. The 
concentration of
citrate will be the same no matter the final volume. You can then dilute that 
down to
whatever your final concentration of citrate needs to be.

If you are looking for the actual method to do the calculations I would suggest 
finding a
chemistry textbook and looking at the chapter on buffering and the 
Henderson-Hasselbalch
equation.

Cheers,
Katherine


On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot mailto:daniel.pi...@ibpc.fr>> wrote:

But you have to be aware that pH depends on the concentration  of the 
buffer. This is
especially the case for phosphate and citrate buffer.
Daniel

Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :

It’s a pain but I usually just make each pH of whatever buffer I’m using 
(if you
make it concentrated then you’ll only have to do it once).  Also, if you 
haven’t
already found it, Hampton has a nice link to calculate volume of components 
while
designing a tray as long as you tell it the concentrations.

http://hamptonresearch.com/make_tray.aspx

Nick

From: Roger Rowlett mailto:rrowl...@colgate.edu>>
Reply-To: Roger Rowlett mailto:rrowl...@colgate.edu>>
Date: Thursday, January 30, 2014 at 7:23 AM
To: "CCP4BB@JISCMAIL.AC.UK " 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

The easiest way to produce repeatable conditions is to titrate a stock 
solution (say
1M) of citric acid with NaOH to the desired pH and use that to mix your 
screen.
That's what Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. 
The pH
won't be linear with mixing ratio, but will be easily repeatable. The 
actual pH of
the final, magic solution can be directly measured if desired. Calculations 
will
never be exactly right; pKa values are ionic strength dependent. Better to 
measure.

Roger Rowlett

On Jan 30, 2014 2:37 AM, "sreetama das" mailto:somon_...@yahoo.co.in>> wrote:

Dear All,
   We have obtained many tiny protein crystals in a condition 
containing
0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too 
small for
mounting in loops.

   We intend to vary the salt concentration & pH to obtain 
larger crystals.

   Could anyone direct us to some links, or provide us with a 
method
(with calculations) to calculate the amounts of citric acid & trisodium 
citrate
required to obtain buffers in a range of pH 3 - 6.5?
   I have come across online buffer calculators and links where 
the
amounts of the components required are mentioned in grams, but none 
explaining
how those values were arrived at.

Thanks & regards,
sreetama






--
"Nil illegitimo carborundum"/- /Didactylos

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Roger Rowlett]

You are correct about certain buffers as interferents. Certain buffer 
species will coordinate with or precipitate silver or mercurous ions 
that are present in the reference electrode compartment of the 
combination pH electrodes. Tris is notorious for clogging the little 
porous frit on the reference electrode, and this, along with reference 
solution cation ion depletion, will drive the electrode crazy until the 
frit is cleared and/or the reference electrode filling solution is 
replenished. Making 1M stock Tris solutions is enough to knock out a pH 
electrode for several hours if you overexpose it to the solution.


Cheers,

___
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 1/30/2014 12:23 PM, Shane Caldwell wrote:

Hi Sreetama,

For most buffers, I use Katherine's method, but in the case of citrate 
I'd recommend just titrating citric acid with NaOH. I've made a pH 
series from citric acid and Na3Cit before, and it's a huge pain. It's 
very difficult to calculate how much of each you'll need, because 
citrate is triprotic and the pKas overlap. When I made a pH series 
this way, I ended up using much more stock than I anticipated, and 
just had an overall unpleasant afternoon.


One other point - high citrate concentrations can cause your pH meter 
to drift, so don't leave the probe in the citrate solution any longer 
than needed, and calibrate it frequently. I'm told this is because 
citrate chelates metals and that throws off the electrode, though 
admittedly I don't know the mechanism and can't find any references to 
back me up - it's just been lab folklore for me. Either way it might 
be worth testing the stability of your electrode over time to get an 
idea.


Shane Caldwell
McGill University


On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel 
mailto:katherine.sip...@gmail.com>> wrote:


Alternatively you could make a stock solution of citric acid (say
1 M for example) and stock solution of sodium citrate (also 1 M).
Mix them in the appropriate ratio to ballpark the right pH and
just adjust up or down with the stock solution. The concentration
of citrate will be the same no matter the final volume. You can
then dilute that down to whatever your final concentration of
citrate needs to be.

If you are looking for the actual method to do the calculations I
would suggest finding a chemistry textbook and looking at the
chapter on buffering and the Henderson-Hasselbalch equation.

Cheers,
Katherine


On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot
mailto:daniel.pi...@ibpc.fr>> wrote:

But you have to be aware that pH depends on the concentration 
of the buffer. This is especially the case for phosphate and

citrate buffer.
Daniel

Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :

It's a pain but I usually just make each pH of whatever
buffer I'm using (if you make it concentrated then you'll
only have to do it once).  Also, if you haven't already found
it, Hampton has a nice link to calculate volume of components
while designing a tray as long as you tell it the concentrations.

http://hamptonresearch.com/make_tray.aspx

Nick

From: Roger Rowlett mailto:rrowl...@colgate.edu>>
Reply-To: Roger Rowlett mailto:rrowl...@colgate.edu>>
Date: Thursday, January 30, 2014 at 7:23 AM
To: "CCP4BB@JISCMAIL.AC.UK "
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

The easiest way to produce repeatable conditions is to
titrate a stock solution (say 1M) of citric acid with NaOH to
the desired pH and use that to mix your screen. That's what
Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and
6.5 buffers. The pH won't be linear with mixing ratio, but
will be easily repeatable. The actual pH of the final, magic
solution can be directly measured if desired. Calculations
will never be exactly right; pKa values are ionic strength
dependent. Better to measure.

