Re: [ccp4bb] FW: endotoxin removal

[Taylor, Jonathan D]

Dear Jerry,

I've just become aware of this spin column to remove endotoxin, marketed by 
Generon, UK.

http://generon.co.uk/index.php?route=product/category&path=238

Good luck,
Jon


On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 wrote:
>
>
> Dear All;
>
> We purified a His tag protein by Ni-NTA and gel-filtration from
> E.coli.
>
>  We tried two endotoxin removal resins from Pierce. However, it is
> hard to remove the endotoxins in the purified protein because the protein
> bound to the resin as well.
>
>  This protein contains quite a few aromatic residues and has a pI
> around 6.
>
>  Any ideas to remove the endotoxins will be highly appreciated.
>
> best regards,
>
> Jerry McCully
>
>

Re: [ccp4bb] Directories & Projects Spring Cleaning

[Enrico Stura]

Mark,

I am faced with the same problem. I work under Linux.
Since everything depends from the .CCP4 directory,
 this directory  can be copied to
a storage location. Example: /oldproj/dotCCP4_2011:
/home/user % cp -r .CCP4   /oldproj/dotCCP4_2011

Once this directory has been effectively backed up
all old projects can be deleted in the ccp4i interface.
The Spring cleaned .CCP4 can be moved
to a new location for example: /projects/dotCCP4_2012
/home/user % mv .CCP4  /projects/dotCCP4_2012
A logical link is established with:
/home/user % ln -s /projects/dotCCP4_2012 .CCP4
The link will point to the new directory. This new directory
will be filled up during 2012 and at the end of the year
backed up in the same way.

To re-use old environments:
/home/user % ln -s /oldproj/dotCCP4_2011 .CCP4
Will allow to return to the 2011 status.

As long as nothing is deleted, nothing is lost.
Caution: Transfered directories may need logical links to ensure that
connectivity to the directories is maintained.

This is not very elegant and I do this by hand since I am not aware of any
script that does it automatically.
Probably in a future version of ccp4i there will be an archive function.
I am sure that many other users would wlecome it too.


Enrico.

On Wed, 07 Mar 2012 19:55:18 +0100, mark Mayer  wrote:

I'd appreciate suggestions about how to reorganize my My  
Directories&ProjectDir listing.
Its currently got several years worth of work and is getting hard to  
work with.
I'd like to archive old projects (say by year) but keep all the CCP4i  
files for each project intact, so that I can easily go back to them in  
the future without having to wade through the current multi year list.


Thanks



--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] setting occupancy for disordered water

[arka chakraborty]

Hi all,

I apologize for being late in expressing my gratitude. Thanks to Prof. Tim
and Prof. Herman, I have fixed the waters and validated the structure.

Cheers,

ARKO

On Wed, Mar 7, 2012 at 8:37 PM, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear ARKO,
>
> you can edit the PDB file by doubling the line of the respective water,
> assign the the "Alternate location indicator" (column 17) to 'A' and 'B'
> (see e.g. http://www.wwpdb.org/documentation/format33/sect9.html#ATOM),
> and change the coordinates from 'B' to the second peak.
>
> Make sure that the water is not near a symmetry axis - there are often
> artefact of density near symmetry elements.
>
> Tim
>
> On 03/07/2012 02:20 PM, arka chakraborty wrote:
> > Hi all,
> >
> > I am having a structure where there is a disordered water .I have fixed
> one
> > water into the density, yet there is a positive density about 1.6 ang
> away.
> > I want to put in another water and attribute 0.50 occupancy to each of
> the
> > the waters while attributing them to  one water in reality. I would like
> to
> > know how to modify the pdb so that phenix.refine accepts it.
> >
> > Thanks in advance,
> >
> > Regards,
> >
> > ARKO
> >
>
> - --
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.10 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
>
> iD8DBQFPV3nDUxlJ7aRr7hoRAhexAKDjXUiCo3NfZBOQF9CICMA+WeJ6WgCgsmyO
> IVxnxkZNnSxXiIF2LNdr8AU=
> =E2g7
> -END PGP SIGNATURE-
>



