[ccp4bb] post doc position Genome Institute of Singapore

[Prasanna Ratnakar KOLATKAR]

Dear all

 

There is at least 1 post doctoral position available at the Genome
Institute of Singapore in the lab of Dr Prasanna R Kolatkar. We are
working primarily with transcription factors related to stem cell and
developmental biology.  Although the attached advertisement is primarily
for a computational position, we will also consider people from
molecular biology/biochemistry background.   The contact email given in
the attached document is [EMAIL PROTECTED] but I would also
be happy to discuss and elaborate on any specifics regarding the science
if you like.

 

Prasanna R Kolatkar

Group Leader

Genome Institute of Singapore

[EMAIL PROTECTED] 

 

 



GIS_cryst_postdoc.doc
Description: GIS_cryst_postdoc.doc

[ccp4bb] Quick-soak

[amit sharma]

Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the presence of
0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
I intend to perform heavy metal derivatization. I read some literature which
suggested that I could carry out quick soak with 0.5M Sodium iodide. I was
wondering if I could soak my crystals with a similar concentration of NaBr
and collect some low resolution data at the in-house source? In addition
should I try to also introduce other heavy metals? it might be worth
mentioning that my protein carries no methionine/cys residues.
I have no prior experience in doing this. So, it would  be of great help if
I could be directed towards literature/protocols pertaining to this. Any
advice/suggestions would be greatly appreciated.

Thanks in advance
-- 
Amit Sharma

Re: [ccp4bb] Quick-soak

[David Briggs]

Hi Amit,

http://www.doe-mbi.ucla.edu/~sawaya/tutorials/Phasing/references.html

And the (IMHO) seminal heavy atom derivative reference:

Petsko, G.A., "Perparation of Isomorphous Heavy-Atom Derivatives"
Methods in Enzymology, Volume 114, , pages 147-157.

should give you all the info you need.

The 0.15M KBr might give you a usable anomalous signal, but the only
way you'll be able to determine that is to go ahead and do it!
the f" of Br @ CuKa is ~1.2e-, with careful data collection, you might
get lucky.

Cheers,

David

2008/9/25 amit sharma <[EMAIL PROTECTED]>:
>
> Dear CCP4bbers,
> I have a protein molecule(~9.0 kDa) that crystallized in the presence of
> 0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
> I intend to perform heavy metal derivatization. I read some literature which
> suggested that I could carry out quick soak with 0.5M Sodium iodide. I was
> wondering if I could soak my crystals with a similar concentration of NaBr
> and collect some low resolution data at the in-house source? In addition
> should I try to also introduce other heavy metals? it might be worth
> mentioning that my protein carries no methionine/cys residues.
> I have no prior experience in doing this. So, it would  be of great help if
> I could be directed towards literature/protocols pertaining to this. Any
> advice/suggestions would be greatly appreciated.
>
> Thanks in advance
> --
> Amit Sharma



-- 

David C. Briggs PhD
Father & Crystallographer
http://drdavidcbriggs.googlepages.com/home
AIM ID: dbassophile

Re: [ccp4bb] Quick-soak

[M T]

2008/9/25 amit sharma <[EMAIL PROTECTED]>
>
> Dear CCP4bbers,
> I have a protein molecule(~9.0 kDa) that crystallized in the presence of 
> 0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present, 
> I intend to perform heavy metal derivatization. I read some literature which 
> suggested that I could carry out quick soak with 0.5M Sodium iodide. I was 
> wondering if I could soak my crystals with a similar concentration of NaBr 
> and collect some low resolution data at the in-house source? In addition 
> should I try to also introduce other heavy metals? it might be worth 
> mentioning that my protein carries no methionine/cys residues.
> I have no prior experience in doing this. So, it would  be of great help if I 
> could be directed towards literature/protocols pertaining to this. Any 
> advice/suggestions would be greatly appreciated.
>
> Thanks in advance
> --
> Amit Sharma

One solution can be soaking or co-crystallization with lanthanide complexes.
More information available in these publications (PubMed):
PMID: 15272192
PMID: 14573945
PMID: 12499547
PMID: 18350532

Re: [ccp4bb] Quick-soak

[Jacob Keller]

You should try collecting a data set at the Br- edge, and perhaps other 
wavelengths for MAD. I would think that you will locate at least one or two 
Br-'s, which should be plenty with only 9 kD. If you want, you could collect 
similar crystals with either KCl or KI, then do either SIRAS or MIRAS.

