[ccp4bb] Long-wavelength MX Beamline at Diamond (1st User Call)

[Armin Wagner]

The long-wavelength MX beamline I23 at Diamond Light Source has been added to 
the current Call for Proposals (deadline 1st Oct) for experiments from April 
2018.

This novel instrument can offer unique opportunities expanding the wavelength 
range for MX towards and beyond the sulphur K edge (lambda ~ 5 A). Potential 
applications are:

-  Native SAD
-  Use of strong anomalous signal from M-edges to phase large complexes
-  Location of sulphur or phosphorus atoms in anomalous difference 
fourier maps to assist model building
-  Identification of light atoms like chlorine, potassium and calcium 
in anomalous difference fourier maps above and below the respective edges

The complete end station and detector are in vacuum, hence all measured signal 
is either from the sample, buffer or sample mount. Special thermally conductive 
sample mounts are required for experiments. We are currently working on the 
logistics on how to make sample mounts accessible for the experiments and 
adaptors to allow fast screening on the existing Diamond beamlines.

If you are interested, we strongly encourage you to read the beamline paper 
(https://doi.org/10.1107/S2059798316001078) which describes the instrument. 
During the extended commissioning phase, we have solved several difficult 
native phasing problems and identified or confirmed the atom type for (unknown) 
peaks present in the electron density for various projects. While the sample 
mounts are different from standard sample holders, we can use adaptors for 
pre-screening samples at our high-throughput beamlines in Diamond. For further 
information, please check our beamline home page.

http://www.diamond.ac.uk/Beamlines/Mx/I23.html

If you are part of an existing Diamond BAG, please request shifts through your 
BAG proposal, for new users we have put some instructions on the page below:

http://www.diamond.ac.uk/Beamlines/Mx/I23/How-to-apply.html

Please do not hesitate to get in touch if you have questions regarding 
potential projects or the application process.

Best regards,

Armin


Armin Wagner
Principal Beamline Scientist
I23 Long-wavelength MX
Diamond Light Source

0044 (0)1235 778560




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Re: [ccp4bb] doubt regarding MR search model

[Eleanor Dodson]

Well - you haven't said what the sequence identity between model and your
protein is, nor if you have a non-crystallographic translation.

With low homology that R factor drop is acceptable and rebuilding can fix
it, However if there is high homology you might expect better.

But this sort of conjecture is pretty pointless - check in all orthorhombic
 space groups as Mark suggests.

Eleanor

On 19 September 2017 at 15:16, Mark J van Raaij 
wrote:

> With Rs of 43/48% I don't think you can be sure that your spacegroup is
> right.
> You should always try all the spacegroup possibilities until you get a
> solution you are sure is right, i.e. that refines to Rs of around 35% or
> preferably even lower.
> More so in the case of screw axes, so try P222, P2122, P2212, P2221,
> P21212, P21221, P22121 and P212121. Phaser can do this automatically for
> you by clicking the right box.
> If necessary, then try lower symmetry like P21 and perhaps P1.
> Programs like Xanuda can help.
>
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
> Editor of Acta Crystallographica F, Structural Biology Communications
> http://journals.iucr.org/f/
>
> On 19 Sep 2017, at 16:01, Satvik Kumar  wrote:
>
> Hello,
>
> Thanks everyone for your explanations.
>
> I have pasted the pointless output to provide more information.
>
> Best Solution:  space group P 21 21 21
>
> Laue group probability:   0.959
>
> Systematic absence probability: 0.818
>
> Total probability: 0.785
>
> Space group confidence:0.751
>
> Laue group confidence   0.951
>
>
> Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00
>
> Also based on L-test, pointless says data does not suggest twinning.
>
> Yes, the R values go down when I refine in both cases. After 20 rounds of 
> restrained refinement using the coordinates generated by monomer as search 
> model, the Rwork and Rfree are 0.43 and 0.48
>
> respectively. Refinement using the coordinates generated by using dimer as
> search model also results in similar R values. I have attached the plots to
> show that the R values indeed reduce in both cases.
>
> Is my space group correct? Do I need to reexamine the space group even
> though the probability is high?
>
> If my space group is indeed correct, how do I decide whether to go ahead
> with the results generated by the monomer search model or the dimer?
>
> Please share your thoughts.
>
>
> Thanks,
> Satvik
>
> On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson  > wrote:
>
>> You need to provide a bit more information.
>>
>> First of all about the data processing..
>>
>> Is the space group correct?
>> ways of being misled are:
>> Non-crystallographic translations with a shift of ~0.5 along an axis -
>> say a.  This will generate absences in the odd h 0 0 reflections and can
>> make the space group appear to be P 21 21 21 whilst it is really P 2 21 21..
>>
>> Perfect twinning can have the same effect. In an orthorhombix space group
>> this can usually only occur if two axes have approximately the same length,
>> but the data processing stats can indicate if that is the case.
>>
>> Then - re PHASER. The packing rejection criteria may be set too severely
>> - that seems the case for your solution.
>>
>> Best check on any MR solution is: does it refine - give it 20 cycles of
>> mindless refinement and see if the R and FreeR go down.
>>
>> Then look at the maps and see if there are obvious corrections to be
>> made..
>>
>> Eleanor
>>
>> On 18 September 2017 at 14:59, Satvik Kumar 
>> wrote:
>>
>>> Dear Crystallographers,
>>>
>>> I am trying to solve a structure in the space group P212121. Based on
>>> Matthews coefficient, there are 4 molecules in the asymmetric unit.
>>>
>>> Based on my limited reading about using of Phaser, I understand that a
>>> single chain should be used as search model even though many copies are
>>> present in asymmetric unit. Am I correct?
>>>
>>> So when I use a single chain as search model and ask Phaser to search
>>> for 4 molecules, Phaser identifies a single solution with a warning "The
>>> top solution from a TF rescoring did not pack" and a warning "Search
>>> request requires more scattering than defined in composition. Composition
>>> increased to accommodate search components". But the final values reported 
>>> "PAK=2
>>> LLG=1065 TFZ==22.6" indicate that phaser has solved the problem.
>>>
>>> Can anyone please explain the meaning of the warning.
>>>
>>> When I inspect the arrangement of the chains (attachment), I observe
>>> minimal contact between the chains and a large cavity in the center. Can a
>>> crystal form this way?
>>>
>>> I have 

[ccp4bb] AW: [ccp4bb] His-6 versus His-10 tag

[Herman . Schreuder]

Dear everybody who reacted,

It seems that when the His6 construct crystallizes, there is a fair chance that 
the his10 construct will crystallize as well. However, as usual in 
crystallography there is no guarantee, so I have added a TEV site to the 
construct in order to be able to remove the tag if necessary.

Thank you all very much!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Savvas 
Savvides
Gesendet: Dienstag, 19. September 2017 16:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] His-6 versus His-10 tag

Hi Herman
the survey article by Carson in Acta D
DOI:

10.1107/S0907444906052024
from a decade ago may provide relevant information/insights.
best wishes
Savvas




On 19 Sep 2017, at 12:10, 
herman.schreu...@sanofi.com wrote:

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman

Re: [ccp4bb] doubt regarding MR search model

[Mark J van Raaij]

With Rs of 43/48% I don't think you can be sure that your spacegroup is right.
You should always try all the spacegroup possibilities until you get a solution 
you are sure is right, i.e. that refines to Rs of around 35% or preferably even 
lower.
More so in the case of screw axes, so try P222, P2122, P2212, P2221, P21212, 
P21221, P22121 and P212121. Phaser can do this automatically for you by 
clicking the right box. 
If necessary, then try lower symmetry like P21 and perhaps P1.
Programs like Xanuda can help.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/

