Re: [ccp4bb] detergent crystals

[Jose Antonio Cuesta Seijo]

Jakob,

That Compacdt disc appearance is very familiar, those were right next to
the hexagonal ones in my experiments. And all were oily when touched. It is
very likely that my "detergent" crystals were in reality detergent-protein
complexes with less than crystalline order. I was doing experiments with
very high detergent concentrations (up to 6%) and in some cases the
crystals would be there in about 50% of the conditions. Their numbers also
correlated better with the detergent concentration than with the protein
concentration, but all that is still compatible with protein-detergent
complexes, of course.
Regarding measuring the final detergent concentration, a fast method is
described in :

"A strategy for identification and quantification of detergents frequently
used in the purification of membrane proteins." Laura R. Eriks, June A.
Mayor and Ronald S. Kaplan
Analytical Biochemistry Volume 323, Issue 2, 15 December 2003, Pages
234-241 

It uses TLC, and the protein crystallization stock can be spotted directly,
water and all. The standard can also be in water. In my hands, the crucial
step was to dry this water thoroughly before running the TLC. I adapted
this to small TLC plates which can be run in a sealed beaker. Running your
sample in between appropriate standards will give you an estimation of your
detergent concentration in as little as 2 hours.

Cheers.

Jose. 



"Jacob Keller"  wrote:
> A recommendation: try looking at the crystals while rotating the
polarizers.
> Often you can get detergent or detergent-protein complex "crystals" which
have
> sharp edges, but are actually liquid crystals. This will be manifest as a
> compact-disc (or vinyl LP, depending on your vintage) appearance which
rotates
> in sync with the rotation of the polarizers. Several colleagues and I
have been
> plagued with these false positives, which are in our experience extremely
hard
> to optimize into real crystals.
> 
> Another possibility: crystallization with a fluorescent or otherwise
detectable
> substrate analogue could also be helpful, at least for determining
whether there
> is protein in the sharp-edged objects.
> 
> The best test, of course, is to mount the objects and put them in the
x-ray
> beam.
> 
> Regards,
> 
> Jacob Keller
> 
> 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> Dallos Laboratory
> F. Searle 1-240
> 2240 Campus Drive
> Evanston IL 60208
> lab: 847.491.2438
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
> 
>   - Original Message - 
>   From: R.M. Garavito 
>   To: CCP4BB@JISCMAIL.AC.UK 
>   Sent: Tuesday, August 04, 2009 12:37 PM
>   Subject: Re: [ccp4bb] detergent crystals
> 
> 
>   Parveen,
> 
> 
>   Bert and Pascal are correct in that most alkyl glycoside detergent are
> notoriously difficult to crystallize in aqueous solution when you have
the
> beta-anomer (what we normally buy).  However, the alpha-anomers can be
quite
> easy to crystallize and can contaminate batches of beta-alkyl glycoside
> detergents.  While the quality control procedures are usually good enough
to
> ensure that the alpha-anomer contamination of DDM, DM, and OG are low, it
may
> not be low enough for all crystallization experiments.  Twenty or so
years ago,
> I was even shown a batch of "pure" beta-OG from a company I shall not
name which
> was insoluble in water.
> 
> 
>   Some people have complained about this, but the impact of alpha-anomer
> contamination on crystal growth and spurious detergent crystallization is
> unknown.  If this persists and you are sure that those are detergent
crystals,
> you might ask to see information about alpha-anomer contamination for
your batch
> of detergent.  Companies like Anatrace will be quite forthcoming with
> information, but larger companies (Sigma or Rohm & Haas) may give you the
run
> around.
> 
> 
>   Good luck,
> 
> 
>   Michael
> 
> 
>   
> 
>   R. Michael Garavito, Ph.D.
> 
>   Professor of Biochemistry & Molecular Biology
> 
>   513 Biochemistry Bldg.   
> 
>   Michigan State University  
> 
>   East Lansing, MI 48824-1319
> 
>   Office:  (517) 355-9724 Lab:  (517) 353-9125
> 
>   FAX:  (517) 353-9334Email:  garav...@msu.edu
> 
>   
> 
> 
> 
> 
> 
>   On Aug 4, 2009, at 12:51 PM, Van Den Berg, Bert wrote:
> 
> 
> Hi Jose,
> 
> how do you know that those crystals were detergent and not protein?
My
> impression is that it is really hard to crystallize DDM, and even harder
for DM
> (solubilities > 20% in water). The easiest (?) way to check this may be
to take
> some crystals, wash them well and run them out on a PAGE gel. If you
don't see
> anything and you've taken enough crystals, then you're probably dealing
with
> pure detergent crystals. As for your second point, you