-- 

*ARKA CHAKRABORTY*
*CAS in Crystallography and Biophysics*
*University of Madras*
*Chennai,India*

[ccp4bb] Professor Dame Louise Johnson FRS

[Elspeth Garman]

Friends and colleagues of Professor Dame Louise Johnson will be very saddened 
to learn that she suffered a serious and very incapacitating heart attack with 
complications in August 2011 and has been in hospital since then. Louise is 
visited daily by members of her family, and, starting very recently, by a small 
number of friends. Cards and messages can be sent to Louise c/o Department of 
Biochemistry, South Parks Road, Oxford, OX1 3QU, from where they will be passed 
on to the family and communicated to Louise.

Please do not reply to this message or email Louise directly. As and when there 
is further news I will let everyone know via the BB.


  Professor Elspeth F. Garman,
  President, British Crystallographic Association
  Director of Systems Biology Programme, Doctoral Training Centre.
Postal address:
  Laboratory of Molecular Biophysics,
  Department of Biochemistry,
  University of Oxford,  Tel: (44)-1865-613297
  South Parks Road,  FAX: (44)-1865-613201
  OXFORD, OX1 3QU, U.K. E-mail: 
elspeth.gar...@bioch.ox.ac.uk
 http://lmb.bioch.ox.ac.uk/www/garman/gindex.html
 -
The University of Oxford's Doctoral Training Centre has three available 
programmes for October 2012 entry: Life Sciences Interface Doctoral Training 
Centre; Systems Biology Doctoral Training Centre; and Systems Approaches to 
Biomedical Science Industrial Doctorate Centre.

To find out more, visit: http://www.dtc.ox.ac.uk/

Re: [ccp4bb] REFMAC Riding Hydrogens

[Martyn Winn]

I have changed the GUI default to be MAKE HYDROGEN ALL. This should be
in the next release.

In any case, the option is in the folder Refinement Parameters, so you
can check.

