Re: [ccp4bb] Substrate/Ligand Induced Oligomerization of enzymes

[Shiva Bhowmik]

Dear All,

Thank you for your responses. Below is the compiled list of examples of
substrate/ligand/co-factor induced oligomerization:
http://www.pnas.org/content/97/13/7096.full.pdf
http://www.ncbi.nlm.nih.gov/pubmed/15710608
http://www.ncbi.nlm.nih.gov/pubmed/17725819
http://www.ncbi.nlm.nih.gov/pubmed/14724278
http://www.ncbi.nlm.nih.gov/pubmed/16338409
http://pubs.acs.org/doi/abs/10.1021/bi301067w
http://pubs.acs.org/doi/abs/10.1021/bi2005637
http://pubs.acs.org/doi/abs/10.1021/bi201381e
http://pubs.acs.org/doi/full/10.1021/bi301593c

Have a great weekend. Cheers,

Shiva



On Thu, Aug 8, 2013 at 5:03 PM, Shiva Bhowmik  wrote:

> Dear All,
>
> I am looking for references and/or example of substrate or ligand induced
> oligomerization of enzymes related to activation.
>
> Any help in this regard would be greatly appreciated.
>
> Thanks.
>
> Shiva
>

[ccp4bb] Substrate/Ligand Induced Oligomerization of enzymes

[Shiva Bhowmik]

Dear All,

I am looking for references and/or example of substrate or ligand induced
oligomerization of enzymes related to activation.

Any help in this regard would be greatly appreciated.

Thanks.

Shiva

[ccp4bb] Ligand Based Macromolecular Crowding or Oligomerization?

[Shiva Bhowmik]

Dear All,

Wish you all a very happy new year. In this new year working on a problem
that I would like get your opinion.

Currently working on an enzyme that turnovers Coenzyme A (CoA)-conjugated
substrate. I have determined the structure of the enzyme and the structure
suggests to be a single domain protein. Based on the chemistry of the
reaction and the position of the active site residues it seems that the CoA
portion of the substrate will be out of the protein during catalysis. Both
the monomeric architecture and the oligomeric (trimeric) assembly of the
protein does not reveal a binding pocket. However, steady state kinetic
expts suggest that the enzyme has preference towards CoA-conjugated
substrate. The enzyme can turnover non-CoA-conjugated substrate but at a
lower substrate specificity.

I was wondering if anyone has encouneterd a similar problem or can shed any
light about the possible interaction of CoA? Is it possible that the CoA
induces macromolecular crowding or oligomerization whereby a binding
pocket is created to accommodate CoA? Any suggestions or previously
published references will be very helpful in this regard.

Thanks for your time.

Shiva

Re: [ccp4bb] do you think it is interesting?

[Shiva Bhowmik]

Hi Anna,

Interesting assembly. What is the function of your protein? Is it known if
your protein forms a fibril-like assembly in solution? Moreover, can your
crystal packing be indexed higher symmetry space group?

Cheers,

Shiva

On Mon, Jun 18, 2012 at 9:03 AM, David Schuller  wrote:

> If the original poster could engineer a few disulfides or other covalent
> linkages in there, I would drop my objections, and be even more impressed.
>
>
> On 06/18/12 11:48, Jacob Keller wrote:
>
>> Okay, I wiki'd it, and according to them seems you're right: it says
>> they are "typically connected by covalent chemical bonds." So either
>> we revert to the etymological use of "polymer," or move onward to
>> "myriomer!" (assuming the cross-bred "multimer" is out of the
>> question!)
>>
>> JPK
>>
>> On Mon, Jun 18, 2012 at 10:37 AM, David Schuller
>>  wrote:
>>
>>> On 06/18/12 11:17, Jacob Keller wrote:
>>>
  But anyway, what is
 wrong with calling her structures "polymers?" Is there a subtle
 covalent insinuation to "polymer?"