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: [EMAIL PROTECTED]
***

  - Original Message - 
  From: amit sharma 
  To: CCP4BB@JISCMAIL.AC.UK 
  Sent: Thursday, September 25, 2008 6:03 AM
  Subject: [ccp4bb] Quick-soak



  Dear CCP4bbers,
  I have a protein molecule(~9.0 kDa) that crystallized in the presence of 
0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present, I 
intend to perform heavy metal derivatization. I read some literature which 
suggested that I could carry out quick soak with 0.5M Sodium iodide. I was 
wondering if I could soak my crystals with a similar concentration of NaBr and 
collect some low resolution data at the in-house source? In addition should I 
try to also introduce other heavy metals? it might be worth mentioning that my 
protein carries no methionine/cys residues.
  I have no prior experience in doing this. So, it would  be of great help if I 
could be directed towards literature/protocols pertaining to this. Any 
advice/suggestions would be greatly appreciated.

  Thanks in advance
  -- 
  Amit Sharma 

Re: [ccp4bb] Quick-soak

[Eric Larson]

Hi Amit,

Dr. Dauter has several excellent papers about solving crystal structures with 
halide ions.  Use this search string in PubMed (without the quotes) to retrieve them: "dauter [AUTH] AND halide".  The anomalous signal of iodide can be detected using a home x-ray source but the anomalous signal of bromide generally requires a synchrotron source.


Good luck,
Eric

__
Eric Larson, PhD
MSGPP Consortium
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

On Thu, 25 Sep 2008, amit sharma wrote:


Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the presence of
0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
I intend to perform heavy metal derivatization. I read some literature which
suggested that I could carry out quick soak with 0.5M Sodium iodide. I was
wondering if I could soak my crystals with a similar concentration of NaBr
and collect some low resolution data at the in-house source? In addition
should I try to also introduce other heavy metals? it might be worth
mentioning that my protein carries no methionine/cys residues.
I have no prior experience in doing this. So, it would  be of great help if
I could be directed towards literature/protocols pertaining to this. Any
advice/suggestions would be greatly appreciated.

Thanks in advance
--
Amit Sharma

[ccp4bb] disulfide bonds, SE sample, and Xray absorption edges

[Michael Jackson]

Hello,
  I had recently collected and solved the phases for a protein molecule using 
CCP4 and the ShelXCDE SAD method in it.  What I was wondering was that the 
peaks for the three SE incorporated methionines are there as expected, but 
there is one peak scored roughly as the second largest where the disulfide is 
based on ShelXD's HA search algorithm. This data was collected at 0.97960 
Angstroms which is close to the peak Xray absorption edge for Se but does 
anyone know if a disulfide has any absorption edge overlapping here?



  

Re: [ccp4bb] disulfide bonds, SE sample, and Xray absorption edges

[George M. Sheldrick]

Unexpected peaks in a S-SAD experiment sometimes turn out to be chloride, 
sulfate or a metal ion. I would suggest that you run shelxd with and 
without the disulfide option (or with different numbers of disulfides)
to see which is best, and also run SHELXE with the -b flag set. This will 
produce an analysis of the anomalous density at the sites that you 
inputted and a list of peaks in the anomalous map. I also suggest that 
you look at the anomalous density by feeding the .pha file from SHELXE
(generated if -b is set) into e.g. Coot.

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Thu, 25 Sep 2008, Michael Jackson wrote:

> Hello,
>   I had recently collected and solved the phases for a protein molecule using 
> CCP4 and the ShelXCDE SAD method in it.  What I was wondering was that the 
> peaks for the three SE incorporated methionines are there as expected, but 
> there is one peak scored roughly as the second largest where the disulfide is 
> based on ShelXD's HA search algorithm. This data was collected at 0.97960 
> Angstroms which is close to the peak Xray absorption edge for Se but does 
> anyone know if a disulfide has any absorption edge overlapping here?
> 
> 
> 
> 

Re: [ccp4bb] disulfide bonds, SE sample, and Xray absorption edges

[Jim Pflugrath]

There is ALWAYS anomalous scattering.  You do not have be at the 
absorption edge to get it.  The question is just whether your experiment 
is good enough to detect it.


So your question of "overlapping" always has the answer Yes, but I would 
remove the words "absorption edge" from your question.