m

On Wed, 2012-03-07 at 23:45 +, Garib N Murshudov wrote:
> Hi
> 
> 
> 1) refmac's default option has been "generate all" starting from
> version 5 (it is around 14 years). The reason is for most tests
> generating hydrogens gave good R or geometry. And if you look at the
> original paper by Ramachandran (I think it was pulished in 1963) you
> see using hard ball approximation and including all atoms generated
> plot is very similar to what we know as Ramachandran plot. A little
> bit more work on included hydrogen plus phi/psi torsion angle
> restraints (in free form, not from Ramachandran plot) should generate
> faithfully Ramchandran plot. It needs to be tested properly and
> carefully. refmac version 4 did not have hydrogen generation option.
> 2) ccp4i's option has always been use hydrogens if present in file (I
> personally do not like this option: this option should be used with
> care)
> 3) If there is no non-bondedn ... others then it is very likely that
> hydrogens were not used. 
> 4) I am not sure that there is a fullproof way to know if hydrogens
> were added or not.
> 
> 
> Regards
> Garib
> 
> 
> 
> On 7 Mar 2012, at 15:00, Paul Smith wrote:
> 
> > Hello CCP4bb,
> > 
> > 
> > Firstly, thanks to all for your comments.  However, I'm still unsure
> > how to sort all of this riding hydrogen business out.
> > 
> > 
> > Robbie's comments seem particularly apt:
> > 
> > 
> > "Because there were some reporting errors in the past
> > (http://proteincrystallography.org/ccp4bb/message18808.html) it is
> > hard to tell from the PDB when refinement with hydrogens became
> > hip."
> > 
> > 
> > Is there any foolproof way to know if a recently deposited file was
> > refined with riding hydrogens in REFMAC, especially since some such
> > structures do not yet have publications associated with them? How
> > about the value of NON-BONDED CONTACTS OTHERS? If that is other than
> > NULL, does that mean riding hydrogens were present?
> > 
> > 
> > Also, how about refmac version numbers?  Is there a clear
> > demarcation when riding hydrogens a) became available, b) became
> > default, or c) became default in the CCP4i GUI?
> > 
> > 
> > Thanks again,
> > 
> > 
> > --Paul
> > 
> > 
> > 
> > 
> > 
> > From: Robbie Joosten 
> > To: CCP4BB@JISCMAIL.AC.UK 
> > Sent: Tuesday, March 6, 2012 4:26 AM
> > Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
> > 
> > 
> > Hi Everyone, 
> >  
> > Pavel’s statement is likely a bit of an exaggeration, but he has a
> > valid (yet hard to prove point). The default in CCP4i was (and is?)
> > to use hydrogens only if present in the input file. This is IMO not
> > a safe default. 
> > Because there were some reporting errors in the past
> > (http://proteincrystallography.org/ccp4bb/message18808.html) it is
> > hard to tell from the PDB when refinement with hydrogens became hip.
> > Discussions on this BB show that at the use of riding hydrogens is
> > still not fully accepted, especially at low resolution (where they
> > actually help most). 
> >  
> > Cheers,
> > Robbie
> >  
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf
> > Of Pavel Afonine
> > Sent: Monday, March 05, 2012 21:53
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] REFMAC Riding Hydrogens
> >  
> > Dear Tim,
> >  
> > good catch, thanks; I could craft that phrase more carefully!
> > Although often it may not be quite fair to take phrases out of
> > context: this newsletter article was written in the context of
> > macromolecular refinement. And yes, "recently" may be a broad term
> > -:)
> >  
> > All the best,
> > Pavel
> > On Mon, Mar 5, 2012 at 12:45 PM, Tim Gruene
> >  wrote:
> > -BEGIN PGP SIGNED MESSAGE-
> > Hash: SHA1
> > 
> > Dear Pavel,
> > 
> > you may want to add to the structures mentioned in [1] one or two
> > organic structures present in the Cambridge Database.
> > 
> > "Until recently it was customary to ignore hydrogen atoms throughout
> > the
> > process of crystallographic X-­‐ray structure determination." [1]
> > 
> > 'recently' as in 1997 [2]? Even though 1997 is probably a poor
> > estimation of the corresponding year...
> > 
> > Cheers,
> > Tim
> > 
> > 
> > [1] "On contribution of hydrogen atoms to X-ray scattering"
> > http://www.phenix-online.org/newsletter/
> > [2] http://shelx.uni-ac.gwdg.de/SHELX/shelx.pdf
> > 
> > On 03/05/2012 09:14 PM, Pavel Afonine wrote:
> > > Hi,
> > >
> > > On Mon, Mar 5, 2012 at 11:52 AM, Matthew Franklin
> > wrote:
> > >
> > >> Adding the riding hydrogens generally gives you some improvement
> > in R
> > >> factors even with a good quality (i.e. stereochemically correct)
> > model.
> > >>
> > >
> > > and here are the results of more or less systematic test that
> > prove this:
> > >
> >

Re: [ccp4bb] Water

[Uma Ratu]

Dear All:

I appreciate for your comments and inputs.

Thank you

Uma

On Thu, Mar 8, 2012 at 12:16 AM, Shekhar Mande wrote:

> Welljust to add, it has been our contention that many of the metal
> ions have been modelled as waters in several structures- due perhaps to the
> lack of sufficiently high resolution data.  We published some of the
> potential metal binding sites in many structures a few years ago:
>
> Proteins. 2008 Mar;70(4):1206-18.
>
> Shekhar
>
>
> On Thu, Mar 8, 2012 at 9:42 AM, Parthasarathy Sampathkumar <
> spart...@gmail.com> wrote:
>
>> Dear Uma,
>>
>> The water pictured in W12-1.jpg: could this be a potential metal ion? If
>> you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4
>> coordination with oxygen atoms. So, provided your crystallization condition
>> or buffer contains metal ion(s), you could attempt to see if it fits better
>> with a refinement cycle.
>>
>> May be a similar situation with the water described in W11-1.jpg as well?
>> Difficult to say from these figures.
>>
>> COOT within the "validate" wizard has an option to search for
>> "hihgly-coordinated waters" like the one you have pictured.
>>
>> Hope this helps,
>> Partha
>>
>> On Wed, Mar 7, 2012 at 4:21 PM, Uma Ratu  wrote:
>>
>>> Dear Roger:
>>>
>>> Thank you very much for your comments. I use them as guideline and
>>> remove many 'false waters".
>>>
>>> Still, I am not clear of some of these 'waters' are real or not. I have
>>> the pic attached.
>>>
>>> In Pic-W11-1, the 'water' is connected to the adjust residues with 4
>>> contacts, which are 'N' or 'O' atoms. I would consider this 'water' is
>>> false. My question is: if these 4 contacts include "C" from residues, will
>>> it be a polar contact or not?
>>>
>>> In Pic-W12-1, the 'water' is connected to the adjust residues with 3
>>> contacts. The 4th is to another 'water'.
>>> Will this 'water' is true or not? Similar case is seen in Pic-W190-1
>>>
>>> In Pic-W109-1, some 'waters' are connected to adjust residues, some not.
>>> Are these 'water' true or not?
>>>
>>> Further more,
>>>  > and the b-factors are not way out of line,
>>>
>>> I am not clear on how to define "out of line".
>>> How to find b-factor of individual residue in Coot? I search the web,
>>> but find no answer.
>>>
>>> Thank you for advice
>>>
>>> Uma
>>>
>>>  On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett wrote:
>>>
 Uma,

 Remember that your structure, ultimately, is a model. A model is your
 best judgment of the true representation of the protein structure in your
 crystal. Your model should make chemical sense. Coot is pretty good at
 placing waters, but it cannot substitute entirely for the experimentalist.
 Coot will miss some waters, and mis-assign others into weak, unmodeled or
 alternate side- or main-chain density, or into density that might be
 attributable to cations and anions or other crystallization materials. Your
 waters should be subjected to inspection and verification. It is really
 helpful to turn on environment distances in Coot when you do this. Even in
 a large protein model, it is possible to inspect all waters for
 reasonableness pretty quickly. If you have no significant positive or
 negative difference density, and the b-factors are not way out of line, and
 hydrogen bonding partners are reasonable, then modeling a water is probably
 a good call.

 Waters should have hydrogen bonding partners with side chains or
 main-chain polar atoms, within reasonable distances, or be withing hydrogen
 bonding distance of other waters that are (chains of waters). If a "water"
 has strong electron density and more than 4 polar contacts, you might
 consider anion or cation occupancy. Most anions and cations will have
 higher electron density, and appropriately different types of polar
 contacts. (e.g. you might find sulfates near a cluster of basic residues).
 Low occupancy anions can often look a lot like water. PEGs can create ugly
 "snakes" of variable density that may be challenging to model. Modeling
 non-protein structural bits is endlessly entertaining for the protein
 crystallographer. ;)

 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 3/7/2012 11:20 AM, Uma Ratu wrote:

 Dear All:

 I try to add water to my model.

 Here is how I did:
 Coot: Find Wates
  Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms:
 2.4 min/3.2 max

 Coot found 270 water molecules.

 I then examed these waters. Most of them had ball shape. Some had two
 or more balls together. Some had irregular sha

[ccp4bb] xds - problem with reference profile

[Florian Schmitzberger]

Dear All,

I am getting a warning message in XDS I have not seen before, when  
trying to integrate a low resolution (~ 7 A) dataset.


!!! WARNING !!! REFERENCE PROFILE #  1 IS EMPTY.
 THE AVERAGE PROFILE IN THIS BATCH IS USED INSTEAD.

and so forth for the other reference profiles.

Indexing works alright with XDS; with likely point group C2. Scaling  
the data integrated with XDS, with Scala fails; apparently most of the  
reflections are rejected.


What are probable causes for the above error message?

