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

[Peter Artymiuk]

If you are willing to include cofactor-induced oligomerization instead of 
substrate-induced, how about imidazoleglycerol-phosphate dehydratase (IGPD) ? 

Inactive trimer:   1RHY
http://www.ncbi.nlm.nih.gov/pubmed/14724278

Active 24-mer  in presence of Mn2+ ions:  2F1D  
http://www.ncbi.nlm.nih.gov/pubmed/16338409

best wishes
Pete





On 9 Aug 2013, at 01:03, 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

Prof Peter Artymiuk
Krebs Institute
Department of Molecular Biology & Biotechnology
University of Sheffield
Sheffield
S10 2TN
ENGLAND

[ccp4bb] Sponsored) access to the integrated facility for structural biology at EMBL Hamburg

[Rob Meijers]

 
Dear all,

the integrated facility at EMBL Hamburg at the PETRA3
synchrotron in Germany consists of two macromolecular crystallography
beamlines, a BioSAXS beamline and a laboratory for sample preparation and
characterization. For those of you who are interested in a comprehensive
characterization of their sample, it might be worth to consider to extend your
synchrotron visit and include additional experiments.
The integrated facility includes:
-   Offline
protein purification and characterization by circular dichroism, dynamic and 
static
light scattering (can be booked with beamtime)
-   Online
gel filtration and light scattering at the BioSAXS beamline
-   A
high-throughput crystallization facility
Samples can be shipped, and experiments can
be monitored remotely through a dedicated web interface
-   Crystal
screening on the crystallography beamlines
-   Quality
control protocols
All incoming samples for crystallization
are tested by mass spectrometry and thermal shift assay, and a report is
returned to the user
-   Sample
optimization
- Inhouse thermofluor screens to optimize
purification, conditioning and crystallizability
- Limited proteolysis
-   Expert
staff that assists in the planning, execution and analysis of experiments 
 
Access
to these facilities is funded by the European Union under the FP7 Biostruct-X
grant. For more information on gaining sponsored access please visit:
 
http://www.biostruct-x.eu/content/apply-funding
 
For
more information on the facilities, please visit:
 
http://www.embl-hamburg.de/facilities/index.html
 
or
send an email to s...@embl-hamburg.de
 
Best
regards,
 
Rob
Meijers
Group Leader
EMBL Hamburg Outstation
P: +49 40 89902 243                EMBL c/o DESY
F: +49 40 89902 149                Notkestrasse 85
      D-22603 Hamburg, Germany

Re: [ccp4bb] TLS refinement and ANISOU records

[Mark J van Raaij]

Omid,
how necessary is it that you do TLS refinement? I.e. are you just doing it to 
improve your Rs a bit or is the density really getting better? If the density 
is not improving, I would just refine without TLS and avoid any posterior 
temperature factor analysis difficulties.
While I am sure there are cases where TLS refinement really helps (cf. 
published examples), my experience in our structures is that it does not really 
make a difference. Also, in my opinion the TLS model you define must make some 
physical sense, i.e. some logical explanation why your structure or domains of 
your structure may have internally consistent anisotropic Bs.
Mark




On 8 Aug 2013, at 23:01, Ethan Merritt wrote:

> On Thursday, August 08, 2013 01:51:34 pm Omid Haji-Ghassemi wrote:
>> Dear Robbie, Marcus and Reginald,
>> 
>> Thanks again for your replies, I truly appreciate the help.
>> 
>> The B-factors was set to 20 when performing TLS refinement so I don't
>> think that is the problem.
>> 
>> I also tried Marcus's suggestion using output from coot, with no luck.
>> 
>> The only thing left to try is to test alternative TLS group as Reginald
>> have suggested.
> 
> You have only told us about an increase in average B, not whether it is
> uniformly inflated. Possibly the output from analysis by the Parvati server
>   http://skuld.bmsc.washington.edu/parvati
> would indicate specific parts of your structure that are behaving
> badly during refinement.
> 
>   Ethan
> 
>> 
>> Cheers
>> Omid
>> 
>>> Hi Omid,
>>> 
>>> Sometimes the choice of TLS groups and to a lesser extent the initial
>>> B-factor matter a lot. You should try a few other TLS group selections and
>>> see if these give nicer results. Things to try: TLSMD, including or
>>> excluding ligands and carbohydrates, other common-sense or gut-feeling
>>> structure partitionings.  If you have a lot of different groupings to
>>> test, you can reset the B-factor and do pure TLS refinement (i.e. 0 cycles
>>> of restrained refinement) for all of them. You can then use the best one
>>> for your 'final' refinement. It's much faster then trying your final
>>> refinement with all TLS groups selections.
>>> 
>>> Cheers,
>>> Robbie
>>> 
>>> Sent from my Windows Phone
>>> 
>>> Van: Omid Haji-Ghassemi
>>> Verzonden: 8-8-2013 21:55
>>> Aan: CCP4BB@JISCMAIL.AC.UK
>>> Onderwerp: Re: [ccp4bb] TLS refinement and ANISOU records
>>> 
>>> Dear Ethan,
>>> 
>>> Thank you for your reply.
>>> 
>>> I will try to review my refinement protocol once more; however, I am still
>>> perplexed at what lies at the heart of the problem.
>>> 
>>> Overestimation of average B-factor using TLS is perfectly sound, but I am
>>> not sure why all my structures the average increases tremendously.
>>> 
>>> In one case it increases from 16.36 to 73.02 for a 2.3Ang structure.
>>> 
>>> I already tried changing weights and number of TLS rounds, which resulting
>>> in only a small change in average B.
>>> 
>>> Omid
>>> 
 On Thursday, August 08, 2013 11:39:22 am Omid Haji-Ghassemi wrote:
> Dear all,
> 
> I was about to deposit a few structures to the pdb when I noticed the
> mean
> B-factors were larger than one might expect.
> 
> All the structures were refined using TLS refinement.
> 
> During refinement in Refmac the average temperature factors for each
> structure is reasonable. For example, a structure at 2.75� has a
> mean
> B-factor of 40; however, after adding the ANISOU records as required by
> the PDB, I noticed the average B-factors double.
 
 Please see my paper:
  E. A. Merritt (2011).
  "Some Beq are more equivalent than others". Acta Cryst. A67, 512-516.
  
 
 In short, the quantity stored in the "B" field of a PDB file after TLS
 refinement is Beq, which overestimates what the isotropic B factor would
 have been if you had refined without TLS.  So in general the "average B"
 after TLS refinement is always higher than the "average B" without TLS.
 The problem is that the two quantities marked "average B" are not
 directly comparable.
 
 Having said that, the overestimate is not usually as much as a factor of
 2.
 So something else may indeed be causing a problem in your case.
 
  Ethan
 
 
> 
> Is this normal?
> 
> Sincerely,
> Omid
> 
> ---
> ---
> Omid Haji-Ghassemi, Graduate Student
> Department of Biochemistry & Microbiology
> University of Victoria
> PO Box 3055 STN CSC
> Victoria, BC, V8W 3P6
> CANADA
> 
> Tel:250-721-8945
> Fax:250-721-8855
> 
 
 --
 Ethan A Merritt
 Biomolecular Structure Center,  K-428 Health Sciences Bldg
 University of Washington, Seattle 98195-

Re: [ccp4bb] TLS refinement and ANISOU records

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi Mark,

does your experience also include the geometry of the model? There
might be improvements, as e.g. reported from molprobity, because of a
better modelling of the temperature factors.

I am asking because you mention improvements of the electron density,
whereas some improvements in the geometry, like the Ramachandran plot,
may not be directly visible in the electron density.