> On 19 Sep 2017, at 16:01, Satvik Kumar  wrote:
> 
> Hello,
> 
> Thanks everyone for your explanations.
> 
> I have pasted the pointless output to provide more information.
> Best Solution:  space group P 21 21 21
>  Laue group probability:   0.959
> Systematic absence probability: 0.818
> Total probability: 0.785
> Space group confidence:0.751
> Laue group confidence   0.951
> 
> Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00
> 
> Also based on L-test, pointless says data does not suggest twinning.
> 
> Yes, the R values go down when I refine in both cases. After 20 rounds of 
> restrained refinement using the coordinates generated by monomer as search 
> model, the Rwork and Rfree are 0.43 and 0.48 
> respectively. Refinement using the coordinates generated by using dimer as 
> search model also results in similar R values. I have attached the plots to 
> show that the R values indeed reduce in both cases. 
> 
> Is my space group correct? Do I need to reexamine the space group even though 
> the probability is high?
> 
> If my space group is indeed correct, how do I decide whether to go ahead with 
> the results generated by the monomer search model or the dimer? 
> 
> Please share your thoughts.
> 
> 
> Thanks,
> Satvik
> 
> On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson  > wrote:
> You need to provide a bit more information.
> 
> First of all about the data processing..
> 
> Is the space group correct?
> ways of being misled are:
> Non-crystallographic translations with a shift of ~0.5 along an axis - say a. 
>  This will generate absences in the odd h 0 0 reflections and can make the 
> space group appear to be P 21 21 21 whilst it is really P 2 21 21..
> 
> Perfect twinning can have the same effect. In an orthorhombix space group 
> this can usually only occur if two axes have approximately the same length, 
> but the data processing stats can indicate if that is the case.
> 
> Then - re PHASER. The packing rejection criteria may be set too severely - 
> that seems the case for your solution.
> 
> Best check on any MR solution is: does it refine - give it 20 cycles of 
> mindless refinement and see if the R and FreeR go down.
> 
> Then look at the maps and see if there are obvious corrections to be made..
> 
> Eleanor
> 
> On 18 September 2017 at 14:59, Satvik Kumar  > wrote:
> Dear Crystallographers,
> 
> I am trying to solve a structure in the space group P212121. Based on 
> Matthews coefficient, there are 4 molecules in the asymmetric unit.
> 
> Based on my limited reading about using of Phaser, I understand that a single 
> chain should be used as search model even though many copies are present in 
> asymmetric unit. Am I correct?
> 
> So when I use a single chain as search model and ask Phaser to search for 4 
> molecules, Phaser identifies a single solution with a warning "The top 
> solution from a TF rescoring did not pack" and a warning "Search request 
> requires more scattering than defined in composition. Composition increased 
> to accommodate search components". But the final values reported "PAK=2 
> LLG=1065 TFZ==22.6" indicate that phaser has solved the problem. 
> 
> Can anyone please explain the meaning of the warning.
> 
> When I inspect the arrangement of the chains (attachment), I observe minimal 
> contact between the chains and a large cavity in the center. Can a crystal 
> form this way?
> 
> I have also tried using the dimer as search model and asking phaser to search 
> for 2 molecules. Even in this case, Phaser finds a single solution but the 
> warning and the advisory still appear as before. The numbers reported reduce 
> a bit to "PAK=1 LLG=722 TFZ==29.2".
> 
> Please help me in understanding these results.
> 
> Thanks,
> Satvik
> 
> 
> 
> 
> 

[ccp4bb] AW: [ccp4bb] doubt regarding MR search model

[Herman . Schreuder]

Dear Satvik,

You only know if the space group is correct AFTER you solved your structure. 
With an Rwork/Rfree of 0.43/0.48, the space group is likely not correct. The 
way to solve this is to run MR in all possible space groups. Most, if not all 
MR programs have an option to do this automatically.

For phaser e.g. you have to give the command: SGALTERNATIVE SELECT ALL
If your search model is not too different from the crystallized protein, this 
should work without a problem. Otherwise, you get into the realm of difficult 
molecular replacement and that will be a different story.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Satvik 
Kumar
Gesendet: Dienstag, 19. September 2017 16:02
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] doubt regarding MR search model

Hello,
Thanks everyone for your explanations.
I have pasted the pointless output to provide more information.