Re: [ccp4bb] detergent crystals

[Jason Hurlbert]

On 8/5/09 5:30 AM, "Jose Antonio Cuesta Seijo"  wrote:

> Jakob,
> 
> That Compacdt disc appearance is very familiar, those were right next to
> the hexagonal ones in my experiments. And all were oily when touched. It is
> very likely that my "detergent" crystals were in reality detergent-protein
> complexes with less than crystalline order. I was doing experiments with
> very high detergent concentrations (up to 6%) and in some cases the
> crystals would be there in about 50% of the conditions. Their numbers also
> correlated better with the detergent concentration than with the protein
> concentration, but all that is still compatible with protein-detergent
> complexes, of course.
> Regarding measuring the final detergent concentration, a fast method is
> described in :
> 
> "A strategy for identification and quantification of detergents frequently
> used in the purification of membrane proteins." Laura R. Eriks, June A.
> Mayor and Ronald S. Kaplan
> Analytical Biochemistry Volume 323, Issue 2, 15 December 2003, Pages
> 234-241 
> 
> It uses TLC, and the protein crystallization stock can be spotted directly,
> water and all. The standard can also be in water. In my hands, the crucial
> step was to dry this water thoroughly before running the TLC. I adapted
> this to small TLC plates which can be run in a sealed beaker. Running your
> sample in between appropriate standards will give you an estimation of your
> detergent concentration in as little as 2 hours.
> 
> Cheers.
> 
> Jose. 
> 
> 
> 
> "Jacob Keller"  wrote:
>> A recommendation: try looking at the crystals while rotating the
> polarizers.
>> Often you can get detergent or detergent-protein complex "crystals" which
> have
>> sharp edges, but are actually liquid crystals. This will be manifest as a
>> compact-disc (or vinyl LP, depending on your vintage) appearance which
> rotates
>> in sync with the rotation of the polarizers. Several colleagues and I
> have been
>> plagued with these false positives, which are in our experience extremely
> hard
>> to optimize into real crystals.
>> 
>> Another possibility: crystallization with a fluorescent or otherwise
> detectable
>> substrate analogue could also be helpful, at least for determining
> whether there
>> is protein in the sharp-edged objects.
>> 
>> The best test, of course, is to mount the objects and put them in the
> x-ray
>> beam.
>> 
>> Regards,
>> 
>> Jacob Keller
>> 
>> 
>> ***
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> Dallos Laboratory
>> F. Searle 1-240
>> 2240 Campus Drive
>> Evanston IL 60208
>> lab: 847.491.2438
>> cel: 773.608.9185
>> email: j-kell...@northwestern.edu
>> ***
>> 
>>   - Original Message -
>>   From: R.M. Garavito
>>   To: CCP4BB@JISCMAIL.AC.UK
>>   Sent: Tuesday, August 04, 2009 12:37 PM
>>   Subject: Re: [ccp4bb] detergent crystals
>> 
>> 
>>   Parveen,
>> 
>> 
>>   Bert and Pascal are correct in that most alkyl glycoside detergent are
>> notoriously difficult to crystallize in aqueous solution when you have
> the
>> beta-anomer (what we normally buy).  However, the alpha-anomers can be
> quite
>> easy to crystallize and can contaminate batches of beta-alkyl glycoside
>> detergents.  While the quality control procedures are usually good enough
> to
>> ensure that the alpha-anomer contamination of DDM, DM, and OG are low, it
> may
>> not be low enough for all crystallization experiments.  Twenty or so
> years ago,
>> I was even shown a batch of "pure" beta-OG from a company I shall not
> name which
>> was insoluble in water.
>> 
>> 
>>   Some people have complained about this, but the impact of alpha-anomer
>> contamination on crystal growth and spurious detergent crystallization is
>> unknown.  If this persists and you are sure that those are detergent
> crystals,
>> you might ask to see information about alpha-anomer contamination for
> your batch
>> of detergent.  Companies like Anatrace will be quite forthcoming with
>> information, but larger companies (Sigma or Rohm & Haas) may give you the
> run
>> around.
>> 
>> 
>>   Good luck,
>> 
>> 
>>   Michael
>> 
>> 
>>   
>> 
>>   R. Michael Garavito, Ph.D.
>> 
>>   Professor of Biochemistry & Molecular Biology
>> 
>>   513 Biochemistry Bldg.
>> 
>>   Michigan State University
>> 
>>   East Lansing, MI 48824-1319
>> 
>>   Office:  (517) 355-9724 Lab:  (517) 353-9125
>> 
>>   FAX:  (517) 353-9334Email:  garav...@msu.edu
>> 
>>   
>> 
>> 
>> 
>> 
>> 
>>   On Aug 4, 2009, at 12:51 PM, Van Den Berg, Bert wrote:
>> 
>> 
>> Hi Jose,
>> 
>> how do you know that those crystals were detergent and not protein?
> My
>> impression is that it is really hard to crystallize DDM, and even harder
> for DM
>> (solubilities > 20% in water). The easiest (?) 