Cheers,

Florian

Re: [ccp4bb] xds - problem with reference profile

[Herman . Schreuder]

Dear Florian,
 
Could it be that you only have spots in the center of your image and that XDS 
is trying to process a lot of image area with only background in it? In this 
case it will try to build profiles from non-existing reflections and start to 
complain. The resulting bad data may cause scala to crash.
Look in the XDS.INP file for an INCLUDE_RESOLUTION_RANGE keyword and set it to 
some sensible value with a high resolution cut-off of say 5 or 6 Å. This should 
solve your problem.
 
Best,
HErman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Florian Schmitzberger
Sent: Thursday, March 08, 2012 3:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] xds - problem with reference profile






Dear All,

I am getting a warning message in XDS I have not seen before, when 
trying to integrate a low resolution (~ 7 A) dataset. 

!!! WARNING !!! REFERENCE PROFILE #  1 IS EMPTY.
 THE AVERAGE PROFILE IN THIS BATCH IS USED INSTEAD.

and so forth for the other reference profiles. 

Indexing works alright with XDS; with likely point group C2. Scaling 
the data integrated with XDS, with Scala fails; apparently most of the 
reflections are rejected. 

What are probable causes for the above error message?

Cheers,

Florian

Re: [ccp4bb] FW: endotoxin removal

[Pius Padayatti]

This is in response to a comment to this thread
Kevin,
Could you explain how that worked?
How do you know your method worked?
Did you estimate the lipopolysaccharide before and after the method?

The method already mentioned here to wash using TritonX100 makes sense.
by washing bound protein sample May able to phase separate into
detergent micellar phase and get rid of endotoxins.

Most widely accepted method to separate is by two phase micellar system.
above the CMC endotoxins will be accomodated by micelles through non-polar
interactions of alkyl chains of lipidA.

Padayatti PS



On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
 wrote:
>o
>
> Dear All;
>
>     We purified a His tag protein by Ni-NTA and gel-filtration from
> E.coli.
>
>  We tried two endotoxin removal resins from Pierce. However, it is
> hard to remove the endotoxins in the purified protein because the protein
> bound to the resin as well.
>
>  This protein contains quite a few aromatic residues and has a pI
> around 6.
>
>  Any ideas to remove the endotoxins will be highly appreciated.
>
>     best regards,
>
> Jerry McCully
>
>



-- 
Pius S Padayatti,PhD,
Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

To Pius,

In my case, the protein/biopolymer is Lysine/amine riched.

1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
then provide positively charge to LPA. Of course, some metal cations
could also be involved in LPA binding.

2. Tris with the NH2 group could also be protonated and positively
charged under the same pH condition. This is why I use high
concentration Tris here.

3. Tris buffer could function as an ionic-exchange competitor for LPA
binding. If you look those commercial available Endotoxin assay kits,
they also use Tris buffer.

4. IPA could change the charge distribution on the surface of protein.
In usual, high% IPA could remove LPA efficiently but may cause protein
denature. In lot of cases, 5~10% IPA could help protein
crystallization to a larger size. As a tradeoff, it may also bring
down the packing quality.

In this case, I used it for endotoxin  removal.

5. NaCl (you can try other salt) would provide additional ion-strength
to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.

6. If you can add EDTA to remove metal cation, it may be even better.
In my case, I could not use EDTA.

I tested the sample (1600EU/ml). After 3 days dialysis, it went down
to < 5EU/ml.  The assay kit for LPA measurement was from Charles River
(?). The procedure is too long to present here. The good thing is that
your sample would not be dehydrated and refold again.

I am saying this will work for protein in all of case, but you try in
an eppendorf tube like amini-prep.

I guess this method may have been  patented.






