Roger Rowlett

On Jan 30, 2014 2:37 AM, "sreetama das"
mailto:somon_...@yahoo.co.in>> wrote:

Dear All,
   We have obtained many tiny protein crystals in
a condition containing 0.1M citric acid pH 3.5, 2M
ammonium sulfate. The crystals are too small for mounting
in loops.

   We intend to vary the salt concentration & pH
to obtain larger crystals.

   Could anyone direct us to some links, or
provide us with a method (with calculation

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Shane Caldwell]

Hi Sreetama,

For most buffers, I use Katherine's method, but in the case of citrate I'd
recommend just titrating citric acid with NaOH. I've made a pH series from
citric acid and Na3Cit before, and it's a huge pain. It's very difficult to
calculate how much of each you'll need, because citrate is triprotic and
the pKas overlap. When I made a pH series this way, I ended up using much
more stock than I anticipated, and just had an overall unpleasant
afternoon.

One other point - high citrate concentrations can cause your pH meter to
drift, so don't leave the probe in the citrate solution any longer than
needed, and calibrate it frequently. I'm told this is because citrate
chelates metals and that throws off the electrode, though admittedly I
don't know the mechanism and can't find any references to back me up - it's
just been lab folklore for me. Either way it might be worth testing the
stability of your electrode over time to get an idea.

Shane Caldwell
McGill University


On Thu, Jan 30, 2014 at 10:40 AM, Katherine Sippel <
katherine.sip...@gmail.com> wrote:

> Alternatively you could make a stock solution of citric acid (say 1 M for
> example) and stock solution of sodium citrate (also 1 M). Mix them in the
> appropriate ratio to ballpark the right pH and just adjust up or down with
> the stock solution. The concentration of citrate will be the same no matter
> the final volume. You can then dilute that down to whatever your final
> concentration of citrate needs to be.
>
> If you are looking for the actual method to do the calculations I would
> suggest finding a chemistry textbook and looking at the chapter on
> buffering and the Henderson-Hasselbalch equation.
>
> Cheers,
> Katherine
>
>
> On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot wrote:
>
>>  But you have to be aware that pH depends on the concentration  of the
>> buffer. This is especially the case for phosphate and citrate buffer.
>> Daniel
>>
>> Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :
>>
>> It's a pain but I usually just make each pH of whatever buffer I'm using
>> (if you make it concentrated then you'll only have to do it once).  Also,
>> if you haven't already found it, Hampton has a nice link to calculate
>> volume of components while designing a tray as long as you tell it the
>> concentrations.
>>
>>  http://hamptonresearch.com/make_tray.aspx
>>
>>  Nick
>>
>>   From: Roger Rowlett 
>> Reply-To: Roger Rowlett 
>> Date: Thursday, January 30, 2014 at 7:23 AM
>> To: "CCP4BB@JISCMAIL.AC.UK" 
>> Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5
>>
>>   The easiest way to produce repeatable conditions is to titrate a stock
>> solution (say 1M) of citric acid with NaOH to the desired pH and use that
>> to mix your screen. That's what Hampton does anyway.
>>
>> If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
>> The pH won't be linear with mixing ratio, but will be easily repeatable.
>> The actual pH of the final, magic solution can be directly measured if
>> desired. Calculations will never be exactly right; pKa values are ionic
>> strength dependent. Better to measure.
>>
>> Roger Rowlett
>> On Jan 30, 2014 2:37 AM, "sreetama das"  wrote:
>>
>>>  Dear All,
>>> We have obtained many tiny protein crystals in a condition
>>> containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are
>>> too small for mounting in loops.
>>>
>>> We intend to vary the salt concentration & pH to obtain
>>> larger crystals.
>>>
>>> Could anyone direct us to some links, or provide us with a
>>> method (with calculations) to calculate the amounts of citric acid &
>>> trisodium citrate required to obtain buffers in a range of pH 3 - 6.5?
>>> I have come across online buffer calculators and links where
>>> the amounts of the components required are mentioned in grams, but none
>>> explaining how those values were arrived at.
>>>
>>>  Thanks & regards,
>>>  sreetama
>>>
>>
>>
>
>
> --
> "Nil illegitimo carborundum"* - *Didactylos
>

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[David Schuller]

RE citrate buffer preparation

The Calbiochem buffers has some generally useful information about 
buffers; pKa and such.


http://www.antibodybeyond.com/books/Calbiochem_Buffers_Booklet_CB0052_E.pdf
http://wolfson.huji.ac.il/purification/PDF/Buffers/Calbiochem_Buffers_Booklet.pdf

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

[ccp4bb] post-doctoral opportunity at the University of Leeds

[Thomas Edwards]

Dear CCP4bb,

If you are a talented and motivated postdoctoral structural biologist, or soon 
to be, or know somebody who is…


Project Title: Structure and function of the essential M2-1 protein of human 
respiratory syncytial virus

Our laboratories are interested in understanding the structural and cellular 
biology of a variety of negative strand RNA viruses. One of these viruses is 
human respiratory syncytial virus (HRSV), which is the leading cause of lower 
respiratory tract illness in young children and the immunocompromised, and has 
been linked to adult asthma. We recently determined the first X-ray crystal 
structure of the HRSV M2-1 protein, which is an essential transcriptional 
anti-terminator (Tanner et al., PNAS, 2014). The current project seeks to 
further understand the essential role of M2-1 in viral mRNA transcription, and 
will involve both X-ray crystallography and cell biological approaches using 
live recombinant virus and genome analog analysis.

