[ccp4bb] recommendations_on_purification

[Petros Giastas]

Dear all,

I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, 
using the standard minimal medium described in the invitrogen manual (plus 
PTM1). Following collection of the culture medium, I am having problems with 
purification of the protein as only a small fraction (~10%) binds to the Ni-NTA 
beads even after extensive buffer exchange (when expressed in full BMGY media 
this is not observed). Could this be attributed to metal ions still present in 
my sample? Is it likely to be due to poor protein quality in this medium? Or 
any other suggestions? 

Thanks in advance
Petros

Re: [ccp4bb] recommendations_on_purification

[Lorenzo Finci]

Petros, 
It has indeed been speculated that high concentrations of Magnesium and/or 
other metals present in the cell lysate effect the binding of the 
Histidine-tag, and thus specific conditions for binding and elution need to be 
optimized for specific elution of your target protein. I believe that the 
standard recommendations when using a Nickel column to bind to your 
Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted 
unspecific binding protein, to try manipulating the binding by lowering the pH 
of the buffers, and by determining the specific concentration of . 
Alternatively, you can also try using another metal column such as Cobalt 
(Talon) with a higher affinity for the Histidine tag.Sincerely, lorenzo

Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard 
Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe 
College of Life SciencesBeijing, China


Date: Mon, 26 Mar 2012 15:04:43 +0300
From: peg...@pasteur.gr
Subject: [ccp4bb] recommendations_on_purification
To: CCP4BB@JISCMAIL.AC.UK

Dear all,

I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, 
using the standard minimal medium described in the invitrogen manual (plus 
PTM1). Following collection of the culture medium, I am having problems with 
purification of the protein as only a small fraction (~10%) binds to the Ni-NTA 
beads even after extensive buffer exchange (when expressed in full BMGY media 
this is not observed). Could this be attributed to metal ions still present in 
my sample? Is it likely to be due to poor protein quality in this medium? Or 
any other suggestions?

Thanks in advance
Petros

Re: [ccp4bb] recommendations_on_purification

[Lorenzo Finci]

Etros, It has indeed been speculated that high concentrations of Magnesium 
and/or other metals present in the cell lysate effect the binding of the 
Histidine-tag, and thus specific conditions for binding and elution need to be 
optimized for specific elution of your target protein. I believe that the 
standard recommendations when using a Nickel column to bind to your 
Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted 
unspecific binding protein, to try manipulating the binding by lowering the pH 
of the buffers, and by determining the specific concentration of Imidazole your 
protein elutes at by using a gradient before progressing to a step-wise 
elution. Alternatively, you can also try using another metal column such as 
Cobalt (Talon) with a higher affinity for the Histidine tag.Sincerely, 
lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard 
Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe 
College of Life SciencesBeijing, China
Date: Mon, 26 Mar 2012 15:04:43 +0300
From: peg...@pasteur.gr
Subject: [ccp4bb] recommendations_on_purification
To: CCP4BB@JISCMAIL.AC.UK













Dear all,



I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, 
using the standard minimal medium described in the invitrogen manual (plus 
PTM1). Following collection of the culture medium, I am having problems with 
purification of the protein as only a small fraction (~10%) binds to the Ni-NTA 
beads even after extensive buffer exchange (when expressed in full BMGY media 
this is not observed). Could this be attributed to metal ions still present in 
my sample? Is it likely to be due to poor protein quality in this medium? Or 
any other suggestions?



Thanks in advance

Petros
  

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Ed Pozharski]

I agree with Eleanor 100%...

In my biased opinion, only the atoms supported by electron density
should be included in deposited models.  To satisfy the "but this will
mess up the electrostatic potential coloring" argument (a valid one, of
course), the "projected model" can be deposited alongside which must be
clearly advertised as the unconstrained interpretation by the
structure's author.

Cheers,

Ed.

On Sun, 2012-03-25 at 08:36 +0100, Eleanor Dodson wrote:
> As Garib says - an atom with occupancy 0.00 is treated as a marker -
> useful 
> for coot - but is not included in any X-ray refinement at all.. Maybe
> it 
> would be more aesthetic to maintain geometry but as crystallographers
> I 
> think we should be interested in the fit of model to experiment -
> right? - 
> and not in reporting a pseudo fit related to geometric parameters
> only.. 
-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

[ccp4bb] Announcement: EMBO Practical Course in protein expression, purification and characterization (PEPC8)

[Rob Meijers]

Dear all,

we are pleased to announce that applications are open for the EMBO Practical 
Course 

"Protein expression, purification and characterization" (PEPC8), which will be 
held at 

EMBL Hamburg from the 3rd of September until the 11th of September 2012.

This is a hands-on course with practicals in cloning, expression (E. coli & 
Baculo virus), 
purification (with and without tags) and characterization (thermofluor, SAXS, 
crystallization and in-situ dynamic light scattering, ITC and thermophoresis).