On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti  wrote:
> This is in response to a comment to this thread
> Kevin,
> Could you explain how that worked?
> How do you know your method worked?
> Did you estimate the lipopolysaccharide before and after the method?
>
> The method already mentioned here to wash using TritonX100 makes sense.
> by washing bound protein sample May able to phase separate into
> detergent micellar phase and get rid of endotoxins.
>
> Most widely accepted method to separate is by two phase micellar system.
> above the CMC endotoxins will be accomodated by micelles through non-polar
> interactions of alkyl chains of lipidA.
>
> Padayatti PS
>
>
>
> On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
>  wrote:
>>o
>>
>> Dear All;
>>
>>     We purified a His tag protein by Ni-NTA and gel-filtration from
>> E.coli.
>>
>>  We tried two endotoxin removal resins from Pierce. However, it is
>> hard to remove the endotoxins in the purified protein because the protein
>> bound to the resin as well.
>>
>>  This protein contains quite a few aromatic residues and has a pI
>> around 6.
>>
>>  Any ideas to remove the endotoxins will be highly appreciated.
>>
>>     best regards,
>>
>> Jerry McCully
>>
>>
>
>
>
> --
> Pius S Padayatti,PhD,
> Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

In my case, this method was developed for large quantity sample
(~100g/batch) purification.

Under cGMP, I could not introduce EDTA or other chemicals like
Triton-X100 into the system. Otherwise, I just solve small problem but
bring into an even large problem to the manufacture line.

I hope you can test my strategy and improve it. If you can give me a
feed back, there will be great!


Kevin

On Thu, Mar 8, 2012 at 1:01 PM, Kevin Jin  wrote:
> To Pius,
>
> In my case, the protein/biopolymer is Lysine/amine riched.
>
> 1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
> then provide positively charge to LPA. Of course, some metal cations
> could also be involved in LPA binding.
>
> 2. Tris with the NH2 group could also be protonated and positively
> charged under the same pH condition. This is why I use high
> concentration Tris here.
>
> 3. Tris buffer could function as an ionic-exchange competitor for LPA
> binding. If you look those commercial available Endotoxin assay kits,
> they also use Tris buffer.
>
> 4. IPA could change the charge distribution on the surface of protein.
> In usual, high% IPA could remove LPA efficiently but may cause protein
> denature. In lot of cases, 5~10% IPA could help protein
> crystallization to a larger size. As a tradeoff, it may also bring
> down the packing quality.
>
> In this case, I used it for endotoxin  removal.
>
> 5. NaCl (you can try other salt) would provide additional ion-strength
> to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.
>
> 6. If you can add EDTA to remove metal cation, it may be even better.
> In my case, I could not use EDTA.
>
> I tested the sample (1600EU/ml). After 3 days dialysis, it went down
> to < 5EU/ml.  The assay kit for LPA measurement was from Charles River
> (?). The procedure is too long to present here. The good thing is that
> your sample would not be dehydrated and refold again.
>
> I am saying this will work for protein in all of case, but you try in
> an eppendorf tube like amini-prep.
>
> I guess this method may have been  patented.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti  wrote:
>> This is in response to a comment to this thread
>> Kevin,
>> Could you explain how that worked?
>> How do you know your method worked?
>> Did you estimate the lipopolysaccharide before and after the method?
>>
>> The method already mentioned here to wash using TritonX100 makes sense.
>> by washing bound protein sample May able to phase separate into
>> detergent micellar phase and get rid of endotoxins.
>>
>> Most widely accepted method to separate is by two phase micellar system.
>> above the CMC endotoxins will be accomodated by micelles through non-polar
>> interactions of alkyl chains of lipidA.
>>
>> Padayatti PS
>>
>>
>>
>> On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
>>  wrote:
>>>o
>>>
>>> Dear All;
>>>
>>>     We purified a His tag protein by Ni-NTA and gel-filtration from
>>> E.coli.
>>>
>>>  We tried two endotoxin removal resins from Pierce. However, it is
>>> hard to remove the endotoxins in the purified protein because the protein
>>> bound to the resin as well.
>>>
>>>  This protein contains quite a few aromatic residues and has a pI
>>> around 6.
>>>
>>>  Any ideas to remove the endotoxins will be highly appreciated.
>>>
>>>     best regards,
>>>
>>> Jerry McCully
>>>
>>>
>>
>>
>>
>> --
>> Pius S Padayatti,PhD,
>> Phone: 216-658-4528