Jim

On Thu, 25 Sep 2008, Michael Jackson wrote:

Hello, ? I had recently collected and solved the phases for a protein 
molecule using CCP4 and the ShelXCDE SAD method in it.? What I was 
wondering was that the peaks for the three SE incorporated methionines 
are there as expected, but there is one peak scored roughly as the 
second largest where the disulfide is based on ShelXD's HA search 
algorithm. This data was collected at 0.97960 Angstroms which is close 
to the peak Xray absorption edge for Se but does anyone know if a 
disulfide has any absorption edge overlapping here?

Re: [ccp4bb] Quick-soak

[Diana Tomchick]

Or you could try crystallizing the protein in the presence of KI or  
NaI, and collect some in-house SAD data. You could also try to boost  
the concentration and number of ordered halide sites by quick soaking  
the crystals with a higher concentration of the iodide salt.


In my limited experience with iodide soaks, the crystals often crack  
with high concentrations (close to 1.0 M) of the iodide salt, but  
lower concentrations (close to 0.1-0.2 M) of the salt don't result in  
a high enough anomalous signal to phase the structure. I've never  
tried it with such a small protein, so perhaps you will be successful.


Diana

On Sep 25, 2008, at 6:03 AM, amit sharma wrote:



Dear CCP4bbers,
I have a protein molecule(~9.0 kDa) that crystallized in the  
presence of 0.15M KBr and HEPES pH 7.0. Since there is no  
homologuous structure present, I intend to perform heavy metal  
derivatization. I read some literature which suggested that I could  
carry out quick soak with 0.5M Sodium iodide. I was wondering if I  
could soak my crystals with a similar concentration of NaBr and  
collect some low resolution data at the in-house source? In addition  
should I try to also introduce other heavy metals? it might be worth  
mentioning that my protein carries no methionine/cys residues.
I have no prior experience in doing this. So, it would  be of great  
help if I could be directed towards literature/protocols pertaining  
to this. Any advice/suggestions would be greatly appreciated.


Thanks in advance
--
Amit Sharma


* * * * * * * * * * * * * * * * * * * * * * * * * * * *
Diana R. Tomchick
Associate Professor
University of Texas Southwestern Medical Center
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214B   
Dallas, TX 75390-8816, U.S.A.   
Email: [EMAIL PROTECTED]
214-645-6383 (phone)
214-645-6353 (fax)

Re: [ccp4bb] disulfide bonds, SE sample, and Xray absorption edges

[Ethan Merritt]

On Thursday 25 September 2008 08:45:10 Michael Jackson wrote:

>   This data was collected at 0.97960 Angstroms which is close to the peak 
>   Xray absorption edge for Se but does anyone know if a disulfide has any 
>   absorption edge overlapping here?   

http://skuld.bmsc.washington.edu/scatter/AS_form.html

-- 
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

Re: [ccp4bb] disulfide bonds, SE sample, and Xray absorption edges

[konstantin v. korotkov]

Your case might be different, but it could also be a true Se signal from 
Se-Cys incorporated into your protein during Se-Met expression. Depending 
on the protocol you used, Se may get incorporated into Cys, especially if 
only source of "sulfur" is Se-Met. We have seen such signals from Cys- 
containing proteins produced in E.coli. Nice thing is that you get extra 
sites to use in phasing!


Konstantin

K.Korotkov,
University of Washington
Department of Biochemistry
Box 357742
Seattle, WA 98195


On Thu, 25 Sep 2008, George M. Sheldrick wrote:



Unexpected peaks in a S-SAD experiment sometimes turn out to be chloride,
sulfate or a metal ion. I would suggest that you run shelxd with and
without the disulfide option (or with different numbers of disulfides)
to see which is best, and also run SHELXE with the -b flag set. This will
produce an analysis of the anomalous density at the sites that you
inputted and a list of peaks in the anomalous map. I also suggest that
you look at the anomalous density by feeding the .pha file from SHELXE
(generated if -b is set) into e.g. Coot.

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Thu, 25 Sep 2008, Michael Jackson wrote:


Hello,
  I had recently collected and solved the phases for a protein molecule using 
CCP4 and the ShelXCDE SAD method in it.  What I was wondering was that the 
peaks for the three SE incorporated methionines are there as expected, but 
there is one peak scored roughly as the second largest where the disulfide is 
based on ShelXD's HA search algorithm. This data was collected at 0.97960 
Angstroms which is close to the peak Xray absorption edge for Se but does 
anyone know if a disulfide has any absorption edge overlapping here?