  subtle? No, it's not subtle.
>>>
>>>
>>> --
>>> ==**==**
>>> ===
>>> All Things Serve the Beam
>>> ==**==**
>>> ===
>>>   David J. Schuller
>>>   modern man in a post-modern world
>>>   MacCHESS, Cornell University
>>>   schul...@cornell.edu
>>>
>>
>>
>>
>
> --
> ==**==**
> ===
> All Things Serve the Beam
> ==**==**
> ===
>   David J. Schuller
>   modern man in a post-modern world
>   MacCHESS, Cornell University
>   schul...@cornell.edu
>

[ccp4bb] Petition to Increase NIH Budget

[Shiva Bhowmik]

Dear CCP4-ers,

Sorry for the offline topic. I would like to bring this the following
petition:

Dear Colleagues,
I wanted to bring your attention to a petition (see link below) started at
WhiteHouse.gov by our colleague Dr. Steve Meltzer
at Johns Hopkins that calls for an increase for the NIH budget. As you
know, the President recently announced his proposed FY2013 budget, which
called for a flat NIH budget. After factoring in inflation, flat funding is
an equivalent of a funding cut. Research by biomedical community is already
heavily stressed, with NIH funding rates at a record low. Many of you would
be interested in supporting this petition, which provides a unique
opportunity to gain the attention of the White House. If the petition
receives 25,000 signatures by March 18th, the White House will review it.
We currently have over 12,000 signatures.

These are troubling times for the research community and medical progress
is at great risk.  Investigators, their institutions, Private research
funding organizations, patient advocate groups, and the biotech/pharma
sector all have a common goal - the betterment of human health. NIH funding
is a critical component for the biomedical research pipeline and increasing
funding for the NIH should be a goal we can all work together on. Please
help us in this endeavor and sign and share this petition with your
friends, family, and colleagues.

The petition link:

http://wh.gov/81O

best regards,
Kerri

Kerri A. Mowen, Ph.D.
Assistant Professor
Departments of Chemical Physiology & Immunology and Microbial Sciences The
Scripps Research Institute 10550 N. Torrey Pines Rd, IMM-11 Immunology
Building Room 315 La Jolla, CA 92037
tel: 858-784-2248

Support bettering human health and improving our economy! Sign this
petition to support  increasing funding for the National Institutes of
Health:
http://wh.gov/81O

Re: [ccp4bb] Iphone app to display protein models

[Shiva Bhowmik]

Dear CCP4-users,

Thank you all for your suggestions. Below is the compiled list of
suggestions for Iphone apps to display models:

1. Import structure directly from itunes in pdb format view in iMolview
(not free).
2. Cuemol for iOS: http://itunes.apple.com/us/**app/cuemol/id496236710?ls=1&;
**mt=8 
3. Jolecule, web based: http://jolecule.appspot.com/

An app for android users under development: NDKmol.
https://market.android.com/**details?id=jp.sfjp.webglmol.**NDKmol

Cheers,

Shiva

On Fri, Jan 27, 2012 at 3:38 AM, Opher Gileadi
wrote:

> Molsoft has a free application:
>
> http://www.molsoft.com/iMolview.html
>

[ccp4bb] Iphone app to display protein models

[Shiva Bhowmik]

Dear All,

I was wondering if there is any IOS5 based app to display protein models,
which are not in public database, on Iphone. There is an app called
Molecules for displaying models but that utilizes coordinates from RCSB and
pubchem.

Thanks,

Shiva

Re: [ccp4bb] UV imaging of crystals

[Shiva Bhowmik]

Would be curious to know the current limitations on UV microscopy employed
for screening protein crystals - such as content of aromatic amino acids,
protein size etc.

Cheers,

Shiva

On Fri, Sep 16, 2011 at 1:19 AM, Klaus Fütterer wrote:

> From the experience when our (commercial) UV imaging system was set up, I
> can confirm that signal-to-noise is a non-trivial parameter for imaging in
> the UV range.
>
> I find the additional info gained from the UV capability very useful, not
> just to distinguish salt from protein crystals, but also to tell protein
> from buffer precipitate, buffer phase separation from protein phase
> separation, etc.
>
> Klaus
>
>
> ===
>
>Klaus Fütterer, Ph.D.
>Reader in Structural Biology
>  Undergraduate Admissions
>
> School of Biosciences P: +44-(0)-121-414 5895
> University of Birmingham  F: +44-(0)-121-414 5925
> Edgbaston E: k.futte...@bham.ac.uk
> Birmingham, B15 2TT, UK   W: http://tinyurl.com/futterer-lab
> ===
>
>
>
>
>
> On 16 Sep 2011, at 04:57, Nagarajan V wrote:
>
> > Typically, what you image is Trp fluorescence by exciting at around 280
> nm and observing at around 350 nm. Standard silicon based detectors do fine
> at the detection wavelength, although, as you can imagine, increased
> sensitivity in the UV means increase in the price of the detector. If your
> excitation and emission light paths do not overlap, you also can get by with
> standard glass (crown, flint, etc.) optics since they do allow some of the
> 350-nm light to get through. Therefore, yes, it is possible to build an
> inexpensive UV imager based on inexpensive excitation light source (Douglas
> Instruments offers a pen light), and standard lab microscope. Of course, for
> increased sensitivity and contrast you need a very good light source, optics
> made of quartz and calcium fluoride that let almost all the UV light
> through, highly discriminating filters and a sensitive detector.
> >
> > V. Nagarajan
> > JANSi
> > http://janscientific.com
> >
> > On Thu, Sep 15, 2011 at 7:07 PM, Edward A. Berry 
> wrote:
> > A "real" UV microscope requires quartz optics, right?
> > Probably conventional microscopes use glass.
> > And you can't see 280 nm (and its not good for your eyes)
> > so you need some kind of phosphor screen to view the image?
> >
> >
> > Bosch, Juergen wrote:
> > I'm replying here to myself :-)
> >
> > So in an off-board discussion it turns out that the "microscope" in
> question was a special
> > emitted light and not a UV microscope. So real UV microscopes might be
> better for the
> > purpose of detecting real crystals.
> >
> > Sorry for the confusion - had too much sun today :-)
> >
> > Jürgen
> >
> > On Sep 15, 2011, at 4:19 PM, Jürgen Bosch wrote:
> >
> > I once tested such a commercial system in Seattle about 4 years ago. It
> did not impress
> > me. In particular the discrimination between salt and protein did not
> work for about 10
> > different proteins from which we already had collected data. sure those
> were small
> > between 10 and 100 micrometer. Excuse was to few tryptophans
> > So in theory it is nice but a cheaper variant might be to add Gfp to your
> protein and
> > screen for something green.
> > Jürgen
> >
> > ..
> > Jürgen Bosch
> > Johns Hopkins Bloomberg School of Public Health
> > Department of Biochemistry & Molecular Biology
> > Johns Hopkins Malaria Research Institute
> > 615 North Wolfe Street, W8708
> > Baltimore, MD 21205
> > Phone: +1-410-614-4742
> > Lab: +1-410-614-4894
> > Fax: +1-410-955-3655
> > http://web.mac.com/bosch_lab/
> >
> > On Sep 15, 2011, at 16:03, Frank von Delft 
> wrote:
> >
> > A while ago I was trying to be cheap, so we played around with it quite
> > a bit in the lab. After rediscovering some of the basics of
> > signal-to-noise and microscope transmission efficiency and that sort of
> > rot, I realised that the commercial systems may not be all that
> > ridiculously overpriced after all. Not if one wants to be able to say
> > something useful about really really small crystals -- the only ones
> > that really matter in the grand scheme of things (big ones are quick to
> > test; little ones must first be optimized = money+time).
> >
> > But maybe I was just being incompetent. Happens.
> > phx.
> >
> >
> >
> >
> > On 15/09/2011 20:50, Andrew Purkiss-Trew wrote:
> > Quoting "Harman, Christine":
> >
> > Hi All,
> > I was curious if any of you have tried or even know if it is
> > possible to adapt a stereoscope (in my case an Olympus SZX10 model)
> > so as to view protein crystals with UV illumination. Basically, I
> > want a cheap manual version of what a Rock UV Imager does. I know
> > this is probably a crazy dream. However, I would greatly appreciate
> > any comments, advice 

Re: [ccp4bb] Strange density in arginine

[Shiva Bhowmik]

Hi Ruheng,

Curious to know what was in the crystallization cocktail, cryoprotectant?
Did you collect the dataset at a synchrotron?