You will have a PhD (or be close to completion) in Structural Molecular Biology 
or a closely allied discipline, together with a proven track record of protein 
structure solution using X-ray crystallography. Experience of working with 
viruses in mammalian cell culture is highly desirable.

University Grade 7 (£30,728 - £36,661 p.a.) Due to funding limitations an 
appointment will not be made above £30,728 p.a.

Informal enquiries may be made to either Dr John N. Barr, email 
j.n.b...@leeds.ac.uk or Dr Thomas Edwards, email 
t.a.edwa...@leeds.ac.uk.

Closing Date: 13 February 2014

http://jobs.leeds.ac.uk/fe/tpl_universityofleeds01.asp?s=4A515F4E5A565B1A&jobid=108922,2351796198&key=9056137&c=344787029814&pagestamp=sedylihlceulaesvjp


Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT
Telephone: 0113 343 3031
http://www.fbs.leeds.ac.uk/staff/tae/
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae
Live as if you were to die tomorrow. Learn as if you were to live forever. 
Mahatma Gandhi

[ccp4bb] Summer School in Chamonix Valley, France: Integrated Structural Cell Biology

[Hugues Nury]

Dear all, 

This july, an exciting Summer School will take place at Les Houches in the 
Chamonix Valley, France. It will deal with Integrated Structural Cell Biology 
by covering a variety of structural, biophysical and computational approaches. 
The school is intended for PhD students, postdocs and early-stage researchers.

The price is 1500 euros for the 4 weeks; selected applications will be 
subsidized.

Learn more about the lectures, the speakers, the place and apply at:
www.leshouches2014.eu

Organisers: Eva Pebay-Peyroula, Christine Ziegler, Francois Parcy, Hugues Nury, 
Rob Ruigrok

--
Hugues Nury
Institut de Biologie Structurale, Grenoble, France

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Katherine Sippel]

Alternatively you could make a stock solution of citric acid (say 1 M for
example) and stock solution of sodium citrate (also 1 M). Mix them in the
appropriate ratio to ballpark the right pH and just adjust up or down with
the stock solution. The concentration of citrate will be the same no matter
the final volume. You can then dilute that down to whatever your final
concentration of citrate needs to be.

If you are looking for the actual method to do the calculations I would
suggest finding a chemistry textbook and looking at the chapter on
buffering and the Henderson-Hasselbalch equation.

Cheers,
Katherine


On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot  wrote:

>  But you have to be aware that pH depends on the concentration  of the
> buffer. This is especially the case for phosphate and citrate buffer.
> Daniel
>
> Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :
>
> It's a pain but I usually just make each pH of whatever buffer I'm using
> (if you make it concentrated then you'll only have to do it once).  Also,
> if you haven't already found it, Hampton has a nice link to calculate
> volume of components while designing a tray as long as you tell it the
> concentrations.
>
>  http://hamptonresearch.com/make_tray.aspx
>
>  Nick
>
>   From: Roger Rowlett 
> Reply-To: Roger Rowlett 
> Date: Thursday, January 30, 2014 at 7:23 AM
> To: "CCP4BB@JISCMAIL.AC.UK" 
> Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5
>
>   The easiest way to produce repeatable conditions is to titrate a stock
> solution (say 1M) of citric acid with NaOH to the desired pH and use that
> to mix your screen. That's what Hampton does anyway.
>
> If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
> The pH won't be linear with mixing ratio, but will be easily repeatable.
> The actual pH of the final, magic solution can be directly measured if
> desired. Calculations will never be exactly right; pKa values are ionic
> strength dependent. Better to measure.
>
> Roger Rowlett
> On Jan 30, 2014 2:37 AM, "sreetama das"  wrote:
>
>>  Dear All,
>> We have obtained many tiny protein crystals in a condition
>> containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are
>> too small for mounting in loops.
>>
>> We intend to vary the salt concentration & pH to obtain
>> larger crystals.
>>
>> Could anyone direct us to some links, or provide us with a
>> method (with calculations) to calculate the amounts of citric acid &
>> trisodium citrate required to obtain buffers in a range of pH 3 - 6.5?
>> I have come across online buffer calculators and links where
>> the amounts of the components required are mentioned in grams, but none
>> explaining how those values were arrived at.
>>
>>  Thanks & regards,
>>  sreetama
>>
>
>


-- 
"Nil illegitimo carborundum"* - *Didactylos

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Daniel Picot]

But you have to be aware that pH depends on the concentration  of the 
buffer. This is especially the case for phosphate and citrate buffer.