Cheers,
Tim

On 08/09/2013 04:27 PM, Mark J van Raaij wrote:
> Omid, how necessary is it that you do TLS refinement? I.e. are you
> just doing it to improve your Rs a bit or is the density really
> getting better? If the density is not improving, I would just
> refine without TLS and avoid any posterior temperature factor
> analysis difficulties. While I am sure there are cases where TLS
> refinement really helps (cf. published examples), my experience in
> our structures is that it does not really make a difference. Also,
> in my opinion the TLS model you define must make some physical
> sense, i.e. some logical explanation why your structure or domains
> of your structure may have internally consistent anisotropic Bs. 
> Mark
> 
> 
> 
> 
> On 8 Aug 2013, at 23:01, Ethan Merritt wrote:
> 
>> On Thursday, August 08, 2013 01:51:34 pm Omid Haji-Ghassemi
>> wrote:
>>> Dear Robbie, Marcus and Reginald,
>>> 
>>> Thanks again for your replies, I truly appreciate the help.
>>> 
>>> The B-factors was set to 20 when performing TLS refinement so I
>>> don't think that is the problem.
>>> 
>>> I also tried Marcus's suggestion using output from coot, with
>>> no luck.
>>> 
>>> The only thing left to try is to test alternative TLS group as
>>> Reginald have suggested.
>> 
>> You have only told us about an increase in average B, not whether
>> it is uniformly inflated. Possibly the output from analysis by
>> the Parvati server http://skuld.bmsc.washington.edu/parvati would
>> indicate specific parts of your structure that are behaving badly
>> during refinement.
>> 
>> Ethan
>> 
>>> 
>>> Cheers Omid
>>> 
 Hi Omid,
 
 Sometimes the choice of TLS groups and to a lesser extent the
 initial B-factor matter a lot. You should try a few other TLS
 group selections and see if these give nicer results. Things
 to try: TLSMD, including or excluding ligands and
 carbohydrates, other common-sense or gut-feeling structure
 partitionings.  If you have a lot of different groupings to 
 test, you can reset the B-factor and do pure TLS refinement
 (i.e. 0 cycles of restrained refinement) for all of them. You
 can then use the best one for your 'final' refinement. It's
 much faster then trying your final refinement with all TLS
 groups selections.
 
 Cheers, Robbie
 
 Sent from my Windows Phone  
 Van: Omid Haji-Ghassemi Verzonden: 8-8-2013 21:55 Aan:
 CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] TLS refinement
 and ANISOU records
 
 Dear Ethan,
 
 Thank you for your reply.
 
 I will try to review my refinement protocol once more;
 however, I am still perplexed at what lies at the heart of
 the problem.
 
 Overestimation of average B-factor using TLS is perfectly
 sound, but I am not sure why all my structures the average
 increases tremendously.
 
 In one case it increases from 16.36 to 73.02 for a 2.3Ang
 structure.
 
 I already tried changing weights and number of TLS rounds,
 which resulting in only a small change in average B.
 
 Omid
 
> On Thursday, August 08, 2013 11:39:22 am Omid Haji-Ghassemi
> wrote:
>> Dear all,
>> 
>> I was about to deposit a few structures to the pdb when I
>> noticed the mean B-factors were larger than one might
>> expect.
>> 
>> All the structures were refined using TLS refinement.
>> 
>> During refinement in Refmac the average temperature
>> factors for each structure is reasonable. For example, a
>> structure at 2.75� has a mean B-factor of 40; however,
>> after adding the ANISOU records as required by the PDB, I
>> noticed the average B-factors double.
> 
> Please see my paper: E. A. Merritt (2011). "Some Beq are
> more equivalent than others". Acta Cryst. A67, 512-516. 
> 
>
>
> 
In short, the quantity stored in the "B" field of a PDB file after TLS
> refinement is Beq, which overestimates what the isotropic B
> factor would have been if you had refined without TLS.  So
> in general the "average B" after TLS refinement is always
> higher than the "average B" without TLS. The problem is
> that the two quantities marked "average B" are not directly
> comparable.
> 
> Having said that, the overestimate is not usually as much
> as a factor of 2. So something else may indeed be causing a
> problem in 

Re: [ccp4bb] TLS refinement and ANISOU records

[Mark J van Raaij]

Hi Tim,
no, in the cases I remember there was no significant difference in geometry - 
of course, we do tend to work on rather stable, rocky proteins...
Mark