Re: [ccp4bb] X-ray diffraction spot

[Zhijie Li]

Was this crystal shot frozen? With new crystals it is always a good idea to 
take some room temperature shots first to get an idea of how good the 
diffraction could possibly be. If you shoot the crystals at room temperature 
and the spots look fine, then the obvious answer is to optimize the freezing. 
If this is the case, one thing often worked for me is to add a little 
cryoprotectant to the crystal growth condition. Or if you are lucky, grow the 
crystal with the full-strength cryoprotectant. This can often reduce the stress 
on the crystal cause by the soaking in the cryoprotectant solution.





From: lysudiezhang1985 
Sent: Wednesday, March 07, 2012 9:45 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] X-ray diffraction spot



Hi  Everyone, Thank you for help.
Recently I get a crystal of my protein , but have poor diffraction data.
The problem is that , just like I show you in the attachment,
many of the diffraction spots look like the comet, who has a long tail.
I wonder to know the reason for this ,
or some advice about what to do with this kind of data , how to optimize
my crystal to improve the diffraction data.


Thanks and have a good day.

Re: [ccp4bb] FW: endotoxin removal

[Pius Padayatti]

Kevin,
thanks.Sine this is of not much interest to many here
please feel free to go off line in the future.

The method of dialysis is confusing here.
How can one achieve this by buffer exchange?

I wonder if an extensive wash of protein on a column would work
instead.

Pius

On Thu, Mar 8, 2012 at 4:01 PM, Kevin Jin  wrote:
> To Pius,
>
> In my case, the protein/biopolymer is Lysine/amine riched.
>
> 1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
> then provide positively charge to LPA. Of course, some metal cations
> could also be involved in LPA binding.
>
> 2. Tris with the NH2 group could also be protonated and positively
> charged under the same pH condition. This is why I use high
> concentration Tris here.
>
> 3. Tris buffer could function as an ionic-exchange competitor for LPA
> binding. If you look those commercial available Endotoxin assay kits,
> they also use Tris buffer.
>
> 4. IPA could change the charge distribution on the surface of protein.
> In usual, high% IPA could remove LPA efficiently but may cause protein
> denature. In lot of cases, 5~10% IPA could help protein
> crystallization to a larger size. As a tradeoff, it may also bring
> down the packing quality.
>
> In this case, I used it for endotoxin  removal.
>
> 5. NaCl (you can try other salt) would provide additional ion-strength
> to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.
>
> 6. If you can add EDTA to remove metal cation, it may be even better.
> In my case, I could not use EDTA.
>
> I tested the sample (1600EU/ml). After 3 days dialysis, it went down
> to < 5EU/ml.  The assay kit for LPA measurement was from Charles River
> (?). The procedure is too long to present here. The good thing is that
> your sample would not be dehydrated and refold again.
>
> I am saying this will work for protein in all of case, but you try in
> an eppendorf tube like amini-prep.
>
> I guess this method may have been  patented.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti  wrote:
>> This is in response to a comment to this thread
>> Kevin,
>> Could you explain how that worked?
>> How do you know your method worked?
>> Did you estimate the lipopolysaccharide before and after the method?
>>
>> The method already mentioned here to wash using TritonX100 makes sense.
>> by washing bound protein sample May able to phase separate into
>> detergent micellar phase and get rid of endotoxins.
>>
>> Most widely accepted method to separate is by two phase micellar system.
>> above the CMC endotoxins will be accomodated by micelles through non-polar
>> interactions of alkyl chains of lipidA.
>>
>> Padayatti PS
>>
>>
>>
>> On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
>>  wrote:
>>>o
>>>
>>> Dear All;
>>>
>>>     We purified a His tag protein by Ni-NTA and gel-filtration from
>>> E.coli.
>>>
>>>  We tried two endotoxin removal resins from Pierce. However, it is
>>> hard to remove the endotoxins in the purified protein because the protein
>>> bound to the resin as well.
>>>
>>>  This protein contains quite a few aromatic residues and has a pI
>>> around 6.
>>>
>>>  Any ideas to remove the endotoxins will be highly appreciated.
>>>
>>>     best regards,
>>>
>>> Jerry McCully
>>>
>>>
>>
>>
>>
>> --
>> Pius S Padayatti,PhD,
>> Phone: 216-658-4528