Daniel

Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :
It’s a pain but I usually just make each pH of whatever buffer I’m 
using (if you make it concentrated then you’ll only have to do it 
once).  Also, if you haven’t already found it, Hampton has a nice link 
to calculate volume of components while designing a tray as long as 
you tell it the concentrations.


http://hamptonresearch.com/make_tray.aspx

Nick

From: Roger Rowlett mailto:rrowl...@colgate.edu>>
Reply-To: Roger Rowlett >

Date: Thursday, January 30, 2014 at 7:23 AM
To: "CCP4BB@JISCMAIL.AC.UK " 
mailto:CCP4BB@JISCMAIL.AC.UK>>

Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

The easiest way to produce repeatable conditions is to titrate a stock 
solution (say 1M) of citric acid with NaOH to the desired pH and use 
that to mix your screen. That's what Hampton does anyway.


If fine sampling pH, you can mix various ratios of pH 3 and 6.5 
buffers. The pH won't be linear with mixing ratio, but will be easily 
repeatable. The actual pH of the final, magic solution can be directly 
measured if desired. Calculations will never be exactly right; pKa 
values are ionic strength dependent. Better to measure.


Roger Rowlett

On Jan 30, 2014 2:37 AM, "sreetama das" > wrote:


Dear All,
   We have obtained many tiny protein crystals in a
condition containing 0.1M citric acid pH 3.5, 2M ammonium sulfate.
The crystals are too small for mounting in loops.

   We intend to vary the salt concentration & pH to obtain
larger crystals.

   Could anyone direct us to some links, or provide us
with a method (with calculations) to calculate the amounts of
citric acid & trisodium citrate required to obtain buffers in a
range of pH 3 - 6.5?
   I have come across online buffer calculators and links
where the amounts of the components required are mentioned in
grams, but none explaining how those values were arrived at.

Thanks & regards,
sreetama

Re: [ccp4bb] Protein Purification Problem

[Anindito Sen]

Dear All,

1st of all , Thanks for the very quick feedback. I will answer your questions 
to make the picture more clear-

Nicolas-

Yes the dimer is co-expressed. Almost all the protein is in the sup and not in 
the pellet. Multiple-step  dialysis has failed. I have been trying various 
controls and methods for the last 2 years but of not great success. I do have 
significant amount of protein in the 1st step all including homo-dimers and the 
hetro-dimer. Only 8% of that amount is hetro-dimer. 

Fiona-

I will look in the pI option. Step dialysis has failed due to the aggregation 
issue. St is for Strep affinity. Yes, there is a lot of protein in the flow 
through from the column, I have checked and those protein are the homo-dimers. 

Roger-

I do need to remove/reduce the salt for cryo-Electron Microscopy (cryo-EM). The 
maximum that can be afforded is 50 mM. Glycerol is a big no for cryo-EM so I am 
not sure how to implement it. You are absolutely correct as the membranes of 
the centrifugal concentration devices are creating one big problem. I will try 
PES (polyethylenesulfone) concentrator. The final pH needs to be 6.8-7.0 at the 
for the EM work. So to make a drop from pH 8.0 to 6.8 will be a additive step 
in purification process. Can you suggest how can we do it without much drop in 
the concentration ?

Bryan-

I agree with you that the plasmid I have in my hand is far from being a good 
candidate for the purification process, so I have to venture more. Your point 
No2 & 3 is well taken, I will look into that. However I cannot have the pH as 
high as 8. It needs to be around 6.8 to 7.0 for EM work. 

Lastly I am not an expert of Protein Purification , my expertise are in Cryo-EM 
and computational  image processing, so I have to go baby-steps of asking such 
questions at the forum. Thanks again. 

Best 

Andy






Dr. Anindito Sen (Ph.D)
Assistant Professor (Project)
Department of Cell Biology & Anatomy
Graduate School of Medicine
University of Tokyo
Tel & fax: +81-3-5841-3339

On Jan 30, 2014, at 10:57 PM, Fiona Whelan  wrote:

> Hi Andy,
> 
> There are lots of things to try. Here are a few:
> Check your protein heterodimer combined pI, make sure you're at least 1 pH 
> unit away from that for your dialysis step. 
> Perhaps your dimer needs high NaCl to maintain its solubility - try dialysing 
> into a range of NaCl concentrations (100mM, 150, 200, 300 mM). Does it need 
> any divalent cations? Check homologs - do they bind divalent cations - try 
> screening these in your dialysis condition. 
> 
> You say 2 column purifications - St - is that Strep affinity? Do you have a 
> lot of protein in the flow through from this column? I've found that using pH 
> 8 is very important for binding at this stage - and high protein 
> concentration.
> 
> I think the BB might be more helpful with a bit more information about your 
> expression method and if you think the protein is lost at some stage during 
> the purification, or only lost in aggregate after dialysis/during 
> concentration.
> 
> Good luck,
> 
> Fiona
> 
> On 30/01/2014 13:17, Anindito Sen wrote:
>> Dear All,
>> 
>> This may be slightly off-the-track question but your feedback will be very 
>> much appreciated. The situation is-
>> 
>> I obtain a very low amount of the protein of my interest (a hetro-dimer) 
>> from the construct I am using (only 8% of the total amount of protein 
>> obtained is the protein of my interest).  After 2 column purifications 
>> (Ni-NTA and St) the concentration of the protein is around 0.24 mg/ml 
>> (volume- ~1.0 ml)  from a litre of bacterial culture and in ~300 mM NaCl 
>> present in the elution  buffer. 
>> 
>> To reduce the high amount of salt I have I use a desalting column which, 
>> further lowers the protein concentration significantly. 
>> 
>> I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 
>> microlts for further experiments. 
>> 
>> As the last resort I try to use high amount of bacterial culture (~6lts) to 
>> scale up the yield and use centricon to concentrate the protein at various 
>> stages.  I am partially successful to obtain 0.56mg/ml of protein 
>> concentration and up to  50 microlts of it. 
>> 
>> 
>> Another problem is that the protein is  notoriously  prone to  aggregation 
>> (> 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt 
>> concentration has failed miserably. 
>> 
>> Please do send your feedback.
>> 
>> Thanks and Best Wishes
>> 
>> 
>> Andy
>> 
>> 
>> 
>> 
>> Dr. Anindito Sen (Ph.D)
>> Department of Cell Biology & Anatomy
>> Graduate School of Medicine
>> University of Tokyo
>> Tel & fax: +81-3-5841-3339
>> 
> 
> 
> -- 
> Dr Fiona Whelan
> Potts Group, L1
> Department of Biology
> The University of York
> 
> Phone: +44 (0) 1904 328673

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Schnicker, Nicholas J]

It’s a pain but I usually just make each pH of whatever buffer I’m using (if 
you make it concentrated then you’ll only have to do it once).  Also, if you 
haven’t already found it, Hampton has a nice link to calculate volume of 
components while designing a tray as long as you tell it the concentrations.

http://hamptonresearch.com/make_tray.aspx

Nick

From: Roger Rowlett mailto:rrowl...@colgate.edu>>
Reply-To: Roger Rowlett mailto:rrowl...@colgate.edu>>
Date: Thursday, January 30, 2014 at 7:23 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5


The easiest way to produce repeatable conditions is to titrate a stock solution 
(say 1M) of citric acid with NaOH to the desired pH and use that to mix your 
screen. That's what Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers. The pH 
won't be linear with mixing ratio, but will be easily repeatable. The actual pH 
of the final, magic solution can be directly measured if desired. Calculations 
will never be exactly right; pKa values are ionic strength dependent. Better to 
measure.

Roger Rowlett

On Jan 30, 2014 2:37 AM, "sreetama das" 
mailto:somon_...@yahoo.co.in>> wrote:
Dear All,
   We have obtained many tiny protein crystals in a condition 
containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too 
small for mounting in loops.

   We intend to vary the salt concentration & pH to obtain larger 
crystals.

   Could anyone direct us to some links, or provide us with a method 
(with calculations) to calculate the amounts of citric acid & trisodium citrate 
required to obtain buffers in a range of pH 3 - 6.5?
   I have come across online buffer calculators and links where the 
amounts of the components required are mentioned in grams, but none explaining 
how those values were arrived at.

Thanks & regards,
sreetama

Re: [ccp4bb] Protein Purification Problem

[Roger Rowlett]

Do you really need to remove the NaCl? Some ionic strength is often 
necessary to stabilize proteins. Our routine purification buffers all 
contain at least 100 mM NaCl. This will not usually interfere with 
crystallization screening.


To minimize the probability of aggregation, you need to (1) ensure that 
the buffer pH is not close to the pI--pH 8.00 is a safe choice for many 
proteins, (2) probably maintain 100-150 mM ionic strength, and (3) 
consider solubility-increasing additives, like glycerol. However, 
solubility additives are going to seriously interfere with 
crystallization, but sometimes you have no choice.


Bear in mind that centrifugal concentrators may bind protein. This may 
become noticeable when using small absolute amounts of protein. Usually 
PES (polyethylenesulfone) concentrator membranes have the least protein 
adsorption.


Good luck!

___
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 1/30/2014 8:17 AM, Anindito Sen wrote:

Dear All,

This may be slightly off-the-track question but your feedback will be 
very much appreciated. The situation is-


I obtain a very low amount of the protein of my interest (a 
hetro-dimer) from the construct I am using (only 8% of the total 
amount of protein obtained is the protein of my interest).  After 2 
column purifications (Ni-NTA and St) the concentration of the protein 
is around 0.24 mg/ml (volume- ~1.0 ml)  from a litre of bacterial 
culture and in ~300 mM NaCl present in the elution  buffer.


To reduce the high amount of salt I have I use a desalting column 
which, further lowers the protein concentration significantly.


I need atleast 1.0mg/ml of protein concentration and to the amount of 
~200 microlts for further experiments.


As the last resort I try to use high amount of bacterial culture 
(~6lts) to scale up the yield and use centricon to concentrate the 
protein at various stages.  I am partially successful to obtain 
0.56mg/ml of protein concentration and up to  50 microlts of it.



Another problem is that the protein is  notoriously  prone to 
 aggregation (> 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce 
the high salt concentration has failed miserably.


Please do send your feedback.