On 9 Aug 2013, at 16:33, Tim Gruene wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Hi Mark,
> 
> does your experience also include the geometry of the model? There
> might be improvements, as e.g. reported from molprobity, because of a
> better modelling of the temperature factors.
> 
> I am asking because you mention improvements of the electron density,
> whereas some improvements in the geometry, like the Ramachandran plot,
> may not be directly visible in the electron density.
> 
> Cheers,
> Tim
> 
> On 08/09/2013 04:27 PM, Mark J van Raaij wrote:
>> Omid, how necessary is it that you do TLS refinement? I.e. are you
>> just doing it to improve your Rs a bit or is the density really
>> getting better? If the density is not improving, I would just
>> refine without TLS and avoid any posterior temperature factor
>> analysis difficulties. While I am sure there are cases where TLS
>> refinement really helps (cf. published examples), my experience in
>> our structures is that it does not really make a difference. Also,
>> in my opinion the TLS model you define must make some physical
>> sense, i.e. some logical explanation why your structure or domains
>> of your structure may have internally consistent anisotropic Bs. 
>> Mark
>> 
>> 
>> 
>> 
>> On 8 Aug 2013, at 23:01, Ethan Merritt wrote:
>> 
>>> On Thursday, August 08, 2013 01:51:34 pm Omid Haji-Ghassemi
>>> wrote:
 Dear Robbie, Marcus and Reginald,
 
 Thanks again for your replies, I truly appreciate the help.
 
 The B-factors was set to 20 when performing TLS refinement so I
 don't think that is the problem.
 
 I also tried Marcus's suggestion using output from coot, with
 no luck.
 
 The only thing left to try is to test alternative TLS group as
 Reginald have suggested.
>>> 
>>> You have only told us about an increase in average B, not whether
>>> it is uniformly inflated. Possibly the output from analysis by
>>> the Parvati server http://skuld.bmsc.washington.edu/parvati would
>>> indicate specific parts of your structure that are behaving badly
>>> during refinement.
>>> 
>>> Ethan
>>> 
 
 Cheers Omid
 
> Hi Omid,
> 
> Sometimes the choice of TLS groups and to a lesser extent the
> initial B-factor matter a lot. You should try a few other TLS
> group selections and see if these give nicer results. Things
> to try: TLSMD, including or excluding ligands and
> carbohydrates, other common-sense or gut-feeling structure
> partitionings.  If you have a lot of different groupings to 
> test, you can reset the B-factor and do pure TLS refinement
> (i.e. 0 cycles of restrained refinement) for all of them. You
> can then use the best one for your 'final' refinement. It's
> much faster then trying your final refinement with all TLS
> groups selections.
> 
> Cheers, Robbie
> 
> Sent from my Windows Phone  
> Van: Omid Haji-Ghassemi Verzonden: 8-8-2013 21:55 Aan:
> CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] TLS refinement
> and ANISOU records
> 
> Dear Ethan,
> 
> Thank you for your reply.
> 
> I will try to review my refinement protocol once more;
> however, I am still perplexed at what lies at the heart of
> the problem.
> 
> Overestimation of average B-factor using TLS is perfectly
> sound, but I am not sure why all my structures the average
> increases tremendously.
> 
> In one case it increases from 16.36 to 73.02 for a 2.3Ang
> structure.
> 
> I already tried changing weights and number of TLS rounds,
> which resulting in only a small change in average B.
> 
> Omid
> 
>> On Thursday, August 08, 2013 11:39:22 am Omid Haji-Ghassemi
>> wrote:
>>> Dear all,
>>> 
>>> I was about to deposit a few structures to the pdb when I
>>> noticed the mean B-factors were larger than one might
>>> expect.
>>> 
>>> All the structures were refined using TLS refinement.
>>> 
>>> During refinement in Refmac the average temperature
>>> factors for each structure is reasonable. For example, a
>>> structure at 2.75� has a mean B-factor of 40; however,
>>> after adding the ANISOU records as required by the PDB, I
>>> noticed the average B-factors double.
>> 
>> Please see my paper: E. A. Merritt (2011). "Some Beq are
>> more equivalent than others". Acta Cryst. A67, 512-516. 
>> 
>> 
>> 
>> 
> In short, the quantity stored in the "B" field of a PDB file after TLS
>> refinement is Beq, which overestimates what the isotropic B
>> factor would have been if you had refined without TLS.  So
>> in gen

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
>

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

[Das, Debanu]

Hi Shiva,

There are some other enzyme systems activated by ligand induced oligomerization 
in addition to the ones on this list:

1) DNA polymerase dimerization on DNA binding
2) Restriction enzyme dimerization on DNA binding (an example is the type IIs 
FokI)
3) membrane enzyme receptors like receptor tyrosine kinase, etc.
4) metallopeptidases with dimerization domain

Thanks,
Debanu.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Shiva 
Bhowmik
Sent: Friday, August 09, 2013 10:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Substrate/Ligand Induced Oligomerization of enzymes

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] 3-way obligatory associating proteins

[Davide Comoletti]

Hi CCP4BB members,

I was wondering if anyone knows of examples of 3-way obligatory associating
complexes. In other words, instead of having protein A binding to protein B
and protein B also binding to protein C, example of complexes where protein
C binds ONLY when proteins A and B are already associated.