Speakers include:

Imre Berger, EMBL Grenoble 
Huseyin Besir, EMBL Heidelberg
Christian Betzel, University of
 Hamburg
Louise Bird, Oxford University
Uwe Bierfreund, GE Healthcare
Richard Burgess, University of Wisconsin-Madison
Stefan Duhr, NanoTemper
David Hacker, EPFL Lausanne
Josan Marquez, EMBL Grenoble
Alex McPherson, University of California
Preben Morth, University of Oslo
Jochen Mueller-Dieckmann, EMBL Hamburg
Joanne Nettleship, University of Oxford
Ilme Schlichting, Max Planck Institute, Munich   

Dmitri Svergun, EMBL Hamburg

Participants are encouraged to bring their own sample to the course for cloning 
and expression in the pOPIN vectors, high-throughput crystallization, SAXS 
and biophysical characterization.

A more extensive characterization of   samples is sponsored in context of the 
course
by the co-sponsor Pcube. 

For more information, please visit our website: 


http://events.embo.org/12-pepc/index.html

or contact us by email: pep...@googlemail.com

Best regards,
Rob Meijers, Stephane Boivin, Annabel Parret & Dmitri Svergun
EMBL Hamburg
Notkestrasse 85
D-22603, Hamburg, Germany

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Gregory Bowman]

But what about the issue of resolution? As was previously pointed out, at say 
3.2 Å resolution, many side chains will fail to fit, but it doesn't seem 
appropriate to trim them all down. The users need to also be aware of the 
quality/resolution of the structures that they are looking at. 

Greg


On Mar 26, 2012, at 9:28 AM, Ed Pozharski wrote:

> I agree with Eleanor 100%...
> 
> In my biased opinion, only the atoms supported by electron density
> should be included in deposited models.  To satisfy the "but this will
> mess up the electrostatic potential coloring" argument (a valid one, of
> course), the "projected model" can be deposited alongside which must be
> clearly advertised as the unconstrained interpretation by the
> structure's author.
> 
> Cheers,
> 
> Ed.
> 
> On Sun, 2012-03-25 at 08:36 +0100, Eleanor Dodson wrote:
>> As Garib says - an atom with occupancy 0.00 is treated as a marker -
>> useful 
>> for coot - but is not included in any X-ray refinement at all.. Maybe
>> it 
>> would be more aesthetic to maintain geometry but as crystallographers
>> I 
>> think we should be interested in the fit of model to experiment -
>> right? - 
>> and not in reporting a pseudo fit related to geometric parameters
>> only.. 
> -- 
> Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
>Julian, King of Lemurs

--
Department of Biophysics
Johns Hopkins University
302 Jenkins Hall
3400 N. Charles St.
Baltimore, MD 21218
Phone: (410) 516-7850 (office)
Phone: (410) 516-3476 (lab)
gdbow...@jhu.edu
http://www.jhu.edu/bowmanlab

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Eleanor Dodson]

 This is a personal preference. I do model at low sigma levels if there IS 
some indication of where to put atoms, always try to keep the correct 
sequence even if some atoms are missing, and just for coot convenience keep 
atoms with occ = 0, rather than delete them altogether. (COOT will refine a 
residue with occs = 0, but not one where atoms are missing.. Paul?? Why 
not!)


At deposition the wwwPDB are welcome to (and I think do) strip out all 
ocs=0..


Eleanor



On Mar 26 2012, Gregory Bowman wrote:

But what about the issue of resolution? As was previously pointed out, at 
say 3.2 Å resolution, many side chains will fail to fit, but it doesn't 
seem appropriate to trim them all down. The users need to also be aware 
of the quality/resolution of the structures that they are looking at.


Greg


On Mar 26, 2012, at 9:28 AM, Ed Pozharski wrote:


I agree with Eleanor 100%...

In my biased opinion, only the atoms supported by electron density
should be included in deposited models.  To satisfy the "but this will
mess up the electrostatic potential coloring" argument (a valid one, of
course), the "projected model" can be deposited alongside which must be
clearly advertised as the unconstrained interpretation by the
structure's author.

Cheers,

Ed.

On Sun, 2012-03-25 at 08:36 +0100, Eleanor Dodson wrote:

As Garib says - an atom with occupancy 0.00 is treated as a marker -
useful 
for coot - but is not included in any X-ray refinement at all.. Maybe
it 
would be more aesthetic to maintain geometry but as crystallographers
I 
think we should be interested in the fit of model to experiment -
right? - 
and not in reporting a pseudo fit related to geometric parameters
only.. 

--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
   Julian, King of Lemurs


--
Department of Biophysics
Johns Hopkins University
302 Jenkins Hall
3400 N. Charles St.
Baltimore, MD 21218
Phone: (410) 516-7850 (office)
Phone: (410) 516-3476 (lab)
gdbow...@jhu.edu
http://www.jhu.edu/bowmanlab




--
Professor Eleanor Dodson
YSNL, Dept of Chemistry
University of York
Heslington YO10 5YW
tel: 00 44 1904 328259
Fax: 00 44 1904 328266

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Herman . Schreuder]

I fully agree. Unfortunately, the perfect model does not exist (at least not 
for protein crystal structures). It is like with Heisenbergs uncertainty 
principle. Either one has a complete model with a number of atoms having a 
coordinate uncertainty of 4-6 Å, or one has a model where the uncertainty of 
all atoms is below say 0.5 Å, but with a lot of truncated side chains with 
clearly contradict available biochemical evidence.
 