Best Solution:  space group P 21 21 21

Laue group probability:   0.959

Systematic absence probability: 0.818

Total probability: 0.785

Space group confidence:0.751

Laue group confidence   0.951


Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00

Also based on L-test, pointless says data does not suggest twinning.

Yes, the R values go down when I refine in both cases. After 20 rounds of 
restrained refinement using the coordinates generated by monomer as search 
model, the Rwork and Rfree are 0.43 and 0.48
respectively. Refinement using the coordinates generated by using dimer as 
search model also results in similar R values. I have attached the plots to 
show that the R values indeed reduce in both cases.
Is my space group correct? Do I need to reexamine the space group even though 
the probability is high?

If my space group is indeed correct, how do I decide whether to go ahead with 
the results generated by the monomer search model or the dimer?
Please share your thoughts.

Thanks,
Satvik

On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson 
> wrote:
You need to provide a bit more information.
First of all about the data processing..
Is the space group correct?
ways of being misled are:
Non-crystallographic translations with a shift of ~0.5 along an axis - say a.  
This will generate absences in the odd h 0 0 reflections and can make the space 
group appear to be P 21 21 21 whilst it is really P 2 21 21..
Perfect twinning can have the same effect. In an orthorhombix space group this 
can usually only occur if two axes have approximately the same length, but the 
data processing stats can indicate if that is the case.
Then - re PHASER. The packing rejection criteria may be set too severely - that 
seems the case for your solution.
Best check on any MR solution is: does it refine - give it 20 cycles of 
mindless refinement and see if the R and FreeR go down.
Then look at the maps and see if there are obvious corrections to be made..
Eleanor

On 18 September 2017 at 14:59, Satvik Kumar 
> wrote:
Dear Crystallographers,
I am trying to solve a structure in the space group P212121. Based on Matthews 
coefficient, there are 4 molecules in the asymmetric unit.
Based on my limited reading about using of Phaser, I understand that a single 
chain should be used as search model even though many copies are present in 
asymmetric unit. Am I correct?
So when I use a single chain as search model and ask Phaser to search for 4 
molecules, Phaser identifies a single solution with a warning "The top solution 
from a TF rescoring did not pack" and a warning "Search request requires more 
scattering than defined in composition. Composition increased to accommodate 
search components". But the final values reported "PAK=2 LLG=1065 TFZ==22.6" 
indicate that phaser has solved the problem.

Can anyone please explain the meaning of the warning.
When I inspect the arrangement of the chains (attachment), I observe minimal 
contact between the chains and a large cavity in the center. Can a crystal form 
this way?

I have also tried using the dimer as search model and asking phaser to search 
for 2 molecules. Even in this case, Phaser finds a single solution but the 
warning and the advisory still appear as before. The numbers reported reduce a 
bit to "PAK=1 LLG=722 TFZ==29.2".
Please help me in understanding these results.
Thanks,
Satvik

Re: [ccp4bb] His-6 versus His-10 tag

[Savvas Savvides]

Hi Herman
the survey article by Carson in Acta D DOI: 10.1107/S0907444906052024 
 from a decade ago may provide 
relevant information/insights.
best wishes
Savvas




> On 19 Sep 2017, at 12:10, herman.schreu...@sanofi.com wrote:
> 
> Dear BB,
>  
> We are planning the production of a protein for crystallization. From 
> literature, we know that the construct with a 6-histidine tag crystallizes. 
> However, for other biophysical measurements, we would prefer to have a 
> 10-histidine tag.
>  
> Does anyone has experience with His-6 versus His-10 tags in terms of 
> crystallization success?
>  
> Thanks for your help!
> Herman

Re: [ccp4bb] His-6 versus His-10 tag

[Artem Evdokimov]

Option 1: make two constructs and compare :)

Option 2: put the his10 on the n-term of the protein with a cleavage site

In my experience it matters. I had cases where his4 crystallized and his6
did not. I also had the opposite experience.