Thanks and Best Wishes


Andy




*Dr. Anindito Sen (Ph.D)*
*Department of Cell Biology & Anatomy*
*Graduate School of Medicine*
*University of Tokyo*
*Tel & fax: +81-3-5841-3339*

Re: [ccp4bb] Protein Purification Problem

[FOOS Nicolas]

hello Andy,

i have different questions about your problem :

Is the hetero-dimer co-expressed ? Or the two partners are express separately ?
Have you controlled "where" are you proteins after the lysis, because if you 
have many of your proteins in the pellet, it's not a good sign for the folding 
state.
If this is the case, you have different solution : change the growing 
conditions, change the buffer for the lysis step...
If you have a low amount of protein in the first step of your purification 
process, you have to control at each step which one is the more limiting. 
Because, it's maybe not really necessary to do two affinity column. You can try 
different condition for the purification and increase the separation. This may 
allow you to skip one step and go directly to the size exclusion column.
Sometimes the problems with the desalting column is the high speed of the 
variation. This high speed is potential source of osmotic stress for the 
protein.
Need you really to reduce the amount of salt ?
If it's absolutely required, you can try to do by dialysis, but by multiple 
steps. 
Another question, as soon as you can, control the good state of your protein ( 
for example with Circular dichroisme or DLS).

Hope to help you.

Nicolas




De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Anindito Sen 
[andysen.to...@gmail.com]
Envoyé : jeudi 30 janvier 2014 14:17
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] Protein Purification Problem

Dear All,

This may be slightly off-the-track question but your feedback will be very much 
appreciated. The situation is-

I obtain a very low amount of the protein of my interest (a hetro-dimer) from 
the construct I am using (only 8% of the total amount of protein obtained is 
the protein of my interest).  After 2 column purifications (Ni-NTA and St) the 
concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml)  from a 
litre of bacterial culture and in ~300 mM NaCl present in the elution  buffer.

To reduce the high amount of salt I have I use a desalting column which, 
further lowers the protein concentration significantly.

I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 
microlts for further experiments.

As the last resort I try to use high amount of bacterial culture (~6lts) to 
scale up the yield and use centricon to concentrate the protein at various 
stages.  I am partially successful to obtain 0.56mg/ml of protein concentration 
and up to  50 microlts of it.


Another problem is that the protein is  notoriously  prone to  aggregation (> 
1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration 
has failed miserably.

Please do send your feedback.

Thanks and Best Wishes


Andy




Dr. Anindito Sen (Ph.D)
Department of Cell Biology & Anatomy
Graduate School of Medicine
University of Tokyo
Tel & fax: +81-3-5841-3339

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Roger Rowlett]

The easiest way to produce repeatable conditions is to titrate a stock
solution (say 1M) of citric acid with NaOH to the desired pH and use that
to mix your screen. That's what Hampton does anyway.

If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
The pH won't be linear with mixing ratio, but will be easily repeatable.
The actual pH of the final, magic solution can be directly measured if
desired. Calculations will never be exactly right; pKa values are ionic
strength dependent. Better to measure.

Roger Rowlett
On Jan 30, 2014 2:37 AM, "sreetama das"  wrote:

> Dear All,
>We have obtained many tiny protein crystals in a condition
> containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are
> too small for mounting in loops.
>
>We intend to vary the salt concentration & pH to obtain larger
> crystals.
>
>Could anyone direct us to some links, or provide us with a
> method (with calculations) to calculate the amounts of citric acid &
> trisodium citrate required to obtain buffers in a range of pH 3 - 6.5?
>I have come across online buffer calculators and links where
> the amounts of the components required are mentioned in grams, but none
> explaining how those values were arrived at.
>
> Thanks & regards,
> sreetama
>

[ccp4bb] Protein Purification Problem

[Anindito Sen]

Dear All,

This may be slightly off-the-track question but your feedback will be very much 
appreciated. The situation is-

I obtain a very low amount of the protein of my interest (a hetro-dimer) from 
the construct I am using (only 8% of the total amount of protein obtained is 
the protein of my interest).  After 2 column purifications (Ni-NTA and St) the 
concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml)  from a 
litre of bacterial culture and in ~300 mM NaCl present in the elution  buffer. 

To reduce the high amount of salt I have I use a desalting column which, 
further lowers the protein concentration significantly. 

I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 
microlts for further experiments. 

As the last resort I try to use high amount of bacterial culture (~6lts) to 
scale up the yield and use centricon to concentrate the protein at various 
stages.  I am partially successful to obtain 0.56mg/ml of protein concentration 
and up to  50 microlts of it. 


Another problem is that the protein is  notoriously  prone to  aggregation (> 
1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration 
has failed miserably. 

Please do send your feedback.

Thanks and Best Wishes


Andy




Dr. Anindito Sen (Ph.D)
Department of Cell Biology & Anatomy
Graduate School of Medicine
University of Tokyo
Tel & fax: +81-3-5841-3339

Re: [ccp4bb] R factor from merged data

[Eleanor Dodson]

I am afraid there is no real solution except to read the paper!
And even that doesnt help if you suspect the merging..
There is lots of discussion about the merit of depositing UNMERGED
data - and that would certainly help developers. Add your voice to the
request!