Cheers,
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Gregory Bowman
Sent: Monday, March 26, 2012 4:17 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] REFMAC5 residues with bad geometry


But what about the issue of resolution? As was previously pointed out, 
at say 3.2 Å resolution, many side chains will fail to fit, but it doesn't seem 
appropriate to trim them all down. The users need to also be aware of the 
quality/resolution of the structures that they are looking at.  

Greg



On Mar 26, 2012, at 9:28 AM, Ed Pozharski wrote:


I agree with Eleanor 100%...

In my biased opinion, only the atoms supported by electron 
density
should be included in deposited models.  To satisfy the "but 
this will
mess up the electrostatic potential coloring" argument (a valid 
one, of
course), the "projected model" can be deposited alongside which 
must be
clearly advertised as the unconstrained interpretation by the
structure's author.

Cheers,

Ed.

On Sun, 2012-03-25 at 08:36 +0100, Eleanor Dodson wrote:


As Garib says - an atom with occupancy 0.00 is treated 
as a marker -


useful 


for coot - but is not included in any X-ray refinement 
at all.. Maybe


it 


would be more aesthetic to maintain geometry but as 
crystallographers


I 


think we should be interested in the fit of model to 
experiment -


right? - 


and not in reporting a pseudo fit related to geometric 
parameters


only.. 


-- 
Oh, suddenly throwing a giraffe into a volcano to make water is 
crazy?
   Julian, King of 
Lemurs






--
Department of Biophysics
Johns Hopkins University
302 Jenkins Hall
3400 N. Charles St.
Baltimore, MD 21218
Phone: (410) 516-7850 (office)
Phone: (410) 516-3476 (lab)
gdbow...@jhu.edu
http://www.jhu.edu/bowmanlab

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Ed Pozharski]

On Mon, 2012-03-26 at 10:17 -0400, Gregory Bowman wrote:
> But what about the issue of resolution? As was previously pointed out,
> at say 3.2 Å resolution, many side chains will fail to fit, but it
> doesn't seem appropriate to trim them all down. 

Why is it inappropriate to trim them down?  Sometimes at low resolution
all one can be confident about is the backbone trace.

Just to be clear, I am talking about atoms whose positions are not
supported by electron density, i.e. where difference map in the absence
of the side chain is featureless.  I assume that is the likely situation
when one would set occupancy to zero. 

Cheers,

Ed.

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Ed Pozharski]

On Mon, 2012-03-26 at 16:30 +0200, herman.schreu...@sanofi.com wrote:
> It is like with Heisenbergs uncertainty principle. Either one has a
> complete model with a number of atoms having a coordinate uncertainty
> of 4-6 Å, or one has a model where the uncertainty of all atoms is
> below say 0.5 Å, but with a lot of truncated side chains with clearly
> contradict available biochemical evidence.

Excellent analogy.  I am not sure why truncated arginine (as long as it
is not renamed to alanine) contradicts biochemical evidence though.
Termini are routinely truncated, no problem there. I have plenty of
biochemical evidence that there are more waters in the crystal than I
model.

If the truncated model contradicts biochemical evidence, the projected
model contradicts crystallographic evidence. I agree that a truncated
model may lead to interpretation problems, and thus the option of
depositing a projected model resolves that.

Cheers,

Ed.

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Herman . Schreuder]

Dear Ed,

In the end it boils down to personal preferences. With the number of crystal 
structures I refine each year, I am not going to go over every flexible surface 
residue to decide whether to truncate the side chain or whether there may be 
some low level density justifying to keep the side chain, so I opt for the 
biochemical evidence. For me the added advantage is that I only have a single 
pdb file to take care of. And again, I see no problem in having a model with 
some atoms with a larger error bar.

You are right that terminii are often truncated. In contrast to a missing side 
chain, here we really have no reasonable hypothesis where the missing residues 
are. They may even have been removed by a protease.

Cheers,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed 
Pozharski
Sent: Monday, March 26, 2012 4:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] REFMAC5 residues with bad geometry

On Mon, 2012-03-26 at 16:30 +0200, herman.schreu...@sanofi.com wrote:
> It is like with Heisenbergs uncertainty principle. Either one has a 
> complete model with a number of atoms having a coordinate uncertainty 
> of 4-6 Å, or one has a model where the uncertainty of all atoms is 
> below say 0.5 Å, but with a lot of truncated side chains with clearly 
> contradict available biochemical evidence.

Excellent analogy.  I am not sure why truncated arginine (as long as it is not 
renamed to alanine) contradicts biochemical evidence though.
Termini are routinely truncated, no problem there. I have plenty of biochemical 
evidence that there are more waters in the crystal than I model.