Shiva

2011/7/4 ruheng 

>  Dear all,
>
> Recently we are working on an archaebacteria protein which was expressed
> and purified from *E.coli *by conventional procedures. After we solved the
> structure, we found that there is an extra density in one of the argninine
> as shown in the attached picture. It seems that the density is larger than a
> methyl group and the atoms in the density are not on one plane. So we are
> curious about whether the density is a kind of posttranslational
> modification of arginine residues in the protein, if it is, what kind of
> modification could it be? And most important, what is the biological
> significance of this kind modification? Any suggestions and dicussions are
> appreciated!
>
> Best,
> Heng
>
> 
>  Strange density in arginine
> 
>   
> 查看幻灯片
> 下载全部
> 添加更多照片
> 此相册包含 1 张照片，并且将在 2011/10/02 之前在 SkyDrive 上提供。
>通过 Hotmail 共享您自己的幻灯片 
>

Re: [ccp4bb] Y-Chi2 running out of chart

[Shiva Bhowmik]

If you had that crystal saved after earlier collection then recollect a
dataset again to see if the same Y-Chi2 profile is observed.

On Mon, Jun 27, 2011 at 4:59 PM, bie gao  wrote:

> Jim, that's the first thing I tried - triclinic gave similar Y-Chi2
> profile.
> Shiva, not sure what you mean by collecting the same crystal again (thaw
> and remount?). But I collected 180 degrees, Chi2 seems to correlate with
> rotation angle.
>
> Cheers,
>
>
> On Fri, Jun 24, 2011 at 1:39 PM, Jim Pflugrath 
> wrote:
>
>> **
>> Instead of an imperfect crystal, this can also occur if one chooses the
>> wrong Bravais lattice type (or spacegroup) to integrate.  For example, if
>> you choose tetragonal when it is really orthorhombic with a ~ b, or if you
>> choose orthorhombic and beta is 90.2, then you can see that trying to force
>> that unit cell will lead to higher residuals during positional
>> refinement.If one reciprocal lattice is oriented mostly along Y, then I
>> think what Bing observes can happen in such cases.  This is easily tested by
>> integrating in triclinic.
>>
>>  ------
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Shiva Bhowmik
>> *Sent:* Friday, June 24, 2011 11:42 AM
>>
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* Re: [ccp4bb] Y-Chi2 running out of chart
>>
>>   Hi Bie,
>>
>> Curious to know what are the cell parameters obtained after scaling? You
>> mention observing perfect Chi2 statistics with lysozyme crystals. But are
>> you observing the same Chi2 statisctis with the crystal that yielded unusual
>> Y-Chi2 if you collect another dataset. If there is a consistency of
>> observing the unusual Y-Chi2 with that crystal again then it is likey that
>> the crystal maybe highly imperfect. If not, then there could have been a one
>> time un-nailed problem occurred during that collection.
>>
>> Cheers,
>>
>> Shiva
>>
>> On Thu, Jun 23, 2011 at 8:59 AM, bie gao  wrote:
>>
>>> Dear colleagues, thank you all for your help! I really appreciate it.
>>> I did perform a lysozyme test after the repair. I collected ~50 degree
>>> (99%). Everything seems to be fine.  Maybe I should have done it for an
>>> entire round.
>>> As for the current collection, as I suspended, it did go out of the chart
>>> but then came down to normal. Overall Rmerge is 7.9% (4% square). As Zbyszek
>>> and others mention, it is probably due to imperfect crystal and also uneven
>>> cooling.
>>> If our field engineer discovers anything else, I'll post it here. Thanks
>>> again for your help.
>>>
>>>
>>>
>>> On Wed, Jun 22, 2011 at 9:00 PM, Artem Evdokimov <
>>> artem.evdoki...@gmail.com> wrote:
>>>
>>>> As a follow up to the excellent suggestions made by others I would
>>>> suggest that a close examination of x-file headers may reveal abnormalities
>>>> in e.g. crystal orientation -- suspecting an unlocked or drifting 
>>>> goniostat.
>>>> It may also indicate a precession around the phi, which should also 
>>>> manifest
>>>> itself in a systematic deviation of average intensities (i.e. scale 
>>>> factors)
>>>> in a similar pattern (assuming uneven illumination of the crystal).
>>>> Sometimes the precession is caused by a bubble or a tiny chunk of ice
>>>> trapped under the pin, it can melt unevenly and re-align the pin a few
>>>> minutes into the run (something similar used to happen a lot at one or two
>>>> beam lines and it drove me nuts until I figured out the need to re-align 
>>>> the
>>>> crystal after the initial screening).
>>>>
>>>> Artem
>>>>
>>>>   On Wed, Jun 22, 2011 at 11:22 AM, bie gao  wrote:
>>>>
>>>>> Dear Colleagues,
>>>>>
>>>>> I'm collecting a dataset on our recently repaired Rigaku home source.
>>>>> Crystal diffracts to 2.2A. Indexing seems to be all fine. However,
>>>>> during integration, I realize Y-Chi2 is increasing constantly (from 2 to
>>>>> 4.5, almost linear) within 60 degree collection, whereas X-Chi2 stays the
>>>>> same. An image is attached. There are still another 60 degree to go.
>>>>> Although the prediction fits the images well so far, I'm afraid the Y-Chi2
>>>>> will eventually run out of the chart.
>>>>> My question is could it be related to any hardware malfunctioning,
>>>>> i.e., goniometer, image plates, etc, which may be a side effect of the
>>>>> recent major repair? Or what else it can be?
>>>>>
>>>>> Thanks,
>>>>> Bing
>>>>>
>>>>
>>>>
>>>
>>
>