Re: [ccp4bb] Quick-soak

[Matt Vetting]

 We have had really outstanding success using trimethyllead and 
samarium acetate as 'quick soak' derivatives. Both are soluble to 100 mM 
in most crystallization solutions and have large anomalous signals at the 
copper-kalpha wavelength used at most home sources. A quick 30 sec to 5 
minute soak and collect as much data (180-360 degrees) as quickly as 
possible. We have been able to solve many datasets by SAD alone on our 
home source (R-AXIS-IV++ - RUH3R generator). Pb and Sm are bound by acidic 
residues. Post mortum of 'quick soak' datasets made in such a manner 
usually indicate 1-3 strong sites which are usually heavy atoms 
coordinated by more than on acidic group, 3-4 medium occupancy sites, and 
5-10 low occupancy sites. Software such as BnP and PHENIX will readily 
find and phase well collected (high accuracy) 'quick soak' datasets.

[ccp4bb] Deglycosylation

[Eugenio De la Mora]

Dear all,
We have some troubles with the cristallization of glycosylated proteins and 
want to try to deglycosate them. We have never done this so we want to know 
what enzymes are the best (efficiency; less protein loss, ... ). And if  it 
gaves better results in crystallization screens.

Thank you

Eugenio De la Mora
Instituto de Biotecnologia 
Universidad NAcional Autonoma de Mexico


  

Re: [ccp4bb] Deglycosylation

[A. Radu Aricescu]

Dear Eugenio,

I believe the paper by Chang VT et al 2007 (PMID: 17355862) might offer the 
answers you're after (assuming N-glycosylation is your main concern). For 
O-linked sugars, the situation is much more complex, reflecting their 
diversity: mucin-type can usually be dealt with by construct design (eliminate 
Ser/Thr/Pro-rich domains), for other types have a look at 
http://www.prozyme.com/pdf/gk80110.pdf for example.

Best wishes,

radu


--
A. Radu Aricescu, PhD
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive, Oxford OX3 7BN
United Kingdom
Phone: +44-1865-287551
Fax: +44-1865-287547


 Original message 
>Date: Thu, 25 Sep 2008 11:30:24 -0700
>From: Eugenio De la Mora <[EMAIL PROTECTED]>  
>Subject: [ccp4bb] Deglycosylation  
>To: CCP4BB@JISCMAIL.AC.UK
>
>Dear all,
>We have some troubles with the cristallization of glycosylated proteins and 
>want to try to deglycosate them. We have never done this so we want to know 
>what enzymes are the best (efficiency; less protein loss, ... ). And if  it 
>gaves better results in crystallization screens.
>
>Thank you
>
>Eugenio De la Mora
>Instituto de Biotecnologia 
>Universidad NAcional Autonoma de Mexico
>
>
>  

[ccp4bb] losing zinc during crystallization

[Sue Roberts]

Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what  
seems to me to be an eternity. The protein contains stoichiometric  
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required  
for activity.  Each crystal we've obtained has lost the zinc and  
contains a disulfide bond between two cysteine residues that should be  
zinc ligands (based on structures of similar proteins).


We've tried crystalizing in the presence of reducing agents,  
crystallizing with substrate analogs, and supplementing the  
crystallization drops with zinc with no success (and combinations of  
these approaches).  We've obtained a variety of crystals and  
determined structures, but none contain any zinc.


Attempts to insert zinc into the crystal (zinc + reducting agent or  
zinc alone) have not been successful.


Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

[ccp4bb] Off-topic: coomassie linearity?

[Jacob Keller]

Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is horribly 
non-linear in intensity per protein concentration, which leads me to two 
questions:


1. Does anybody have a reference for quantitation of coomassie's linearity 
(and possibly other stains), i.e. where and how extensive the linear range 
is, and

2. Can anybody suggest a more linear protein gel stain?

Thanks,

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: [EMAIL PROTECTED]
***

Re: [ccp4bb] Deglycosylation

[Joao Dias]

Hi Eugenio,
It will depend on the expressing system you are using: mamalian  
cells, insect cells, yeast.
For enzymatic deglycosylation I suggest you to do first do a small  
scale test, using the following enzymes:

PNGase F is extremely efficient.
EndoH (you can try EndoHf which is cheaper and )
Neuraminidase to remove the sialic acid (specially useful for  
mammalian cells).
You can check these enzymes at NEB or Prozyme (like Radu mentioned  
before. Check also his great paper Chang et al, 2007.).

http://www.neb.com/nebecomm/default.asp

You can also use glycosylation inhibitors like tunicamycin,  
kifunensine, swainsonine, etc...


Another approach is site-directed mutagenesis.

And yes, "usually" (this means not always...) it gives you better  
crystals which will diffract at higher resolution.