Artem


On Sep 19, 2017 6:11 AM,  wrote:

Dear BB,



We are planning the production of a protein for crystallization. From
literature, we know that the construct with a 6-histidine tag crystallizes.
However, for other biophysical measurements, we would prefer to have a
10-histidine tag.



Does anyone has experience with His-6 versus His-10 tags in terms of
crystallization success?



Thanks for your help!

Herman

Re: [ccp4bb] His-6 versus His-10 tag

[Mohinder Pal]

Dear Herman,
I have crystallised the same protein with 6xHis and 12xHis and they both 
diffracted to high resolution. However, I have not seen these tags in the 
structure. 

Best wishes,

Mohinder 

Sent from my iPhone

> On 19 Sep 2017, at 12:10, herman.schreu...@sanofi.com wrote:
> 
> Dear BB,
>  
> We are planning the production of a protein for crystallization. From 
> literature, we know that the construct with a 6-histidine tag crystallizes. 
> However, for other biophysical measurements, we would prefer to have a 
> 10-histidine tag.
>  
> Does anyone has experience with His-6 versus His-10 tags in terms of 
> crystallization success?
>  
> Thanks for your help!
> Herman
>  
>  

Re: [ccp4bb] doubt regarding MR search model

[Satvik Kumar]

Hello,

Thanks everyone for your explanations.

I have pasted the pointless output to provide more information.

Best Solution:  space group P 21 21 21

Laue group probability:   0.959

Systematic absence probability: 0.818

Total probability: 0.785

Space group confidence:0.751

Laue group confidence   0.951


Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00

Also based on L-test, pointless says data does not suggest twinning.

Yes, the R values go down when I refine in both cases. After 20 rounds
of restrained refinement using the coordinates generated by monomer as
search model, the Rwork and Rfree are 0.43 and 0.48

respectively. Refinement using the coordinates generated by using dimer as
search model also results in similar R values. I have attached the plots to
show that the R values indeed reduce in both cases.

Is my space group correct? Do I need to reexamine the space group even
though the probability is high?

If my space group is indeed correct, how do I decide whether to go ahead
with the results generated by the monomer search model or the dimer?

Please share your thoughts.


Thanks,
Satvik

On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson 
wrote:

> You need to provide a bit more information.
>
> First of all about the data processing..
>
> Is the space group correct?
> ways of being misled are:
> Non-crystallographic translations with a shift of ~0.5 along an axis - say
> a.  This will generate absences in the odd h 0 0 reflections and can make
> the space group appear to be P 21 21 21 whilst it is really P 2 21 21..
>
> Perfect twinning can have the same effect. In an orthorhombix space group
> this can usually only occur if two axes have approximately the same length,
> but the data processing stats can indicate if that is the case.
>
> Then - re PHASER. The packing rejection criteria may be set too severely -
> that seems the case for your solution.
>
> Best check on any MR solution is: does it refine - give it 20 cycles of
> mindless refinement and see if the R and FreeR go down.
>
> Then look at the maps and see if there are obvious corrections to be made..
>
> Eleanor
>
> On 18 September 2017 at 14:59, Satvik Kumar 
> wrote:
>
>> Dear Crystallographers,
>>
>> I am trying to solve a structure in the space group P212121. Based on
>> Matthews coefficient, there are 4 molecules in the asymmetric unit.
>>
>> Based on my limited reading about using of Phaser, I understand that a
>> single chain should be used as search model even though many copies are
>> present in asymmetric unit. Am I correct?
>>
>> So when I use a single chain as search model and ask Phaser to search for
>> 4 molecules, Phaser identifies a single solution with a warning "The top
>> solution from a TF rescoring did not pack" and a warning "Search request
>> requires more scattering than defined in composition. Composition
>> increased to accommodate search components". But the final values reported 
>> "PAK=2
>> LLG=1065 TFZ==22.6" indicate that phaser has solved the problem.
>>
>> Can anyone please explain the meaning of the warning.
>>
>> When I inspect the arrangement of the chains (attachment), I observe
>> minimal contact between the chains and a large cavity in the center. Can a
>> crystal form this way?
>>
>> I have also tried using the dimer as search model and asking phaser to
>> search for 2 molecules. Even in this case, Phaser finds a single solution
>> but the warning and the advisory still appear as before. The numbers
>> reported reduce a bit to "PAK=1 LLG=722 TFZ==29.2".
>>
>> Please help me in understanding these results.
>>
>> Thanks,
>> Satvik
>>
>>
>>
>