Re: [ccp4bb] How to get rid of Tubulin derived from insect cell expression

[Shiva Bhowmik]

Amonium sulfate precipitation? Considering the fact that you tried to
seperate tubulin contamination both by charge and mass difference amonium
sulfate pption may work.

Shiva

On Thu, Jun 23, 2011 at 4:05 AM, Seungil Han  wrote:

> All,
> I am sorry that this is off topic.
> My target protein expressed in insect cell expression system is eluted with
> tubulin contamination. We have tried ion exchange & sizing column to get
> ride of tubulin from our target protein for structural studies.
>
> Has anyone had similar problem? Any suggestion?
>
> Thanks in advance.
> Seungil
>
>
> Seungil Han, Ph.D.
> Pfizer Inc.
> Eastern Point Road, MS8118W-228
> Groton, CT 06340
> Tel: 860-686-1788,  Fax: 860-686-2095
> Email: 
> seungil@pfizer.com

Re: [ccp4bb] Y-Chi2 running out of chart

[Shiva Bhowmik]

Hi Bie,

Curious to know what are the cell parameters obtained after scaling? You
mention observing perfect Chi2 statistics with lysozyme crystals. But are
you observing the same Chi2 statisctis with the crystal that yielded unusual
Y-Chi2 if you collect another dataset. If there is a consistency of
observing the unusual Y-Chi2 with that crystal again then it is likey that
the crystal maybe highly imperfect. If not, then there could have been a one
time un-nailed problem occurred during that collection.

Cheers,

Shiva

On Thu, Jun 23, 2011 at 8:59 AM, bie gao  wrote:

> Dear colleagues, thank you all for your help! I really appreciate it.
> I did perform a lysozyme test after the repair. I collected ~50 degree
> (99%). Everything seems to be fine.  Maybe I should have done it for an
> entire round.
> As for the current collection, as I suspended, it did go out of the chart
> but then came down to normal. Overall Rmerge is 7.9% (4% square). As Zbyszek
> and others mention, it is probably due to imperfect crystal and also uneven
> cooling.
> If our field engineer discovers anything else, I'll post it here. Thanks
> again for your help.
>
>
>
> On Wed, Jun 22, 2011 at 9:00 PM, Artem Evdokimov <
> artem.evdoki...@gmail.com> wrote:
>
>> As a follow up to the excellent suggestions made by others I would suggest
>> that a close examination of x-file headers may reveal abnormalities in e.g.
>> crystal orientation -- suspecting an unlocked or drifting goniostat. It may
>> also indicate a precession around the phi, which should also manifest itself
>> in a systematic deviation of average intensities (i.e. scale factors) in a
>> similar pattern (assuming uneven illumination of the crystal). Sometimes the
>> precession is caused by a bubble or a tiny chunk of ice trapped under the
>> pin, it can melt unevenly and re-align the pin a few minutes into the run
>> (something similar used to happen a lot at one or two beam lines and it
>> drove me nuts until I figured out the need to re-align the crystal after the
>> initial screening).
>>
>> Artem
>>
>>   On Wed, Jun 22, 2011 at 11:22 AM, bie gao  wrote:
>>
>>> Dear Colleagues,
>>>
>>> I'm collecting a dataset on our recently repaired Rigaku home source.
>>> Crystal diffracts to 2.2A. Indexing seems to be all fine. However, during
>>> integration, I realize Y-Chi2 is increasing constantly (from 2 to 4.5,
>>> almost linear) within 60 degree collection, whereas X-Chi2 stays the same.
>>> An image is attached. There are still another 60 degree to go. Although the
>>> prediction fits the images well so far, I'm afraid the Y-Chi2 will
>>> eventually run out of the chart.
>>> My question is could it be related to any hardware malfunctioning, i.e.,
>>> goniometer, image plates, etc, which may be a side effect of the recent
>>> major repair? Or what else it can be?
>>>
>>> Thanks,
>>> Bing
>>>
>>
>>
>

[ccp4bb] Two different asymmetric units from two different crystallization conditions

[Shiva Bhowmik]

Dear All,

I am working on a protein structure that yielded comparable diffraction
quality crystals from two different crystallization condition. One of the
crystallization condition conatins high conc. of  salt pptant whereas the
oher one contains high conc. of organic pptant. There are some minor
differences in the stucture with respect to the backbone but what most
surprising is the oligomeric structure. AnSEC study suggest the protein to
be a tetramer in solution and a tetrameric assembly is observed in the
asymmetric unit of the space group of the crystal obtained from high conc.
of organic pptant. However, the crystal structure from the high conc. of
salt pptant does not reveal any oligomeric assembly - symmetry operations of
the space group does not suggest any oligomeric assembly. I believe the high
salt conc. disrupted the tetrameric assembly and enabled crystallization of
the protein as a monomer.

Curious to know if there has been any similar precedence before.

Cheers,

Shiva

Re: [ccp4bb] unknown density

[Shiva Bhowmik]

My suggestion would be to refine with full glu and then put either O and C
atoms at the region corresponidng to the extra electron density.

On Wed, May 18, 2011 at 1:41 PM, Borhani, David <
david.borh...@deshawresearch.com> wrote:

>  Glutamate methyl ester? (or ethyl ester, which I’m not sure has ever been
> documented…hard to tell how many extra heavy atoms given the view direction
> in the image you supplied). Or the methyl/ethyl amides.
>
>
>
> Dave
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Bosch,
> Juergen
> *Sent:* Wednesday, 18 May 2011 14:06
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] unknown density
>
>
>
> I take the lysine back, just realized wrong color coding it's an oxygen and
> not a nitrogen closer to the extra density :-)
>
> Jürgen
>
>
>
> On May 18, 2011, at 2:03 PM, Bosch, Juergen wrote:
>
>
>
>  But that's a lysine, so maybe a PTM ? Acetylated perhaps ?
>
>
>
> Jürgen
>
>
>
> On May 18, 2011, at 1:56 PM, Shiva Bhowmik wrote:
>
>
>
>  Hi Cedric,
>
>
>
> I presume you collected the datset at a synchrtron source. It could be free
> radical generated due to X-ray radiation has modified the glutamate
> residues. I have seen this happening for Ser residues.
>
>
>
> Cheers,
>
>
>
> Shiva
>
> On Wed, May 18, 2011 at 9:08 AM, cedric bauvois 
> wrote:
>
> Hi, everyone,
>
> I am working on the refinement of a structure  at about 1.4 Ang and found
> some clear extra density connected to a glutamate (see figure), but I don't
> know what it should be. The crystal came out from Tris-HCl buffer with
> peg600, and protein buffer contains sodium-phosphate.
>
> Does anyone figure out what this density can be ?
> Thanks for any help you can give.
>
>  Cédric
>
>
>
>
>
> ..
>
> Jürgen Bosch
>
> Johns Hopkins Bloomberg School of Public Health
>
> Department of Biochemistry & Molecular Biology
>
> Johns Hopkins Malaria Research Institute
>
> 615 North Wolfe Street, W8708
>
> Baltimore, MD 21205
>
> Phone: +1-410-614-4742
>
> Lab:  +1-410-614-4894
>
> Fax:  +1-410-955-3655
>
> http://web.mac.com/bosch_lab/
>
>
>
>
>
>
>
>
>
> ..
>
> Jürgen Bosch
>
> Johns Hopkins Bloomberg School of Public Health
>
> Department of Biochemistry & Molecular Biology
>
> Johns Hopkins Malaria Research Institute
>
> 615 North Wolfe Street, W8708
>
> Baltimore, MD 21205
>
> Phone: +1-410-614-4742
>
> Lab:  +1-410-614-4894
>
> Fax:  +1-410-955-3655
>
> http://web.mac.com/bosch_lab/
>
>
>
>
>
>
>