Thanks for your help,

Davide

[ccp4bb] TLS update

[Omid Haji-Ghassemi]

Dear all,

Thanks again for all the suggestions.

I should perhaps add that I am working on glycosylated proteins and they
which tend to be quite flexible in some areas.

The website suggested by Ethan:

http://skuld.bmsc.washington.edu/parvati

did not show any areas that appeared alarming, or at least that is how I
interpreted it.

I realized TLS refinement can be avoided entirely in the structure that
appeared with with the most extreme increase in mean B-factor (~ 80),
doing so only resulted in a slight increase of R-factors. I can live with
that.

However, this is not the case for my other structures. Without TLS
refinement in one of my structures the R-free goes from 24.5% to 29.6%.

As for impact on density or geometry, its hard to to say. I have only
observed few areas which displayed improved density following TLS
refinement. Though it must be said, the improvements were marginal.

Hope these discussion helps others in the future

Best Regards

Omid


> Hi Tim,
> no, in the cases I remember there was no significant difference in
> geometry - of course, we do tend to work on rather stable, rocky
> proteins...
> Mark
>
>
>
> On 9 Aug 2013, at 16:33, Tim Gruene wrote:
>
>> -BEGIN PGP SIGNED MESSAGE-
>> Hash: SHA1
>>
>> Hi Mark,
>>
>> does your experience also include the geometry of the model? There
>> might be improvements, as e.g. reported from molprobity, because of a
>> better modelling of the temperature factors.
>>
>> I am asking because you mention improvements of the electron density,
>> whereas some improvements in the geometry, like the Ramachandran plot,
>> may not be directly visible in the electron density.
>>
>> Cheers,
>> Tim
>>
>> On 08/09/2013 04:27 PM, Mark J van Raaij wrote:
>>> Omid, how necessary is it that you do TLS refinement? I.e. are you
>>> just doing it to improve your Rs a bit or is the density really
>>> getting better? If the density is not improving, I would just
>>> refine without TLS and avoid any posterior temperature factor
>>> analysis difficulties. While I am sure there are cases where TLS
>>> refinement really helps (cf. published examples), my experience in
>>> our structures is that it does not really make a difference. Also,
>>> in my opinion the TLS model you define must make some physical
>>> sense, i.e. some logical explanation why your structure or domains
>>> of your structure may have internally consistent anisotropic Bs.
>>> Mark
>>>
>>>
>>>
>>>
>>> On 8 Aug 2013, at 23:01, Ethan Merritt wrote:
>>>
 On Thursday, August 08, 2013 01:51:34 pm Omid Haji-Ghassemi
 wrote:
> Dear Robbie, Marcus and Reginald,
>
> Thanks again for your replies, I truly appreciate the help.
>
> The B-factors was set to 20 when performing TLS refinement so I
> don't think that is the problem.
>
> I also tried Marcus's suggestion using output from coot, with
> no luck.
>
> The only thing left to try is to test alternative TLS group as
> Reginald have suggested.

 You have only told us about an increase in average B, not whether
 it is uniformly inflated. Possibly the output from analysis by
 the Parvati server http://skuld.bmsc.washington.edu/parvati would
 indicate specific parts of your structure that are behaving badly
 during refinement.