Good luck,
Joao

João M. Dias, Ph. D.
Ollmann Saphire Lab
The Scripps Research Institute
10550 North Torrey Pines Rd. IMM-2
La Jolla, CA 92037 USA
tel (858)784-8925
http://www.scripps.edu/~jmdias/
On Sep 25, 2008, at 1:40 PM, A. Radu Aricescu wrote:


Dear Eugenio,

I believe the paper by Chang VT et al 2007 (PMID: 17355862) might  
offer the answers you're after (assuming N-glycosylation is your  
main concern). For O-linked sugars, the situation is much more  
complex, reflecting their diversity: mucin-type can usually be  
dealt with by construct design (eliminate Ser/Thr/Pro-rich  
domains), for other types have a look at http://www.prozyme.com/pdf/ 
gk80110.pdf for example.


Best wishes,

radu


--
A. Radu Aricescu, PhD
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive, Oxford OX3 7BN
United Kingdom
Phone: +44-1865-287551
Fax: +44-1865-287547


 Original message 

Date: Thu, 25 Sep 2008 11:30:24 -0700
From: Eugenio De la Mora <[EMAIL PROTECTED]>
Subject: [ccp4bb] Deglycosylation
To: CCP4BB@JISCMAIL.AC.UK

Dear all,
We have some troubles with the cristallization of glycosylated  
proteins and want to try to deglycosate them. We have never done  
this so we want to know what enzymes are the best (efficiency;  
less protein loss, ... ). And if  it gaves better results in  
crystallization screens.


Thank you

Eugenio De la Mora
Instituto de Biotecnologia
Universidad NAcional Autonoma de Mexico

Re: [ccp4bb] losing zinc during crystallization

[Roger Rowlett]

Based on my own experience with zinc-metalloenzymes with thiolate 
ligands, it's usually more a problem to get the zinc OUT than get it IN. 
Zinc is pretty thiophilic, so removing it once ligated in a multiple Cys 
environment is often difficult. Have you tried TCEP as a reducing agent 
for protecting properly zinc-populated protein?


Cheers,

--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]


Sue Roberts wrote:

Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what
seems to me to be an eternity. The protein contains stoichiometric
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required
for activity.  Each crystal we've obtained has lost the zinc and
contains a disulfide bond between two cysteine residues that should be
zinc ligands (based on structures of similar proteins).

We've tried crystalizing in the presence of reducing agents,
crystallizing with substrate analogs, and supplementing the
crystallization drops with zinc with no success (and combinations of
these approaches).  We've obtained a variety of crystals and
determined structures, but none contain any zinc.

Attempts to insert zinc into the crystal (zinc + reducting agent or
zinc alone) have not been successful.

Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]
  

Re: [ccp4bb] Deglycosylation

[Mark Mayer]

One thing to watch out for: in addition to frustrating crystallographers those 
sugars play 
other roles in protein chemistry, and its not uncommon for the solubility of 
proteins to 
change dramatically after deglycosylation. 

Good luck!

MLM

Re: [ccp4bb] losing zinc during crystallization

[Eric]

We had a zinc-finger containing protein that we were soaking with different 
heavy atom compounds.  It turns out KAu(CN)2 provided the best diffraction 
of several soaks.  We found out it was because the gold had replaced the 
zinc ion and was coordinated by the Cys and His's.  The lab nickednamed the 
protein Goldfinger!  Although we didn't have the problem with disulfides, 
perhaps a similar gold soak might help if you tried to crystallize the 
protein in the presence of TCEP as well.


HTH

- Original Message - 
From: "Sue Roberts" <[EMAIL PROTECTED]>

To: 
Sent: Thursday, September 25, 2008 4:35 PM
Subject: losing zinc during crystallization



Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what
seems to me to be an eternity. The protein contains stoichiometric
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required
for activity.  Each crystal we've obtained has lost the zinc and
contains a disulfide bond between two cysteine residues that should be
zinc ligands (based on structures of similar proteins).

We've tried crystalizing in the presence of reducing agents,
crystallizing with substrate analogs, and supplementing the
crystallization drops with zinc with no success (and combinations of
these approaches).  We've obtained a variety of crystals and
determined structures, but none contain any zinc.

Attempts to insert zinc into the crystal (zinc + reducting agent or
zinc alone) have not been successful.

Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

Re: [ccp4bb] losing zinc during crystallization

[Jennifer Han-Chun Tsai]

I don't know if anyone had experience of TCEP inducing zinc acetate to
precipitate. This paper mentioned this.