If the truncated model contradicts biochemical evidence, the projected model 
contradicts crystallographic evidence. I agree that a truncated model may lead 
to interpretation problems, and thus the option of depositing a projected model 
resolves that.

Cheers,

Ed.

--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Katherine Sippel]

I agree with Herman about personal preference but it also boils to our job
as crystallographers to educate non-structural end-users. The fact of the
matter is that a lot of researchers use structures without looking at the
nuances of the PDB. It's actually pretty common among biologists to
download a PDB file and build hypothesis or throw the coordinates into a
downstream program like APBS or AutoDock without looking. They don't even
realize that density exists or that they should check it, which makes the
odds of them reading a header file for missing atoms or understanding the
concept of b-factors and occupancy almost nil. Realizing that renders the
argument moot until crystallographic data is demystified across the other
sciences.

I will say that in principle I do like the idea of a data model + a
projected model because it seems like something an end-user could wrap
their head around, but in practice this would probably refuel the "what
constitutes modelable density" debate all over again.

Cheers,

Katherine

On Mon, Mar 26, 2012 at 10:19 AM,  wrote:

> Dear Ed,
>
> In the end it boils down to personal preferences. With the number of
> crystal structures I refine each year, I am not going to go over every
> flexible surface residue to decide whether to truncate the side chain or
> whether there may be some low level density justifying to keep the side
> chain, so I opt for the biochemical evidence. For me the added advantage is
> that I only have a single pdb file to take care of. And again, I see no
> problem in having a model with some atoms with a larger error bar.
>
> You are right that terminii are often truncated. In contrast to a missing
> side chain, here we really have no reasonable hypothesis where the missing
> residues are. They may even have been removed by a protease.
>
> Cheers,
> Herman
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed
> Pozharski
> Sent: Monday, March 26, 2012 4:50 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] REFMAC5 residues with bad geometry
>
> On Mon, 2012-03-26 at 16:30 +0200, herman.schreu...@sanofi.com wrote:
> > It is like with Heisenbergs uncertainty principle. Either one has a
> > complete model with a number of atoms having a coordinate uncertainty
> > of 4-6 Å, or one has a model where the uncertainty of all atoms is
> > below say 0.5 Å, but with a lot of truncated side chains with clearly
> > contradict available biochemical evidence.
>
> Excellent analogy.  I am not sure why truncated arginine (as long as it is
> not renamed to alanine) contradicts biochemical evidence though.
> Termini are routinely truncated, no problem there. I have plenty of
> biochemical evidence that there are more waters in the crystal than I model.
>
> If the truncated model contradicts biochemical evidence, the projected
> model contradicts crystallographic evidence. I agree that a truncated model
> may lead to interpretation problems, and thus the option of depositing a
> projected model resolves that.
>
> Cheers,
>
> Ed.
>
> --
> Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
>Julian, King of Lemurs
>

[ccp4bb] JME editor in PRODRG

[Shya Biswas]

Hi all,

I was wondering if anyone had problems with drawing molecule using JME
editor in PRODRG? If yes then how do I fix it? I could not draw molecule in
the JME editor in the PRODRG webpage recently and JAVA is already installed
in my computer.
thanks,
Shya

[ccp4bb] DDM

[Katarzyna Rudzka]

Hi All,
Has anyone had any luck purifying membrane proteins with DDM 
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC 
is very low: 0.009%). I would like to keep it as low as possible, so I don't 
have too much DDM around when I get to the crystallization step. I wonder If 
the amount of detergent sufficient for the protein extraction has to be 
determined experimentally for each protein or maybe there are some good rules 
of thumb.  I appreciate your help. Thanks.
Kasia
 

Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA

Re: [ccp4bb] DDM

[yybbll]

Hi, 

I used 0.017% or 0.012%. My protein is very stable at this concentration. 

Good luck.

Lin


2012-03-26 



yybbll 



 
Hi All,
Has anyone had any luck purifying membrane proteins with DDM 
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC 
is very low: 0.009%). I would like to keep it as low as possible, so I don't 
have too much DDM around when I get to the crystallization step. I wonder If 
the amount of detergent sufficient for the protein extraction has to be 
determined experimentally for each protein or maybe there are some good rules 
of thumb.  I appreciate your help. Thanks.
Kasia


Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA

Re: [ccp4bb] DDM

[Kelly Daughtry]

I generally use 1 - 2% DDM for extraction only, but lower the concentration
to 0.01% for following steps (i.e. NiNTA and gel filtration). The excess
DDM is washed away by using a lower concentration in your wash and elution
buffers.