[ccp4bb] Restraint hydrogen bond Refmac

[Kristof Van Hecke]

Dear,

When (trying) to refine a DNA-structure (resolution 2.5) using  
Refmac_5.5.0072 (CCP4 6.1.1), some of the H-bonds between Watson- 
Crick bases are becoming too large.


Reducing the Matrix weighting term to tighten the geometry, doesn't  
effect these H-bond distances much.

Reducing the "VDW SIGMA HBOND" also doesn't solve the problem.

Adding "external distance restraints" does the trick, but some B- 
factors (not the ones involved in H-bonding!) blow up completely.


Hence, what's actually the best way of tighten H-bond restraints in  
Refmac, or am I overlooking some other issues here..?



Thank you very much.

Regards

Kristof Van Hecke

--
Kristof Van Hecke, PhD
Biomoleculaire Architectuur
Celestijnenlaan 200 F
B-3001 Heverlee (Leuven)
Tel: +32(0)16327477
--

Re: [ccp4bb] Restraint hydrogen bond Refmac

[Ian Tickle]

Hi Kristof

AFAIK there's no attractive H-bond term in Refmac, only a repulsive one,
and even that is probably between the N/O atoms not between the H atoms
themselves, which only 'ride' on their parent atoms (again this is to my
knowledge).  This is all related to the fact that H atoms don't scatter
X-rays very well!  So it all depends on the bases refining to their true
positions which obviously depends on resolution, data quality, thermal
motion and disorder etc.

HTH (though probably not very helpful!)

-- Ian

> -Original Message-
> From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk]
On
> Behalf Of Kristof Van Hecke
> Sent: 05 August 2009 10:53
> To: bulletin_ccp4
> Subject: Restraint hydrogen bond Refmac
> 
> Dear,
> 
> When (trying) to refine a DNA-structure (resolution 2.5) using
> Refmac_5.5.0072 (CCP4 6.1.1), some of the H-bonds between Watson-Crick
> bases are becoming too large.
> 
> Reducing the Matrix weighting term to tighten the geometry, doesn't
effect
> these H-bond distances much.
> Reducing the "VDW SIGMA HBOND" also doesn't solve the problem.
> 
> Adding "external distance restraints" does the trick, but some
B-factors
> (not the ones involved in H-bonding!) blow up completely.
> 
> Hence, what's actually the best way of tighten H-bond restraints in
> Refmac, or am I overlooking some other issues here..?
> 
> 
> Thank you very much.
> 
> Regards
> 
> Kristof Van Hecke
> 
> 
> --
> Kristof Van Hecke, PhD
> Biomoleculaire Architectuur
> Celestijnenlaan 200 F
> B-3001 Heverlee (Leuven)
> Tel: +32(0)16327477
> --
> 
> 



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[ccp4bb] question

[Chandra Verma]

Dear all

Can anyone suggest examples where the same peptide is bound to the same 
receptor site in two or more different (very different) conformations 
and this binding has been resolved crystallographically as well as using 
ITC. In the extreme cases this could be two different proteins as well 
interacting through the same interface but with very different surfaces.


the more diverse the interfacial conformations, the better.