Monomer_searchmodel.pdf
Description: Adobe PDF document


Dimer_searchmodel.pdf
Description: Adobe PDF document

Re: [ccp4bb] His-6 versus His-10 tag

[Parthasarathy Sampathkumar]

Hi Herman,

I worked on a kinase, where moved from 6-His in the literature to 8-His,
and it didn't impact crystallization or diffraction (which around 3Angs).

Good Luck
Partha
On Tue, Sep 19, 2017 at 9:43 AM Oganesyan, Vaheh 
wrote:

> Hi Herman,
>
>
>
> I haven’t done His-6 versus His-10 for the same protein, but have done
> that for different ones with success. However, if in His-6 containing
> protein structure the packing or folding is such that you don’t see His-6
> then it shouldn’t matter it is 6 or 10. Just an opinion.
>
>
>
> *Regards,*
>
>
>
> *Vaheh Oganesyan*
>
> *www.medimmune.com *
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *
> herman.schreu...@sanofi.com
> *Sent:* Tuesday, September 19, 2017 6:11 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] His-6 versus His-10 tag
>
>
>
> Dear BB,
>
>
>
> We are planning the production of a protein for crystallization. From
> literature, we know that the construct with a 6-histidine tag crystallizes.
> However, for other biophysical measurements, we would prefer to have a
> 10-histidine tag.
>
>
>
> Does anyone has experience with His-6 versus His-10 tags in terms of
> crystallization success?
>
>
>
> Thanks for your help!
>
> Herman
>
>
>
>
> To the extent this electronic communication or any of its attachments
> contain information that is not in the public domain, such information is
> considered by MedImmune to be confidential and proprietary. This
> communication is expected to be read and/or used only by the individual(s)
> for whom it is intended. If you have received this electronic communication
> in error, please reply to the sender advising of the error in transmission
> and delete the original message and any accompanying documents from your
> system immediately, without copying, reviewing or otherwise using them for
> any purpose. Thank you for your cooperation.
>

Re: [ccp4bb] His-6 versus His-10 tag

[Oganesyan, Vaheh]

Hi Herman,

I haven't done His-6 versus His-10 for the same protein, but have done that for 
different ones with success. However, if in His-6 containing protein structure 
the packing or folding is such that you don't see His-6 then it shouldn't 
matter it is 6 or 10. Just an opinion.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
herman.schreu...@sanofi.com
Sent: Tuesday, September 19, 2017 6:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] His-6 versus His-10 tag

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman


To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.

[ccp4bb] AW: [ccp4bb] question regarding sequence numbering

[Herman . Schreuder]

Hi Dave and Tony,

Upon submission, the pdb checks the sequence and automatically generates 
comments about sequences derived from the expression vector. So you do not have 
to do anything. Given the issues many programs have with non-sequentially 
numbered residues, I would also number them 7,8,9.

Best,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Briggs, 
David C
Gesendet: Dienstag, 19. September 2017 14:24
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] question regarding sequence numbering

Hi Tony,
When I've had similar issues, I've numbered them sequentially (i.e.  7,8,9) and 
remarked in the PDB header that they are vector-derived sequence.
I believe that is what the PDB ask you to do in situations like this (maybe 
they can comment?).
If they are not numbered sequentially, then often graphics and refinement 
software won't treat them as linked.
Dave
--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: Antonio Ariza
Sent: Tuesday 19 September, 13:15
Subject: [ccp4bb] question regarding sequence numbering
To: ccp4bb@jiscmail.ac.uk

Hi all,
Here's a problem I haven't come across before. I'm working on a structure whose 
expression plasmid was designed to remove the first 9 amino acids from the 
protein of interest and to which an N-terminal tag was added. After cleaving 
the tag I am left with 3 amino acids (GPM) followed by the original sequence. 
Obviously the residues of interest should follow the numbering of the original 
sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?
Cheers,
Tony
--
Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
Structural insights into the function of 

 
ZRANB3
 in replication stress 
response

Re: [ccp4bb] question regarding sequence numbering

[Briggs, David C]

Hi Tony,

When I've had similar issues, I've numbered them sequentially (i.e.  7,8,9) and 
remarked in the PDB header that they are vector-derived sequence.