Kelly

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Mon, Mar 26, 2012 at 1:17 PM, Katarzyna Rudzka wrote:

> Hi All,
> Has anyone had any luck purifying membrane proteins with DDM
> (n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its
> CMC is very low: 0.009%). I would like to keep it as low as possible, so I
> don't have too much DDM around when I get to the crystallization step. I
> wonder If the amount of detergent sufficient for the protein extraction has
> to be determined experimentally for each protein or maybe there are some
> good rules of thumb.  I appreciate your help. Thanks.
> Kasia
>
>
> Katarzyna Rudzka, Postdoctoral Fellow
> Department of Biophysics and Biophysical Chemistry
> Johns Hopkins University, School of Medicine
> Baltimore, Maryland 21205 USA
>

Re: [ccp4bb] DDM

[Das, Debanu]

Hi Katarzyna,

Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value 
is very low, at the extraction step you need to use a much higher concentration 
up to ~100x CMC. During different rounds of purification, you can bring the CMC 
level down to 1-2x CMC and even try detergent exchange in the last step, 
although it can be difficult to totally exchange out the DDM due to its low 
CMC. Also use a 100 kDa MWCO concentrator since the DDM micelle size is large.

In general, DDM is a reasonable choice for extraction, purification and 
crystallization setups, but it will be worthwhile to screen a panel of 
different detergents for extraction and purification for your particular target.
In 2005, we set up a fast protocol for screening a panel of 18 detergents in 
48-72 hours, 
http://smb.slac.stanford.edu/~debanu/posters/012605_PPCW2005_DDAS.pdf, which 
includes suggestions for starting concentrations for extraction.

There are also some papers on detergent screening, for example:
http://www.ncbi.nlm.nih.gov/pubmed/18988031
http://www.nature.com/nprot/journal/v4/n5/full/nprot.2009.27.html (Stroud lab)

Thanks,
Debanu.


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna 
Rudzka
Sent: Monday, March 26, 2012 10:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] DDM

Hi All,
Has anyone had any luck purifying membrane proteins with DDM 
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC 
is very low: 0.009%). I would like to keep it as low as possible, so I don't 
have too much DDM around when I get to the crystallization step. I wonder If 
the amount of detergent sufficient for the protein extraction has to be 
determined experimentally for each protein or maybe there are some good rules 
of thumb.  I appreciate your help. Thanks.
Kasia
 

Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA

Re: [ccp4bb] DDM

[Joao Dias]

Kasia,
A lot of people uses DDM to purify membrane proteins, not a lot of people 
crystallises them.
If you want to crystallise a protein purified in DDM, then you should use LCP.
If you go for vapour diffusion, you should exchange the DDM for a detergent 
with a smaller micelle size otherwise you might get crystals but it is 
difficult to get good diffraction. Try mixed micelles for example.
Typically use 0.05% DDM during purification and use 100kDa cut-off membranes in 
order to prevent detergent concentration.
For extraction it depends on your protein and expression system but you can see 
in the literature values between 0.5-2% being used successfully.
Good luck.
Cheers,
Joao

Joao Dias, Ph.D.

Senior Scientist
Heptares Therapeutics Ltd
BioPark, Broadwater Road,
Welwyn Garden City,
Herts, AL7 3AX
UK

This document contains company confidential and/or proprietary information. It 
is intended for the exclusive attention of the addressee(s) above and should 
not be copied or disclosed to any other. If you have received this transmission 
in error, please make no use of its contents and contact the sender.



[cid:image6c7082.GIF@43484746.4ab9695e]




 The information in this e-mail is confidential and may be legally privileged. 
It is intended for the exclusive attention of the addressee stated above and 
should not be copied or disclosed to any other. If you have received this 
transmission in error, please make no use of its contents and contact the 
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BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katarzyna 
Rudzka
Sent: 26 March 2012 18:17
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] DDM

Hi All,
Has anyone had any luck purifying membrane proteins with DDM 
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC 
is very low: 0.009%). I would like to keep it as low as possible, so I don't 
have too much DDM around when I get to the crystallization step. I wonder If 
the amount of detergent sufficient for the protein extraction has to be 
determined experimentally for each protein or maybe there are some good rules 
of thumb.  I appreciate your help. Thanks.
Kasia


Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA




<>

Re: [ccp4bb] DDM

[Edward A. Berry]

If I recall correctly cytochrome oxidase, which I believe was
the first protein purified with DDM, requires about 10x cmc
in column buffers to keep it soluble. Check for papers from 1970's or 80's
by S. Ferguson-Miller.

Cytochrme bc1 complex, on the other hand, is perfectly clear in 1 cmc
and can be diluted from that into detergent-free buffer for
spectroscopywithout becoming turbid, at least within an huor.

My rule of thumb for solubilizing with DDM is 1 g/g protein (+ 1 cmc).
If protein is say 10-20 g./l, the (+ 1 cmc) becomes completely insignificant.
(Total protein, not the protein of interest. Probably depends on the
amount of lipid too, but this is just a rule of thumb to start with.)

eab

Katarzyna Rudzka wrote:

Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ?
(Its CMC is very low: 0.009%). I would like to keep it as low as
possible, so I don't have too much DDM around when I get to the
crystallization step. I wonder If the amount of detergent sufficient for
the protein extraction has to be determined experimentally for each
protein or maybe there are some good rules of thumb. I appreciate your
help. Thanks.
Kasia

Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA

[ccp4bb] Unable to reproduce robot tray hits in hand trays

[Matthew Lalonde]

Dear All,

I have searched the archives and would like more information about
reproducing robot tray hits using 24-well hand trays. I reproducibly get
crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
precipitate in 1-2 ul sitting drops in 24-well hand plates.