The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution
*

Paul C. Smith, Stuart Firestein and John F. Hunt

Journal of Molecular
Biology
Volume 319, Issue
3,
7 June 2002, Pages 807-821
*

I saw if protein sample containing TCEP would either form precipitation or
weird and rounded shape of small molecule crystal but not salt. I have tried
to crush that rounded shape crystal. It's quite soft, very similar to the
way protein crystals got crushed.
Does anyone have this experience? Or actually this kind precipitation could
get nice crystal after optimization?

Thanks,
Jennifer Tsai

On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED]> wrote:

> We had a zinc-finger containing protein that we were soaking with different
> heavy atom compounds.  It turns out KAu(CN)2 provided the best diffraction
> of several soaks.  We found out it was because the gold had replaced the
> zinc ion and was coordinated by the Cys and His's.  The lab nickednamed the
> protein Goldfinger!  Although we didn't have the problem with disulfides,
> perhaps a similar gold soak might help if you tried to crystallize the
> protein in the presence of TCEP as well.
>
> HTH
>
> - Original Message - From: "Sue Roberts" <[EMAIL PROTECTED]>
> To: 
> Sent: Thursday, September 25, 2008 4:35 PM
> Subject: losing zinc during crystallization
>
>
>
> Hello Everyone
>>
>> I've been trying to crystallize a zinc-containing enzyme for what
>> seems to me to be an eternity. The protein contains stoichiometric
>> zinc  (1 zinc/ protein monomer) when isolated and the zinc is required
>> for activity.  Each crystal we've obtained has lost the zinc and
>> contains a disulfide bond between two cysteine residues that should be
>> zinc ligands (based on structures of similar proteins).
>>
>> We've tried crystalizing in the presence of reducing agents,
>> crystallizing with substrate analogs, and supplementing the
>> crystallization drops with zinc with no success (and combinations of
>> these approaches).  We've obtained a variety of crystals and
>> determined structures, but none contain any zinc.
>>
>> Attempts to insert zinc into the crystal (zinc + reducting agent or
>> zinc alone) have not been successful.
>>
>> Does anyone have any tricks to suggest that might help?
>>
>> Thanks in advance.
>>
>> Sue
>>
>> Dr. Sue A. Roberts
>> Biochemistry & Molecular Biophysics
>> University of Arizona
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Re: [ccp4bb] losing zinc during crystallization

[Artem Evdokimov]

Dear Sue,

This is a very interesting case! Normally the Zn-S bond is quite strong - so
it's an unusual situation to have. Did you already attempt to purify the
protein in the presence of tiny quantities of Zn? Also, what buffers and
other components are you using during purification, and what is the chemical
composition of the crystallization solution (if you don't mind telling)?
Finally, does the purified protein have high activity before
crystallization? In other terms - does it have full Zn occupancy before you
crystallize it - or does the disulphide form during expression or
purification? This would change some of the other solutions that you might
attempt.

Cheers,

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sue
Roberts
Sent: Thursday, September 25, 2008 5:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] losing zinc during crystallization

Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what  
seems to me to be an eternity. The protein contains stoichiometric  
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required  
for activity.  Each crystal we've obtained has lost the zinc and  
contains a disulfide bond between two cysteine residues that should be  
zinc ligands (based on structures of similar proteins).

We've tried crystalizing in the presence of reducing agents,  
crystallizing with substrate analogs, and supplementing the  
crystallization drops with zinc with no success (and combinations of  
these approaches).  We've obtained a variety of crystals and  
determined structures, but none contain any zinc.

Attempts to insert zinc into the crystal (zinc + reducting agent or  
zinc alone) have not been successful.

Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

Re: [ccp4bb] losing zinc during crystallization

[Artem Evdokimov]

Please note that TCEP decomposes and one of the decomposition products is
phosphate. Enough TCEP and you might have Zinc Phosphate crystals which can
sometimes look very odd and ‘protein looking’.

 

Artem

 

  _  

From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Jennifer Han-Chun Tsai
Sent: Thursday, September 25, 2008 6:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] losing zinc during crystallization

 

 

I don't know if anyone had experience of TCEP inducing zinc acetate to
precipitate. This paper mentioned this. 

The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution 

Paul C. Smith, Stuart Firestein and John F. Hunt

Journal of 
Molecular Biology
Volume
  319, Issue 3, 7 June 2002, Pages 807-821 

 

I saw if protein sample containing TCEP would either form precipitation or
weird and rounded shape of small molecule crystal but not salt. I have tried
to crush that rounded shape crystal. It's quite soft, very similar to the
way protein crystals got crushed.