-- 
Pius S Padayatti,PhD,
Phone: 216-658-4528

Re: [ccp4bb] FW: endotoxin removal

[Kevin Jin]

Sure, we can talk about this off line.

-- Kevin

On Thu, Mar 8, 2012 at 7:48 PM, Pius Padayatti  wrote:
> Kevin,

> thanks.Sine this is of not much interest to many here


> please feel free to go off line in the future.
>
> The method of dialysis is confusing here.
> How can one achieve this by buffer exchange?
>
> I wonder if an extensive wash of protein on a column would work
> instead.
>
> Pius
>
> On Thu, Mar 8, 2012 at 4:01 PM, Kevin Jin  wrote:
>> To Pius,
>>
>> In my case, the protein/biopolymer is Lysine/amine riched.
>>
>> 1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
>> then provide positively charge to LPA. Of course, some metal cations
>> could also be involved in LPA binding.
>>
>> 2. Tris with the NH2 group could also be protonated and positively
>> charged under the same pH condition. This is why I use high
>> concentration Tris here.
>>
>> 3. Tris buffer could function as an ionic-exchange competitor for LPA
>> binding. If you look those commercial available Endotoxin assay kits,
>> they also use Tris buffer.
>>
>> 4. IPA could change the charge distribution on the surface of protein.
>> In usual, high% IPA could remove LPA efficiently but may cause protein
>> denature. In lot of cases, 5~10% IPA could help protein
>> crystallization to a larger size. As a tradeoff, it may also bring
>> down the packing quality.
>>
>> In this case, I used it for endotoxin  removal.
>>
>> 5. NaCl (you can try other salt) would provide additional ion-strength
>> to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.
>>
>> 6. If you can add EDTA to remove metal cation, it may be even better.
>> In my case, I could not use EDTA.
>>
>> I tested the sample (1600EU/ml). After 3 days dialysis, it went down
>> to < 5EU/ml.  The assay kit for LPA measurement was from Charles River
>> (?). The procedure is too long to present here. The good thing is that
>> your sample would not be dehydrated and refold again.
>>
>> I am saying this will work for protein in all of case, but you try in
>> an eppendorf tube like amini-prep.
>>
>> I guess this method may have been  patented.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti  wrote:
>>> This is in response to a comment to this thread
>>> Kevin,
>>> Could you explain how that worked?
>>> How do you know your method worked?
>>> Did you estimate the lipopolysaccharide before and after the method?
>>>
>>> The method already mentioned here to wash using TritonX100 makes sense.
>>> by washing bound protein sample May able to phase separate into
>>> detergent micellar phase and get rid of endotoxins.
>>>
>>> Most widely accepted method to separate is by two phase micellar system.
>>> above the CMC endotoxins will be accomodated by micelles through non-polar
>>> interactions of alkyl chains of lipidA.
>>>
>>> Padayatti PS
>>>
>>>
>>>
>>> On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully
>>>  wrote:
o

 Dear All;

     We purified a His tag protein by Ni-NTA and gel-filtration from
 E.coli.

  We tried two endotoxin removal resins from Pierce. However, it is
 hard to remove the endotoxins in the purified protein because the protein
 bound to the resin as well.

  This protein contains quite a few aromatic residues and has a pI
 around 6.

  Any ideas to remove the endotoxins will be highly appreciated.

     best regards,

 Jerry McCully


>>>
>>>
>>>
>>> --
>>> Pius S Padayatti,PhD,
>>> Phone: 216-658-4528
>
>
>
> --
> Pius S Padayatti,PhD,
> Phone: 216-658-4528