What parameters should I vary to reproduce crystals in hand plates?
References that discuss this procedure exhaustively would also be
appreciated.

Thanks,
Matt

Re: [ccp4bb] JME editor in PRODRG

[Tom Oldfield]

Shya

There have been a number of issues of the releases of the JVM, particularly
with browser IE9 - most resulting in security exceptions when accessing 
data files.


You should be able to update your Java on your computer as these problems
appear to have been finally resolved through the normal Java updates.

Regards
Tom


Hi all,

I was wondering if anyone had problems with drawing molecule using JME 
editor in PRODRG? If yes then how do I fix it? I could not draw 
molecule in the JME editor in the PRODRG webpage recently and JAVA is 
already installed in my computer.

thanks,
Shya

Re: [ccp4bb] recommendations_on_purification

[Carlos Kikuti]

90% of the protein could be aggregated and hiding the His tag from the resin. 
Metal ions might be removed from the starting sample by adding up to 10 mM EDTA 
in the first buffer exchange cycle, but just after reading "10% of the protein 
binds" I wouldn't bet much on this. Rather increase the culture volume and move 
on.

Carlos Kikuti


Em 26/03/2012, às 14:39, Lorenzo Finci escreveu:

> Petros, 
> 
> It has indeed been speculated that high concentrations of Magnesium and/or 
> other metals present in the cell lysate effect the binding of the 
> Histidine-tag, and thus specific conditions for binding and elution need to 
> be optimized for specific elution of your target protein. I believe that the 
> standard recommendations when using a Nickel column to bind to your 
> Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted 
> unspecific binding protein, to try manipulating the binding by lowering the 
> pH of the buffers, and by determining the specific concentration of . 
> Alternatively, you can also try using another metal column such as Cobalt 
> (Talon) with a higher affinity for the Histidine tag.
> Sincerely, 
> lorenzo
> 
> 
> Lorenzo Ihsan FInci, Ph.D.
> Postdoctoral Scientist, Wang Laboratory
> Harvard Medical School
> Dana-Farber Cancer Institute
> Boston, MA 
> Peking University
> The College of Life Sciences
> Beijing, China
> 
> 
> 
> Date: Mon, 26 Mar 2012 15:04:43 +0300
> From: peg...@pasteur.gr
> Subject: [ccp4bb] recommendations_on_purification
> To: CCP4BB@JISCMAIL.AC.UK
> 
> Dear all,
> 
> I am expressing a 6xHis tagged secreted protein in a fermentor in P. 
> pastoris, using the standard minimal medium described in the invitrogen 
> manual (plus PTM1). Following collection of the culture medium, I am having 
> problems with purification of the protein as only a small fraction (~10%) 
> binds to the Ni-NTA beads even after extensive buffer exchange (when 
> expressed in full BMGY media this is not observed). Could this be attributed 
> to metal ions still present in my sample? Is it likely to be due to poor 
> protein quality in this medium? Or any other suggestions?
> 
> Thanks in advance
> Petros 

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[Nian Huang]

I have seen people only use robot to optimize their crystal and get
good diffraction (~2 A). If you keep having trouble, you can try this
method instead, even though generally the case is the bigger the drop
the bigger crystal.

I remember the archive suggest us to use higher concentration of
protein to reproduce the result when using bigger volume.

Best,

Nian

On Mon, Mar 26, 2012 at 12:57 PM, Matthew Lalonde  wrote:
> Dear All,
>
> I have searched the archives and would like more information about
> reproducing robot tray hits using 24-well hand trays. I reproducibly get
> crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
> precipitate in 1-2 ul sitting drops in 24-well hand plates.
>
> What parameters should I vary to reproduce crystals in hand plates?
> References that discuss this procedure exhaustively would also be
> appreciated.
>
> Thanks,
> Matt

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[Ed Pozharski]

On Mon, 2012-03-26 at 11:57 -0600, Matthew Lalonde wrote:
> What parameters should I vary to reproduce crystals in hand plates? 

First of all, protein concentration.  It also does not hurt diluting
your reservoir since you are getting precipitates.  If your goal is to
get bigger crystals (which is not always better), consider using
crystals that grow on intelliplates for seeding.

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] recommendations_on_purification

[Artem Evdokimov]

Hi Petros,

It's possible but unlikely that your media contains enough metal ions
to matter. More likely options include protein issues like
overgkycosylation, proteolysis (removal of the tag) or severe
aggregation. Since you are not experiencing this in BMGY, I wonder why
not to supplement your synthetic medium with yeast extract and/or
peptone and the like?