Eleanor

On 30 January 2014 08:26, Frank von Delft  wrote:
> I think the name says it all:
> "Rmerge"
> "merged data"
> So no, there wouldn't be.  You'll find it in the header.
>
> On 30/01/2014 08:18, Fulvio Saccoccia wrote:
>
> Dear ccp4 users,
> does anyone know if there is a way to (re)calculate the Rmerge from a
> deposited .cif file in PDB?
> In this case is an already merged structure factor file.
>
> I know that the EDS from Uppsala makes it, providing the PDB entry, but I
> need a procedure in order to do it by myself.
> I see that ccp4i utilities only accept unmerged .mtz; have you got any
> advice?
> Thanks in advance
>
> Fulvio
> --
> Fulvio Saccoccia, PhD
> Dept. of Biochemical Sciences
> Sapienza University of Rome
> 00185, Rome (Italy)
> Phone: +39 06 4991 0556
>
>

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Eleanor Dodson]

Insulin can take very different structures for 20% of the residues in
different conditions, and there are several examples of "T3R3"
hexamers, with one "T" and one "R" in the asymmetric unit. There are
even observations of the transformation occurring within the crystal,
which looked battered but still diffracted to some extent. Examples
are: 2tci  3mth  1mpj

Eleanor Dodson



On 30 January 2014 11:06, Bernhard Rupp  wrote:
> There is one additional point perhaps worth making: As already noted in the
> thread, if you have a NCS homo-oligomer, the different copies in general
> have different environment, and proper inspection of the contacts reveals
> the details. On multiple occasions during inspections and review I have
> noticed that such is not always interpreted or welcome as a functionally
> necessary manifestation of the plasticity of the protein in question. In
> case of binding sites, in contrast it occurs that the 'best' one where a
> ligand actually exists or assumes a pose/environment perceived as useful for
> the proposed hypothesis is picked as the representative (figure), and from
> there the story evolves. This misses the point, and somewhat reeks of
> confirmation bias (the neglect of negative or 'unsuitable' results) leading
> straight down the road to scientific serfdom in terms of becoming a slave of
> one's own (pre)conceptions
>
> Best regards, BR
>
>
> On Wed, Jan 29, 2014 at 10:24 AM, Kay Diederichs
>  wrote:
>>
>> Hi Shane,
>>
>> some crystal forms of trimeric AcrB (a multi-drug resistance secondary
>> transporter) have 3 (or 6) monomers in the ASU and these are substantially
>> different, which suggests how the protein functions.
>> One reference is e.g.  Seeger et al. (2006) "Structural Asymmetry of AcrB
>> Trimer Suggests a Peristaltic Pump Mechanism " Science 313, 1295-1298
>> DOI: 10.1126/science.1131542 (sorry for the self-plug!)
>>
>> best,
>>
>> Kay
>>
>>
>>
>> On Mon, 27 Jan 2014 13:08:33 -0500, Shane Caldwell
>>  wrote:
>>
>> >Hi ccp4bb,
>> >
>> >I'm putting together a talk for some peers that highlights strengths and
>> >weaknesses of structural models for the outsider. For one point, I'd like
>> >to find some examples of proteins that show very different conformations
>> >between different copies in the ASU. One example I know of is c-Abl
>> > (1OPL),
>> >which crystallizes with both autoinhibited and active forms in the ASU,
>> >with dramatically different domain organization. I'd like to find some
>> >additional examples - can anyone suggest some other structures that have
>> >multiple copies with large structural variations?
>> >
>> >Thanks in advance!
>> >
>> >Shane Caldwell
>> >McGill University
>> >
>
>
>
>
> --
> -
> Bernhard Rupp (Hofkristallrat a. D)
> 001 (925) 209-7429
> +43 (676) 571-0536
> b...@ruppweb.org
> hofkristall...@gmail.com
> http://www.ruppweb.org/
> -
> The hard part about playing chicken
> is to know when to flinch
> -

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Bernhard Rupp]

There is one additional point perhaps worth making: As already noted in the
thread, if you have a NCS homo-oligomer, the different copies in general
have different environment, and proper inspection of the contacts reveals
the details. On multiple occasions during inspections and review I have
noticed that such is not always interpreted or welcome as a functionally
necessary manifestation of the plasticity of the protein in question. In
case of binding sites, in contrast it occurs that the 'best' one where a
ligand actually exists or assumes a pose/environment perceived as useful
for the proposed hypothesis is picked as the representative (figure), and
from there the story evolves. This misses the point, and somewhat reeks of
confirmation bias (the neglect of negative or 'unsuitable' results) leading
straight down the road to scientific serfdom in terms of becoming a slave
of one's own (pre)conceptions

Best regards, BR


On Wed, Jan 29, 2014 at 10:24 AM, Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Hi Shane,
>
> some crystal forms of trimeric AcrB (a multi-drug resistance secondary
> transporter) have 3 (or 6) monomers in the ASU and these are substantially
> different, which suggests how the protein functions.
> One reference is e.g.  Seeger et al. (2006) "Structural Asymmetry of AcrB
> Trimer Suggests a Peristaltic Pump Mechanism " Science 313, 1295-1298
> DOI: 10.1126/science.1131542 (sorry for the self-plug!)
>
> best,
>
> Kay
>
>
>
> On Mon, 27 Jan 2014 13:08:33 -0500, Shane Caldwell <
> shane.caldwel...@gmail.com> wrote:
>
> >Hi ccp4bb,
> >
> >I'm putting together a talk for some peers that highlights strengths and
> >weaknesses of structural models for the outsider. For one point, I'd like
> >to find some examples of proteins that show very different conformations
> >between different copies in the ASU. One example I know of is c-Abl
> (1OPL),
> >which crystallizes with both autoinhibited and active forms in the ASU,
> >with dramatically different domain organization. I'd like to find some
> >additional examples - can anyone suggest some other structures that have
> >multiple copies with large structural variations?
> >
> >Thanks in advance!
> >
> >Shane Caldwell
> >McGill University
> >
>