Does anyone have this experience? Or actually this kind precipitation could
get nice crystal after optimization?

 

Thanks,

Jennifer Tsai

On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED]> wrote:

We had a zinc-finger containing protein that we were soaking with different
heavy atom compounds.  It turns out KAu(CN)2 provided the best diffraction
of several soaks.  We found out it was because the gold had replaced the
zinc ion and was coordinated by the Cys and His's.  The lab nickednamed the
protein Goldfinger!  Although we didn't have the problem with disulfides,
perhaps a similar gold soak might help if you tried to crystallize the
protein in the presence of TCEP as well.

HTH

- Original Message - From: "Sue Roberts" <[EMAIL PROTECTED]>
To: 
Sent: Thursday, September 25, 2008 4:35 PM
Subject: losing zinc during crystallization 





Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what
seems to me to be an eternity. The protein contains stoichiometric
zinc  (1 zinc/ protein monomer) when isolated and the zinc is required
for activity.  Each crystal we've obtained has lost the zinc and
contains a disulfide bond between two cysteine residues that should be
zinc ligands (based on structures of similar proteins).

We've tried crystalizing in the presence of reducing agents,
crystallizing with substrate analogs, and supplementing the
crystallization drops with zinc with no success (and combinations of
these approaches).  We've obtained a variety of crystals and
determined structures, but none contain any zinc.

Attempts to insert zinc into the crystal (zinc + reducting agent or
zinc alone) have not been successful.

Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]

 

Re: [ccp4bb] Off-topic: coomassie linearity?

[Juergen Bosch]

On 25 Sep 2008, at 14:35, Jacob Keller wrote:


Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is  
horribly non-linear in intensity per protein concentration, which  
leads me to two questions:


1. Does anybody have a reference for quantitation of coomassie's  
linearity (and possibly other stains), i.e. where and how extensive  
the linear range is, and

2. Can anybody suggest a more linear protein gel stain?

Silver stain would be an option. Not quite sure how the linearity is  
when using fluorescent probes e.g. SyproOrange for protein staining.


Jürgen


Thanks,

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: [EMAIL PROTECTED]
***


-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone:   +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

Re: [ccp4bb] losing zinc during crystallization

[Engin Ozkan]

Here is my two cents...

How strong zinc is captured by the protein is very protein dependent: I 
always thought that a great case for this variability was the protection 
of RING ubiquitin ligases against NEM. Cysteine-catalyzing HECT 
ubiquitin ligase are killed by NEM; RING finger ligases, where zinc is 
assumed to be only structural, are thought to be NEM resistant... Well, 
until you actually do the experiment and see that some RING ligases are 
also inactivated by NEM! My theory was that I had a case of weak zinc 
coordination, where oxidation of the zinc-coordinating cysteines was a 
big issue. DTT made it worse, TCEP kept the protein unaggregated the 
longest.


That's just one protein, though.

Engin

Artem Evdokimov wrote:


Please note that TCEP decomposes and one of the decomposition products 
is phosphate. Enough TCEP and you might have Zinc Phosphate crystals 
which can sometimes look very odd and ‘protein looking’.


Artem



*From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf 
Of *Jennifer Han-Chun Tsai

*Sent:* Thursday, September 25, 2008 6:18 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] losing zinc during crystallization

I don't know if anyone had experience of TCEP inducing zinc acetate to 
precipitate. This paper mentioned this.


The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution

*Paul C. Smith, Stuart Firestein and John F. Hunt*

*Journal of Molecular Biology 

Volume 319, Issue 3 
, 
7 June 2002, Pages 807-821 *


I saw if protein sample containing TCEP would either form 
precipitation or weird and rounded shape of small molecule crystal but 
not salt. I have tried to crush that rounded shape crystal. It's quite 
soft, very similar to the way protein crystals got crushed.


Does anyone have this experience? Or actually this kind precipitation 
could get nice crystal after optimization?


Thanks,

Jennifer Tsai

On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED] 
> wrote:


We had a zinc-finger containing protein that we were soaking with 
different heavy atom compounds. It turns out KAu(CN)2 provided the 
best diffraction of several soaks. We found out it was because the 
gold had replaced the zinc ion and was coordinated by the Cys and 
His's. The lab nickednamed the protein Goldfinger! Although we didn't 
have the problem with disulfides, perhaps a similar gold soak might 
help if you tried to crystallize the protein in the presence of TCEP 
as well.