Artem


On Mon, Mar 26, 2012 at 7:04 AM, Petros Giastas  wrote:
> Dear all,
>
> I am expressing a 6xHis tagged secreted protein in a fermentor in P.
> pastoris, using the standard minimal medium described in the invitrogen
> manual (plus PTM1). Following collection of the culture medium, I am having
> problems with purification of the protein as only a small fraction (~10%)
> binds to the Ni-NTA beads even after extensive buffer exchange (when
> expressed in full BMGY media this is not observed). Could this be attributed
> to metal ions still present in my sample? Is it likely to be due to poor
> protein quality in this medium? Or any other suggestions?
>
> Thanks in advance
> Petros

Re: [ccp4bb] recommendations_on_purification

[Kendall Nettles]

I suspect that sometimes the protein chaperones the tag, which is solvent 
exposed some fraction  of the time.  Try very slow loading or batch binding.

Kendall Nettles

On Mar 26, 2012, at 8:15 AM, "Petros Giastas" 
mailto:peg...@pasteur.gr>> wrote:


Dear all,

I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, 
using the standard minimal medium described in the invitrogen manual (plus 
PTM1). Following collection of the culture medium, I am having problems with 
purification of the protein as only a small fraction (~10%) binds to the Ni-NTA 
beads even after extensive buffer exchange (when expressed in full BMGY media 
this is not observed). Could this be attributed to metal ions still present in 
my sample? Is it likely to be due to poor protein quality in this medium? Or 
any other suggestions?

Thanks in advance
Petros

Re: [ccp4bb] DDM

[Ho Leung Ng]

Actually, DDM is the most successfully used detergent for membrane
protein crystallization. See Newstead et al, Protein Sci. 17:466. But
yes, the rule of thumb is that detergents that form smaller micelles
give better diffracting crystals, but are more destabilizing.


Ho

Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
h...@hawaii.edu


On Mon, Mar 26, 2012 at 1:06 PM, CCP4BB automatic digest system
 wrote:
> Date:    Mon, 26 Mar 2012 17:39:57 +
> From:    Joao Dias 
> Subject: Re: DDM
>
> Kasia,
> A lot of people uses DDM to purify membrane proteins, not a lot of people 
> crystallises them.
> If you want to crystallise a protein purified in DDM, then you should use LCP.
> If you go for vapour diffusion, you should exchange the DDM for a detergent 
> with a smaller micelle size otherwise you might get crystals but it is 
> difficult to get good diffraction. Try mixed micelles for example.
> Typically use 0.05% DDM during purification and use 100kDa cut-off membranes 
> in order to prevent detergent concentration.
> For extraction it depends on your protein and expression system but you can 
> see in the literature values between 0.5-2% being used successfully.
> Good luck.
> Cheers,
> Joao
>
> Joao Dias, Ph.D.
>
> Senior Scientist
> Heptares Therapeutics Ltd
> BioPark, Broadwater Road,
> Welwyn Garden City,
> Herts, AL7 3AX
> UK

[ccp4bb] a question about protein sequences in the PDB

[Francois Berenger]

Dear list,

If I take all the fasta files for proteins in the PDB,
are the sequences complete?

I mean, do they have holes sometimes (missing amino acids)?

Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with the FASTA files.

Regards,
Francois.

Re: [ccp4bb] a question about protein sequences in the PDB

[Bosch, Juergen]

I think that depends on what the depositor considered complete.
Just as an example the construct you cloned say from residue 20 - 380 would you 
consider that complete or would you consider only the sequence complete if it 
contained the first 20residues ?
Regarding the gaps in terms of missing residues in the structure because they 
were not observed you shouldn't be worried as they are included in the FASTA 
sequence.
3TGH just as an example, would you consider that complete ?

Jürgen


On Mar 26, 2012, at 10:54 PM, Francois Berenger wrote:

Dear list,

If I take all the fasta files for proteins in the PDB,
are the sequences complete?

I mean, do they have holes sometimes (missing amino acids)?

Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with the FASTA files.

Regards,
Francois.

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/

Re: [ccp4bb] a question about protein sequences in the PDB

[Ethan Merritt]

On Monday, 26 March 2012, Francois Berenger wrote:
> Dear list,
> 
> If I take all the fasta files for proteins in the PDB,
> are the sequences complete?
> 
> I mean, do they have holes sometimes (missing amino acids)?

In theory the SEQRES records describe the sequence of the
entity that was crystallized, whether or not it is all visible
in the electron density or present in the deposited model.
So normally there should not be any "missing" internal
residues.  But if the expression construct was a not the full
gene sequence, e.g. an N-terminal truncation, then those
N- or C- terminal residues (or whole domains) will not be
listed.

So goes the theory. There are always corner cases.
I remember having a dispute with the PDB long ago about
whether a peptide chain that was known to have undergone
loop cleavage was properly described with a single
chain identifier or with two chain identifiers.  And if the
cleavage involved excission of one or more residues, would
they appear in the SEQRES records anyhow?


> Sorry for the maybe stupid question but I know that sometimes
> the PDB files have missing residues, I am hoping that
> it is not the case with the FASTA files.