Re: [ccp4bb] unknown density

[Shiva Bhowmik]

Hi Cedric,

I presume you collected the datset at a synchrtron source. It could be free
radical generated due to X-ray radiation has modified the glutamate
residues. I have seen this happening for Ser residues.

Cheers,

Shiva

On Wed, May 18, 2011 at 9:08 AM, cedric bauvois  wrote:

> Hi, everyone,
>
> I am working on the refinement of a structure  at about 1.4 Ang and found
> some clear extra density connected to a glutamate (see figure), but I
> don't know what it should be. The crystal came out from Tris-HCl buffer with
> peg600, and protein buffer contains sodium-phosphate.
>
> Does anyone figure out what this density can be ?
> Thanks for any help you can give.
>
>  Cédric

Re: [ccp4bb] protein turns brown

[shiva bhowmik]

That's a possibility. Do you know if the same coloration would arise if there 
is TCEP instead of DTT or b-ME? I usually have TCEP as the reducing agent.

Thanks Fillip. 

Cheers,

Shiva

--- On Fri, 9/24/10, Filip Van Petegem  wrote:

From: Filip Van Petegem 
Subject: Re: [ccp4bb] protein turns brown
To: CCP4BB@JISCMAIL.AC.UK
Date: Friday, September 24, 2010, 2:50 PM


I also have a similar observation for proteins purified by Ni-NTA column. After 
concentrating the sample eluted from the Ni-NTA column, I see a 
brownish-yellowish tinge closer to the bottom of the filter with colorless 
buffer on top. This is observed even for non-metal binding and non-FeS cluster 
proteins and also for proteins after removal of the His-Tag. This is presumably 
arising due to difference in viscosity where the protein gets concentrated 
closer to the bottom of the filter.



Or more likely:  Nickel bleeding from the column was complexing bME or DTT in 
your sample: Ni-bME forms a tight brown complex.

Filip Van Petegem





  

Re: [ccp4bb] protein turns brown

[shiva bhowmik]

Hi All,

I also have a similar observation for proteins purified by Ni-NTA column. After 
concentrating the sample eluted from the Ni-NTA column, I see a 
brownish-yellowish tinge closer to the bottom of the filter with colorless 
buffer on top. This is observed even for non-metal binding and non-FeS cluster 
proteins and also for proteins after removal of the His-Tag. This is presumably 
arising due to difference in viscosity where the protein gets concentrated 
closer to the bottom of the filter.

Cheers,

Shiva


--- On Fri, 9/24/10, sandeep  wrote:

From: sandeep 
Subject: [ccp4bb] protein turns brown
To: CCP4BB@JISCMAIL.AC.UK
Date: Friday, September 24, 2010, 4:34 AM

Dear all,



I have purified protein from E.coli. expression system. the protein has been 
purified with three independant columns. Now during concentration step using 
amicon, the protein shows brown colour. what could be the reason.



best regards and Thanks,

sandy