 Ethan

>
> Cheers Omid
>
>> Hi Omid,
>>
>> Sometimes the choice of TLS groups and to a lesser extent the
>> initial B-factor matter a lot. You should try a few other TLS
>> group selections and see if these give nicer results. Things
>> to try: TLSMD, including or excluding ligands and
>> carbohydrates, other common-sense or gut-feeling structure
>> partitionings.  If you have a lot of different groupings to
>> test, you can reset the B-factor and do pure TLS refinement
>> (i.e. 0 cycles of restrained refinement) for all of them. You
>> can then use the best one for your 'final' refinement. It's
>> much faster then trying your final refinement with all TLS
>> groups selections.
>>
>> Cheers, Robbie
>>
>> Sent from my Windows Phone 
>> Van: Omid Haji-Ghassemi Verzonden: 8-8-2013 21:55 Aan:
>> CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] TLS refinement
>> and ANISOU records
>>
>> Dear Ethan,
>>
>> Thank you for your reply.
>>
>> I will try to review my refinement protocol once more;
>> however, I am still perplexed at what lies at the heart of
>> the problem.
>>
>> Overestimation of average B-factor using TLS is perfectly
>> sound, but I am not sure why all my structures the average
>> increases tremendously.
>>
>> In one case it increases from 16.36 to 73.02 for a 2.3Ang
>> structure.
>>
>> I already tried changing weights and number of TLS rounds,
>> which resulting in only a small change in average B.
>>
>> Omid
>>
>>> On Thursday, A

Re: [ccp4bb] 3-way obligatory associating proteins

[Das, Debanu]

An easy example is heterotrimeric G-proteins. In the termination cycle, G-alpha 
bound to GTP will hydrolyze the GTP, which then allows it to associate again 
with the G-beta/G-gamma dimer.

Many membrane receptors (GPCRs, Chemotaxis, etc) can oligomerize and can be 
viewed like this, i.e., one dimer (protein dimer unit C) binds to another 
already associated dimer (units A and B).

Also in DNA replication, transcription and mRNA splicing and translation  
complexes where recruitment of third or more protein occurs only after initial 
partners are already in complex.

Thanks,
Debanu.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Davide 
Comoletti
Sent: Friday, August 09, 2013 10:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 3-way obligatory associating proteins

Hi CCP4BB members,


I was wondering if anyone knows of examples of 3-way obligatory associating 
complexes. In other words, instead of having protein A binding to protein B and 
protein B also binding to protein C, example of complexes where protein C binds 
ONLY when proteins A and B are already associated.



Thanks for your help,

Davide

[ccp4bb] Xtal formed during purification

[ZHIZHI WANG]

Hi all,
   After I purified my target protein by ion exchange, I left the fractions 
with high protein concentrations overnight @4C. Now I saw a lot of small needle 
crystals inside the EP tubes this morning. 
I wonder whether there is any technique or method to get bigger crystals from 
this?

ZZ

Re: [ccp4bb] Xtal formed during purification

[Tomas Malinauskas]

Dear Zhizhi,
we had a case like that. I would switch to slightly different buffer
(e.g. different pH) so crystals do not appear overnight, and then do
crystallisation screening. I bet you will have many hits in different
conditions, likely with bigger crystals.
Good luck!
Best wishes,
Tomas




On Fri, Aug 9, 2013 at 8:31 PM, ZHIZHI WANG  wrote:
> Hi all,
>After I purified my target protein by ion exchange, I left the fractions 
> with high protein concentrations overnight @4C. Now I saw a lot of small 
> needle crystals inside the EP tubes this morning.
> I wonder whether there is any technique or method to get bigger crystals from 
> this?
>
> ZZ

Re: [ccp4bb] X-rays gas scattering picture needed

[James Holton]

I put some pictures of scattering from random things (including "air") here:

http://bl831.als.lbl.gov/~jamesh/pickup/images/


It is actually more difficult than you might think to get a clean image of
"air scatter" on a well-maintained camera.  This is because we usually try
to minimize the contribution from air.  Generally, air scatter is reduced
to the point where it becomes less than the most bothersome "other thing",
such as fluorescence from slits and pinholes, etc.  To demonstrate this, I
took two 60s shots of "air scatter" with the cryostream set at 100K and
200K and then subtracted them.  The result is
"N2_gas_100K-200K_0_001.img".  The reason why there is so much more "air
scatter" with the stream at 100K vs 200K is because N2 gas at 100K is twice
as dense as it is at 200K (and ~3x denser than the ambient air).