HTH

- Original Message - From: "Sue Roberts" 
<[EMAIL PROTECTED] >

To: mailto:CCP4BB@JISCMAIL.AC.UK>>
Sent: Thursday, September 25, 2008 4:35 PM
Subject: losing zinc during crystallization



Hello Everyone

I've been trying to crystallize a zinc-containing enzyme for what
seems to me to be an eternity. The protein contains stoichiometric
zinc (1 zinc/ protein monomer) when isolated and the zinc is required
for activity. Each crystal we've obtained has lost the zinc and
contains a disulfide bond between two cysteine residues that should be
zinc ligands (based on structures of similar proteins).

We've tried crystalizing in the presence of reducing agents,
crystallizing with substrate analogs, and supplementing the
crystallization drops with zinc with no success (and combinations of
these approaches). We've obtained a variety of crystals and
determined structures, but none contain any zinc.

Attempts to insert zinc into the crystal (zinc + reducting agent or
zinc alone) have not been successful.

Does anyone have any tricks to suggest that might help?

Thanks in advance.

Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED] 

[ccp4bb] Reading the old literature / truncate / refinement programs

[Dunten, Pete W.]

 

I mentioned previously phenix.refine tosses your weak data if IMEAN, SIGIMEAN 
are chosen during refinement.

 

I'm wondering if this omission of weak Fobs from the Fobs-Fcalc difference map 
explains why the difference maps out of refmac seem to be more helpful in 
showing where to move atoms.  

 

D. Crowfoot et al. in The Chemistry of Penicillin (1949) explain why this might 
be so, and Stout & Jenson elaborate the argument.  Briefly, the calculated 
phase will be closest to the phase of the vector difference Fcalc - Fobs when 
|Fcalc| > |Fobs|.  

 

I leave it to the reader to try calculating some maps with and without the weak 
Fobs in phenix.refine or refmac, and perhaps making some deliberate rotamer 
errors, to see if using the complete data with weak Fobs helps.

Re: [ccp4bb] Off-topic: coomassie linearity?

[Gregory Alushin]

The Bio-Rad Flamingo Fluorescent Gel Stain has a very linear profile  
with protein concentration (if you believe the standard curves in the  
manual).  In my hands it gives nice results for binding assays.


Cheers,
-Greg Alushin

On Sep 25, 2008, at 2:35 PM, Jacob Keller wrote:


Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is  
horribly non-linear in intensity per protein concentration, which  
leads me to two questions:


1. Does anybody have a reference for quantitation of coomassie's  
linearity (and possibly other stains), i.e. where and how extensive  
the linear range is, and

2. Can anybody suggest a more linear protein gel stain?

Thanks,

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: [EMAIL PROTECTED]
***

Re: [ccp4bb] Off-topic: coomassie linearity?

[Steven Darnell]

Jacob,

Coomassie fluorescence is linear over a wide range of concentrations 
(citation below).  You need access to an infrared scanner in order to 
use this technique.  I've used a Li-cor Odyssey scanner with good 
results (http://www.licor.com/bio/odyssey/index.jsp).



Anal Biochem. 2006 Mar 15;350(2):233-8.
Quantitation of protein on gels and blots by infrared fluorescence of 
Coomassie blue and Fast Green.

Luo S, Wehr NB, Levine RL.

Coomassie blue staining of gels and blots is commonly employed for 
detection and quantitation of proteins by densitometry. We found that 
Coomassie blue or Fast Green FCF bound to protein fluoresces in the 
near infrared. We took advantage of this property to develop a rapid 
and sensitive method for detection and quantitation of proteins in 
gels and on blots. The fluorescence response is quantitative for 
protein content between 10 ng and 20 microg per band or spot. Staining 
and destaining require only 30 min, and the method is compatible with 
subsequent immunodetection.


PMID: 16336940

Regards,
Steve

--
Steven Darnell
University of Wisconsin-Madison
Madison, WI USA

Jacob Keller said the following, on 09/25/2008 04:35 PM:

Dear Crystallographers,

I remember having seen on this listserve that coomassie stain is 
horribly non-linear in intensity per protein concentration, which 
leads me to two questions:


1. Does anybody have a reference for quantitation of coomassie's 
linearity (and possibly other stains), i.e. where and how extensive 
the linear range is, and

2. Can anybody suggest a more linear protein gel stain?

Thanks,

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: [EMAIL PROTECTED]
***