I believe that is what the PDB ask you to do in situations like this (maybe 
they can comment?).

If they are not numbered sequentially, then often graphics and refinement 
software won't treat them as linked.

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs



From: Antonio Ariza
Sent: Tuesday 19 September, 13:15
Subject: [ccp4bb] question regarding sequence numbering
To: ccp4bb@jiscmail.ac.uk


Hi all,

Here's a problem I haven't come across before. I'm working on a structure whose 
expression plasmid was designed to remove the first 9 amino acids from the 
protein of interest and to which an N-terminal tag was added. After cleaving 
the tag I am left with 3 amino acids (GPM) followed by the original sequence. 
Obviously the residues of interest should follow the numbering of the original 
sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?

Cheers,

Tony

--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE
e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
Structural insights into the function of 
 
ZRANB3 in replication stress 
response

[ccp4bb] question regarding sequence numbering

[Antonio Ariza]

Hi all,

Here's a problem I haven't come across before. I'm working on a structure whose 
expression plasmid was designed to remove the first 9 amino acids from the 
protein of interest and to which an N-terminal tag was added. After cleaving 
the tag I am left with 3 amino acids (GPM) followed by the original sequence. 
Obviously the residues of interest should follow the numbering of the original 
sequence (i.e. 10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?

Cheers,

Tony

--

Dr. Antonio Ariza
University of Oxford
Sir William Dunn School of Pathology
South Parks Road
Oxford
OX1 3RE

e-mail: antonio.ar...@path.ox.ac.uk
Tel: 00 +44 1865 285655

Links to my public profiles:
ResearchGate
LinkedIn
GoogleScholar
Twitter

Check out my latest paper!!!
Structural insights into the function of ZRANB3 in replication stress 
response

[ccp4bb] Kendrew Symposium, 16-17 Nov 2017, EMBL Heidelberg, Germany - Registration deadline October 5, 2017

[Christoph Mueller]

Dear Colleagues,

The 2-day Kendrew Symposium: "Revolutions in Structural Biology: 
Celebrating the 100th Anniversary of Sir John Kendrew" will take place 
on 16-17 November 2017 at EMBL Heidelberg.


The aim of this event is to celebrate Sir John Kendrew 100th anniversary 
with outstanding leaders in the field of structural biology. The talks 
will explore how revolutionary developments in structural biology 
techniques have maintained structural biology at the forefront of biology.


To view the programme and speakers, please visit:
https://www.embl.de/training/events/2017/JKS17-01/index.html

The registration deadline for the Kendrew Symposium is October 5, 2017. 
So please register now and join us at this exciting conference!


Best regards,
Christoph Müller

--

Dr. Christoph W. Müller
Head of Structural and Computational Biology Unit

EMBL
Meyerhofstrasse 1
69117 Heidelberg, Germany

email: cmuel...@embl.de
phone: 0049-6221-387-8320
fax: 0049-6221-387-519
http://www.embl.de
http://www.embl.de/research/units/scb/mueller_christoph/index.html
-

[ccp4bb] His-6 versus His-10 tag

[Herman . Schreuder]

Dear BB,

We are planning the production of a protein for crystallization. From 
literature, we know that the construct with a 6-histidine tag crystallizes. 
However, for other biophysical measurements, we would prefer to have a 
10-histidine tag.

Does anyone has experience with His-6 versus His-10 tags in terms of 
crystallization success?

Thanks for your help!
Herman