-- 
-
Bernhard Rupp (Hofkristallrat a. D)
001 (925) 209-7429
+43 (676) 571-0536
b...@ruppweb.org
hofkristall...@gmail.com
http://www.ruppweb.org/
-
The hard part about playing chicken
is to know when to flinch
-

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Markus Wiederstein]

Hi Shane,

another curious case is the crystal structure of a signal receiver domain of 
Desulfovibrio desulfuricans (PDB code: 3cg0, Patskovsky et al., to be 
published), as discussed in Sippl (2009) Curr. Opin. Struct. Biol.

Best,
-Markus

> Hi ccp4bb,
> 
> I'm putting together a talk for some peers that highlights strengths and 
> weaknesses of structural models for the outsider. For one point, I'd like to 
> find some examples of proteins that show very different conformations between 
> different copies in the ASU. One example I know of is c-Abl (1OPL), which 
> crystallizes with both autoinhibited and active forms in the ASU, with 
> dramatically different domain organization. I'd like to find some additional 
> examples - can anyone suggest some other structures that have multiple copies 
> with large structural variations?
> 
> Thanks in advance!
> 
> Shane Caldwell
> McGill University
> 
> 

Re: [ccp4bb] Scripting error running Phaser_EP/HYSS?

[Randy Read]

Dear Roger,

The Phaser developers contribute Phaser to both CCP4 and Phenix, so it's better 
to contact us directly rather than, say, going through the phenix help, 
especially if it's a CCP4 specific issue.

The syntax of the call to HySS in the Phaser SAD phasing pipeline hasn't 
changed for at least two years and it used to work, so someone working on HySS 
must recently have tidied up the syntax (which could be more consistent 
throughout Phenix in the use of hyphens and underscores), without realising 
that it would break the SAD phasing pipeline in ccp4i.  The documentation on 
the Phenix website is actually inconsistent on this point, with one page 
(http://www.phenix-online.org/download/documentation/cci_apps/hyss/examples.html)
 still giving the syntax with hyphens.

We'll fix the syntax of the HySS call for the next CCP4 update.  In the 
meantime, you can choose the SHELXC/D option to find sites or run HySS 
separately and provide the substructure PDB file to the pipeline.

Thanks for reporting the problem!

Best wishes,

Randy Read

On 29 Jan 2014, at 17:33, Roger Rowlett  wrote:

> Is there a scripting error in 
> ccp4-6.4.0/share/ccp4i/scripts/phaser_EP.script? CCP4i jobs fail with an 
> argument format error, e.g.,
> 
> Error interpreting command line argument as a parameter definition: 
> "data-label=F_whatever(+)"
> Improper definition name "data-label"
> 
> I think phenix requires underscores, not hyphens, e.g. "data_label", 
> "pdb_only" etc. Even the phenix documentation page is conflicted, showing 
> both "data-label" and "data_label". Maybe is isn't supposed to matter, but in 
> Phenix 1.8.4, it apparently does matter.
> 
> Is this something that could be fixed in the next CCP4 update? Or should I 
> direct this to the phenix developers?
> 
> Cheers,
> 
> ___
> 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

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] R factor from merged data

[Frank von Delft]

I think the name says it all:
"Rmerge"
"merged data"
So no, there wouldn't be.  You'll find it in the header.

On 30/01/2014 08:18, Fulvio Saccoccia wrote:

Dear ccp4 users,
does anyone know if there is a way to (re)calculate the Rmerge from a 
deposited .cif file in PDB?

In this case is an already merged structure factor file.

I know that the EDS from Uppsala makes it, providing the PDB entry, 
but I need a procedure in order to do it by myself.
I see that ccp4i utilities only accept unmerged .mtz; have you got any 
advice?

Thanks in advance

Fulvio
--
Fulvio Saccoccia, PhD
Dept. of Biochemical Sciences
Sapienza University of Rome
00185, Rome (Italy)
Phone: +39 06 4991 0556

[ccp4bb] R factor from merged data

[Fulvio Saccoccia]

Dear ccp4 users,
does anyone know if there is a way to (re)calculate the Rmerge from a
deposited .cif file in PDB?
 In this case is an already merged structure factor file.

I know that the EDS from Uppsala makes it, providing the PDB entry, but I
need a procedure in order to do it by myself.
I see that ccp4i utilities only accept unmerged .mtz; have you got any
advice?
Thanks in advance

Fulvio
-- 
Fulvio Saccoccia, PhD
Dept. of Biochemical Sciences
Sapienza University of Rome
00185, Rome (Italy)
Phone: +39 06 4991 0556