I was assuming that the FASTA files you refer to are just
conversions of the SEQRES records.  If not, then all bets are
off.  If the FASTA files are retrieved by gene ID from Uniprot
or some other sequence data base, then they will be complete in
one sense but may not perfectly match what was in the deposited
crystal structure due to cloning artifacts, strain variation,
allelic non-uniformity, etc.

Ethan

> Regards,
> Francois.
> 

Re: [ccp4bb] a question about protein sequences in the PDB

[Francois Berenger]

On 03/27/2012 12:20 PM, Ethan Merritt wrote:

On Monday, 26 March 2012, Francois Berenger wrote:

Dear list,

If I take all the fasta files for proteins in the PDB,
are the sequences complete?

I mean, do they have holes sometimes (missing amino acids)?


In theory the SEQRES records describe the sequence of the
entity that was crystallized, whether or not it is all visible
in the electron density or present in the deposited model.
So normally there should not be any "missing" internal
residues.  But if the expression construct was a not the full
gene sequence, e.g. an N-terminal truncation, then those
N- or C- terminal residues (or whole domains) will not be
listed.

So goes the theory. There are always corner cases.
I remember having a dispute with the PDB long ago about
whether a peptide chain that was known to have undergone
loop cleavage was properly described with a single
chain identifier or with two chain identifiers.  And if the
cleavage involved excission of one or more residues, would
they appear in the SEQRES records anyhow?



Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with the FASTA files.


I was assuming that the FASTA files you refer to are just
conversions of the SEQRES records.  If not, then all bets are
off.  If the FASTA files are retrieved by gene ID from Uniprot
or some other sequence data base, then they will be complete in
one sense but may not perfectly match what was in the deposited
crystal structure due to cloning artifacts, strain variation,
allelic non-uniformity, etc.


OK, thanks for the answers.
I'll try to find out more about the FASTA files present in the database 
then.


Regards,
F.


Ethan


Regards,
Francois.

Re: [ccp4bb] a question about protein sequences in the PDB

[Chad Simmons]

The total model that fits the observable electron density should be the 
standard for the PDB FASTA file with the deposited structure factors, however, 
not all depositions contain a link to the expression construct details because 
many are not published, and I believe that it should be explicitly detailed in 
the submission.

Chad


On 3/26/12 7:54 PM, "Francois Berenger"  wrote:

Dear list,

If I take all the fasta files for proteins in the PDB,
are the sequences complete?

I mean, do they have holes sometimes (missing amino acids)?

Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with the FASTA files.

Regards,
Francois.


***
Chad R. Simmons
Researcher
Biodesign Institute
at Arizona State University
P.O. Box 875601
Tempe, AZ  85287
Office: (480) 727-6495
Lab:(480) 727-0428
Fax:(480) 727-0396

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[David Briggs]

Hi Matt,

This doesn't really answer your question directly, but sidesteps
around the issue -
I wrote a little something on this exact subject not so long ago -
http://xtaldave.wordpress.com/2012/02/23/on-protein-crystallisation/
(You can ignore the first 5 paragraphs of intro - it was written with
a lay audience in mind)

Basically, I setup fine gradient screens in deep well blocks by
titrating stock solutions across a row - which I can then put straight
into use with our Phoenix crystallisation robot. In most situations I
would hope to get decent diffracting crystals from a standard 3-well
intelliplate setup this way.

HTH

Dave

David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

Webs : http://flavors.me/xtaldave
Twitter: @xtaldave
Skype: DocDCB




On 26 March 2012 18:57, Matthew Lalonde  wrote:
> Dear All,
>
> I have searched the archives and would like more information about
> reproducing robot tray hits using 24-well hand trays. I reproducibly get
> crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
> precipitate in 1-2 ul sitting drops in 24-well hand plates.
>
> What parameters should I vary to reproduce crystals in hand plates?
> References that discuss this procedure exhaustively would also be
> appreciated.
>
> Thanks,
> Matt

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[Richardson, Brian C.]

Most of the labs sharing our Phoenix have had enough trouble with exactly
this that our standard procedure is to now to use 1 µl drops (.5/.5
protein/well) in our initial screens – those scale up much more reliably to
the 24-well format and seem less finicky in general, reducing the chances of
an unusable hit.  We often get a somewhat different set of hits than we get
at the lower volume, as well, so it might be worth rescreening while you
work on the existing hit.  

 

Of course we also try the usual tricks with optimizing anyway (my first
crystallized protein precipitated in sitting drops but not hanging drops,
etc). J

 

 



Dr. Brian C. Richardson

Weill Institute for Cell and Molecular Biology

Cornell University

(609)933-4548



 

 

From: Matthew Lalonde [mailto:mattc...@gmail.com] 
Sent: Monday, March 26, 2012 1:57 PM
Subject: Unable to reproduce robot tray hits in hand trays

 

Dear All,

I have searched the archives and would like more information about
reproducing robot tray hits using 24-well hand trays. I reproducibly get
crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
precipitate in 1-2 ul sitting drops in 24-well hand plates.

What parameters should I vary to reproduce crystals in hand plates?
References that discuss this procedure exhaustively would also be
appreciated.

Thanks,
Matt