In fact, you can actually confirm the sensitivity of your detector doing
this, provided you know your beamline's flux.  This is because the
scattering from any gas in the "forward" direction (near the beamstop) is:

I_gas = F^2*r_e^2/kB*P/T*thickness*flux*exposure*(pixel_size/distance)^2

where:
I_gas = the number of photons hitting one pixel due to the gas
F = 14 electrons (7 for atomic nitrogen, 14 for N2)
r_e^2 = 7.94e-30 m^2 (Thomson cross section)
kB = 1.38e-23 kg*m^2/s/K (Boltzmann constant)
P = 101325 kg*m/s^2 (ambient pressure)
T = 100 K or 200 K
thickness = ~8e-3 m (width of cryojet nozzle)
flux = 2.4e11 photons/s
exposure = 60 s
pixel_size = 102.5e-6 m (ADSC Q315r in binned mode)
distance = 150e-3 m (150 mm sample to detector)

Note that P/T/kB is just the number of molecules per cubic meter of the
gas.  Using the above numbers, the difference between 100K and 200K is 3074
photons/pixel falling on the detector near the beamstop.  (at higher
angles, you need to use a smaller "F").

You can see in the N2_gas_100K-200K*.img file there are ~3600
"counts"/pixel near the beamstop, wich is pretty close, but actually a
coincidence.  The "gain" of this detector is not unity, so the "counts" on
the image are not photons.  For a Q315r in hardware-binned mode (my
default), the digitizer is set to 4 electrons per ADU (Area Detector Unit =
one pixel level on the image).  A single 11 keV photon will generate an
average of 7.3 electrons in the CCD (according to the manufacturer), which
gives us a total "gain" of 1.8 ADU/photon. Yes, this is the number you give
to MOSFLM as the "GAIN", and yes it does mean that one "photon" actually
generates almost two ADU.

Given, this, my N2_gas_100K-200K image should have 5611 ADU/pixel.  So, now
its too high.  This is because not all 8 mm of the cryo stream width is at
the lowest temperature.  There is a bit of a "gradient", which you can see
in this image:

http://bl831.als.lbl.gov/~jamesh/pickup/images/cryojet_net.img

This is a difference image taken with the cryojet "on" vs "off" with the
collimator and beamstop removed and an 0.3 mm selenium foil inserted 30 cm
upstream from the sample.  This is just thick enough to "stop" the main
beam, so this is literally an "x-ray" of the sample area from a "point"
source of Se Kalpha fluorescence.  You can see the outline of the cryojet
nozzle on the left and the sample magnet on the right.  If you use the ADXV
"line" tool you can see how the gas of the cryo stream is actually denser
(colder) in the middle, and somewhat denser toward the bottom.  Note that
the "beam center" in the image is actually the point where the x-ray beam
passes through the cryo stream, which can be useful for alignment.

If I take the "relative density" across this image of the stream (assuming
"1" at the center and "0" beyond the edges), I get an average of 60%, which
brings 5611 ADU/pixel down to 3366 ADU/pixel, vs the observed 3600
ADU/pixel.  Not too bad, I think.

-James Holton
MAD Scientist


On Thu, Aug 1, 2013 at 9:31 AM, Pietro Roversi <
pietro.rove...@bioch.ox.ac.uk> wrote:

> Dear all,
>
> together with two fellow crystallographers,
> I am writing a pamphlet to introduce schoolchildren
> to X-ray crystallography.
>
> For the introductory chapter,
> we would need a picture of X-rays scattered by air.
> Or by any gas for that matter.
>
> I have tried Google images without much luck.
>
> Can anyone either kindly donate, or point me to,
> an image of X-rays scattered by a gas?
>
> Thanks!
>
> Pietro
>
> PS of course a picture of X-ray scattering by a liquid would also be very
> welcome
>

[ccp4bb] Looking for heated goniometer heads

[Chris Waddling]

I'm wondering if anyone has any still-functioning heated goniometer heads
that they no longer need.  If so, please let me know offline from the list.

Thanks,

Chris

__

DR. CHRISTOPHER A. WADDLING, PHD
Crystallography Facility Manager
University of California, San Francisco
Department of Biochemistry & Biophysics MC:2140
600 16TH Street  
Room S126C
San Francisco, CA 94158
(415) 476-8288 (office)
(415) 810-7556 (cell)
waddl...@msg.ucsf.edu
http://www.msg.ucsf.edu/XRayLab/
http://tiny.ucsf.edu/waddling