thanks

chandra

[ccp4bb] Postdoctoral position in x-ray crystallography at Birkbeck

[Gabriel Waksman]

ref 10289
Full time and fixed term until 31 March 2013

The School of Crystallography/Institute of Structural and Molecular  
Biology is seeking a Post-doctoral Research Assistant to carry out  
structural analysis (x-ray crystallography or cryo-electron  
microscopy) of complexes formed during type IV secretion. The group is  
led by Professor Gabriel Waksman and has over the years produced  
numerous high profile publications in the highest impact journals. The  
research programme is funded by a grant from the Wellcome Trust to  
Prof Gabriel Waksman. Further details on the research group and on  
type IV secretion can be found at http://people.cryst.bbk.ac.uk/~ubcg54a/ 
.


Applicants should have a PhD in Structural Molecular Biology,  
Biophysics or a related area, postdoctoral research experience and  
relevant research publications.


Salary range will be from £33,501 to £40,558 per annum inclusive of  
London Allowance on Grade 7 or 8, initial salary will be dependent on  
the skills and experience of the successful applicant.


Closing date: Monday 31st August 2009

To apply for this post and for further information please visit: www.bbk.ac.uk/jobs 
  (Search using reference number 10289)

Re: [ccp4bb] Restraint hydrogen bond Refmac

[Anastassis Perrakis]

Hi Kristof -

I think that you are overlooking at the fact that you do not know what  
the hydrogen bond lengths are between the base pairs.
You should make sure that the covalent geometry is correct (tighten or  
loosen the matrix as you do!) and then when you
have a well refined structure, analyze your DNA for deviations from  
'ideality' eg ideal A or B DNA.


Longer-than-text-book hydrogen bonds, as well as the often ignored  
pucker amplitudes of your sugars, carry valuable

information about DNA conformation, likely showing:
a. the strain due of your bound protein (I am assuming here you have  
protein bound to it).
b. the properties of the particular sequence (assuming no protein  
bound).


The B-DNA-like sugar puckers, must be a very common mistake in the  
PDB, especially for bend DNA, legacy of an 'energy term'
causing the sugar puckers to be more tightly restrained to B-DNA than  
covalent bonds to Engh-Huber values, in (older versions?) of CNS.
That, together with retraining the hydrogen bonds in DNA as far as I  
recall, made DNA look good, but did not necessarily made it more  
correct.
Good old PROTIN/PROLSQ had an even worse bug ignoring chiral centers,  
but then not many people refined DNA with PROTIN/PROLSQ.


Refmac, Phenix and Buster seem to handle DNA geometry right (for my  
taste, eg they leave puckers and h bonds unrestrained)

(I am not sure what CNS does these days).

Sorry for the more general review. The short answer is that I would  
leave the H-bonds alone, make sure the rest is Ok,
and see what is the underlying reason they are longer than usual: this  
is likely to be informative at the end.


Tassos

On Aug 5, 2009, at 11:52, Kristof Van Hecke wrote:


Dear,

When (trying) to refine a DNA-structure (resolution 2.5) using  
Refmac_5.5.0072 (CCP4 6.1.1), some of the H-bonds between Watson- 
Crick bases are becoming too large.


Reducing the Matrix weighting term to tighten the geometry, doesn't  
effect these H-bond distances much.

Reducing the "VDW SIGMA HBOND" also doesn't solve the problem.

Adding "external distance restraints" does the trick, but some B- 
factors (not the ones involved in H-bonding!) blow up completely.


Hence, what's actually the best way of tighten H-bond restraints in  
Refmac, or am I overlooking some other issues here..?



Thank you very much.

Regards

Kristof Van Hecke

--
Kristof Van Hecke, PhD
Biomoleculaire Architectuur
Celestijnenlaan 200 F
B-3001 Heverlee (Leuven)
Tel: +32(0)16327477
--





P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

[ccp4bb] Postdoctoral Position Announcement

[Raquel Lieberman]

Dear All,

The Lieberman Lab, located in the School of Chemistry & Biochemistry  
at the Georgia Institute of Technology (Atlanta, GA) invites highly  
motivated individuals interested in the field of protein misfolding  
diseases and/or membrane protein crystallography to join our research  
team. The successful Postdoctoral Research Fellow candidate will work  
on a multidisciplinary project involving protein expression,  
biochemical and biophysical characterization, and crystallization.


Individuals should have research experience in a related field  
(biochemistry/molecular biophysics) or technique (macromolecular  
crystallography) and hold a Ph.D. degree. The initial appointment will  
be for one year, with renewal up to three years subject to  
satisfactory progress and mutual agreement.  The position is available  
immediately, but timing can be flexible.


To apply, please email a (1) a cover letter describing your interest  
in the position, (2) the names and contact information for three  
references, (3) a curriculum vita (including publications) to Raquel  
L. Lieberman, PhD: raquel.lieber...@chemistry.gatech.edu.


The Lieberman laboratory is supported by grants from the National  
Science Foundation, American Federation for Aging, Glaucoma Research  
Foundation, and the American Health Assistance Foundation. The  
position will provide competitive salaries and benefits. Georgia Tech  
is a member of SER-CAT at APS, and is an equal opportunity employer.


Thank you for your interest.

Raquel L. Lieberman, Ph.D.
Assistant Professor
School of Chemistry and Biochemistry
Georgia Institute of Technology
901 Atlantic Drive, NW
Atlanta, GA 30332-0400
e-mail: raquel.lieber...@chemistry.gatech.edu
Phone: (404) 385-3663
Faculty website: http://www.chemistry.gatech.edu/faculty/Lieberman
Laboratory website: http://web.chemistry.gatech.edu/~lieberman/liebermanlab/

[ccp4bb] Making choice on screw axes on resolution with HA sites.

[Francis E Reyes]

Hi all

I did some HA searching and found some sites in C222 that seem to be  
NCS related. Any ideas on how to determine whether or not the space  
group is really C2221 from these sites?


Thanks

FR

-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] Making choice on screw axes on resolution with HA sites.

[Eleanor Dodson]

I would expect that if you found HA sites in C222 then they must obey 
C222 symmetry?


Why not just repeat the search in C2221 and see if you get a better answer?


Going back a stage:
Arent you able to guess whether there is a screw axis from your absences 
along 00l?


Pointless gives a good analysis of likely SGs from these sorts of 
considerations.


The results are good if
a) you have actually measured the 00l line, and
b) you dont have a non-crystallographic translation with coordinates, 
x,y,1/2. 

Both ctruncate (part of the scale and merge task) or sfcheck will test 
for this.


 Eleanor





Francis E Reyes wrote:

Hi all

I did some HA searching and found some sites in C222 that seem to be 
NCS related. Any ideas on how to determine whether or not the space 
group is really C2221 from these sites?


Thanks

FR

-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

[ccp4bb] Protein Biochemistry Positions at Merck

[Soisson, Stephen M]

Hello All-

I figured I would post this here since there are some protein
biochemists who frequent the site, and many of the crystallographers may
know of protein biochemists who might be looking for a job.  Please pass
this along to any potentially interested parties.

Thanks!

Steve

P.S. Please do not respond to me directly if interested in the position,
our HR group requires that you apply through the online tool.

---
Stephen M. Soisson, Ph.D.
Global Structural Biology
Merck Research Laboratories
Phone: (215) 652-6185


Merck & Co. Inc., established in 1891, is a global research-driven
pharmaceutical company dedicated to putting patients first. 
Join us and experience our culture first-hand - one of strong ethics &
integrity, diversified experiences and a resounding passion for
improving human health. As part of our global team, you'll have the
opportunity to collaborate with talented and dedicated colleagues while
developing and expanding your career. 
This exciting position within Merck Global Structural Biology at the
West Point, PA site will enable drug lead discovery and optimization
through the production and characterization of proteins for use in X-ray
crystallography and screening. The incumbent will perform protein
purification and characterization of recombinant proteins from
prokaryotic and eukaryotic hosts. Duties will include exploring new
technologies to optimize purification methods, and communicating results
at departmental meetings.
Education Requirement: 
BS in Biochemistry or related discipline with 4+ years experience 
MS in Biochemistry or related discipline with 3+ years experience 
Required: 
Demonstrated success in high-quality purification of recombinant
proteins (milligram scale) from both eukaryotic and prokaryotic hosts,
using state-of-the-art technologies (e.g. AKTA FPLC/Purifier/Express) is
essential. 
Experience working with both untagged and affinity-tagged (e.g. His,
GST, FLAG, HA etc) recombinant proteins is required. 
Experience with protein characterization (e.g. SDS-PAGE, Western
analysis) and protein concentration determination via standard
spectroscopic methods (Bradford, A280nm etc). 
Demonstrated ability to scientifically plan and execute multiple
purification projects simultaneously, to ensure the team goals are met. 
Excellent communication skills (written, presentation, and oral). 
Ability to work in a team-oriented environment within timelines. 
Desired: 
Experience in the application of biophysical characterization methods
(e.g. Biacore, Thermofluor, mass spectrometry, protein NMR) and activity
analysis (e.g. enzyme kinetics) is a strong plus. 
Knowledge of molecular biology, structural biology or HTS is a plus. 
Familiarity with protein crystallization is a plus. 
Industry experience is a plus. 
Consistently cited as a great place to work, we discover, develop,
manufacture and market a wide range of vaccines and medicines to address
unmet medical needs. Each of our employees is joined by an extraordinary
sense of purpose - bringing Merck's finest achievements to people around
the world. 
We offer an excellent salary and an industry-ranked benefits program,
including tuition reimbursement, work-life balance initiatives and
developmental programs at all levels. Merck's retirement package
includes a pension plan and one of the best 401(k) plans in the nation. 
To be considered for this position, please visit our career site at
www.merck.com/careers to create a profile and submit your resume for
requisition # BIO002197. Merck is an equal opportunity employer, M/F/D/V
- proudly embracing diversity in all of its manifestations. 
Our work is someone's hope. Join us. 
Where patients come first - Merck 




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[ccp4bb] Zinc or Iron binding protein, that is a question!

[Xuan Yang]

Dear Sir or Madam,

The ICP-ES results indicated that 1 molar my protein purified from E.coli
Origami(DE3) contained about a half molar Zinc and nearly a quarter
molar Iron (whether II or III was not available). The protein carried a MBP
tag on the N-terminal and the situation was similar with or without His tag
at the C terminal. I want to determine whether my protein really bind Zinc
or Iron. Does anyone have any experience about such problems?

Specifically, now I want to compare the binding efficiency on various IMAC,
i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or CuSO4(control).
However,  considering the instability of Fe(II) in solution, the design
still seemed problematic.

Sincerely,

Xuan Yang

National Laboratory of Biomacromolecules and
Center for Infection and Immunity,
Institute of Biophysics,
Chinese Academy of Sciences,
Room 1617, 15 DaTun Road,Chaoyang District,
Beijing, China, 100101
Tel: 86-10-64884329
Academic email: ya...@moon.ibp.ac.cn
We will either find a way or make one.

Re: [ccp4bb] Zinc or Iron binding protein, that is a question!

[Guenter Fritz]

Hi Xuan,
I guess your protein is not an E.coli protein. There are several 
examples that eukaryotic Zn-proteins expressed in E.coli contain Fe 
instead of Zn. I am sceptic whether IMAC with different metal ions will 
give the solution of the problem. If you really want to get information 
on the metal ion binding properties you will have to do some matallo 
biochemistry: preparing apo protein, reconstitution with metal ions, 
UV-Vis spectroscopy, EPR would be great, ...



Dear Sir or Madam,
 
The ICP-ES results indicated that 1 molar my protein purified from 
E.coli Origami(DE3) contained about a half molar Zinc and nearly a 
quarter molar Iron (whether II or III was not available). The protein 
carried a MBP tag on the N-terminal and the situation was similar with 
or without His tag at the C terminal. I want to determine whether my 
protein really bind Zinc or Iron. Does anyone have any experience 
about such problems?
 
Specifically, now I want to compare the binding efficiency on various 
IMAC, i.e. 50mM ZnSO4, FeSO4, Fe2(SO4)3, NiSO4(control), or 
CuSO4(control). However,  considering the instability of Fe(II) in 
solution, the design still seemed problematic.
 
Sincerely,
 
Xuan Yang
 
National Laboratory of Biomacromolecules and

Center for Infection and Immunity,
Institute of Biophysics,
Chinese Academy of Sciences,
Room 1617, 15 DaTun Road,Chaoyang District,
Beijing, China, 100101
Tel: 86-10-64884329
Academic email: ya...@moon.ibp.ac.cn 
We will either find a way or make one.