Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[David Briggs]

If you "multi-chicken" your 2fo-fc map in coot...

(Extensions > maps > multi-chicken)

... You might be able to pick out where different atoms are by comparing
the peak height with the near-by protein atoms (ADPs & occupancy
notwithstanding). At your resolution you will be able to see that O > N > C
and anything bigger like P or S atoms will appear enormous.

Maybe the nature of the enzyme will help you out here? Does it have a known
substrate or co-factor that might have been pulled through purification?

HTH,

Dave


On Sat, 4 Feb 2017, 21:24 Kevin Jin,  wrote:

> According to your first fig. The Ser may carry a dual conformation. If
> then, the occ ration could be ~ 7:3
>
> According to your 1st, 2nd and 3rd figures, the geometry of the density
>  is tetrahedral. Can it be PO4 with the same occ (~70%) ?
>
> If there were more figures available, the geometry of the unknown density
> could be more clear.
>
> Can You try Glucose phosphate withe occ of 70%?
>
> On Sat, Feb 4, 2017 at 7:03 AM, sharifah nur hidayah syed mazlan <
> shn.hida...@gmail.com> wrote:
>
> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>
>
>
> --
> Kevin Jin
>
> Sharing knowledge each other is always very joyful..
>
> Website: http://www.jinkai.org/
>
>
-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Kevin Jin]

According to your first fig. The Ser may carry a dual conformation. If
then, the occ ration could be ~ 7:3

According to your 1st, 2nd and 3rd figures, the geometry of the density  is
tetrahedral. Can it be PO4 with the same occ (~70%) ?

If there were more figures available, the geometry of the unknown density
could be more clear.

Can You try Glucose phosphate withe occ of 70%?

On Sat, Feb 4, 2017 at 7:03 AM, sharifah nur hidayah syed mazlan <
shn.hida...@gmail.com> wrote:

> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

Website: http://www.jinkai.org/

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Eleanor Dodson]

It is always worth doing a anomalous difference Fourier especially with
such high resolution data. Very easy if you are using CCP4 GUI2 - just ask
for it as part of your refinement run.

It is likely the high peaks could be S - but you can check by comparing
their height to that of a MET S peak . But remember MET are often somewhat
floppy and may well be less well resolved..
Eleanor


On 4 February 2017 at 19:40, Artem Evdokimov 
wrote:

> Hard to see but maybe a transesterified serine like ser-o-AMP
>
> Can you share the nature of the enzyme?
>
> Artem
> www.harkerbio.com
> "Zoidberg was here"
>
>
>
>
> On Feb 4, 2017 10:14 AM, "sharifah nur hidayah syed mazlan" <
> shn.hida...@gmail.com> wrote:
>
>> Dear All,
>>
>> I am working on a structure with an unknown blob extended from the gamma
>> O of the catalytic serine residue. The resolution of the dataset is 1.38 A.
>> I have no idea whether the residue is modified or the blob belongs to other
>> molecule.
>>
>> The protein was expressed in Rosettagami (DE3), purified using
>> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
>> chromatography). The crystallization formulation used contain 15% PEG 8000,
>> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
>> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
>> protease inhibitor was used (eg: PMSF)
>>
>> I have tried to fit in diethylene glycol as shown in one of the attached
>> figures, but as observed, it is not really fit and the molecule is in
>> incorrect conformation.
>>
>> Kindly help me with this matter.
>>
>>
>>
>> Thanks and regards,
>>
>> Sharifah Nur Hidayah
>> Universiti Putra Malaysia,
>> Malaysia
>>
>>
>>

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Artem Evdokimov]

Hard to see but maybe a transesterified serine like ser-o-AMP

Can you share the nature of the enzyme?

Artem
www.harkerbio.com
"Zoidberg was here"




On Feb 4, 2017 10:14 AM, "sharifah nur hidayah syed mazlan" <
shn.hida...@gmail.com> wrote:

> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Eleanor Dodson]

That is some blob!  What is the expectd ligand?  It is hard to see in these
pictures but are there rings? other chemical features? to be seen

You could let Arp/War build dummy atoms into it - and see what it makes.

Eleanor

On 4 February 2017 at 15:03, sharifah nur hidayah syed mazlan <
shn.hida...@gmail.com> wrote:

> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>

[ccp4bb] Unknown blob extended from catalytic serine residue

[Jim Pflugrath]

I didn't see the figure, but I would not be surprised if cacodylate donates
a methyl group to make O-methylserine.  Diethylene glycol is quite a bit
bigger than a methyl though.

Cacodylate is quite labile and undergoes radiolysis.  It should probably
not be used for crystallization.  If you either grow in a different buffer
or harvest into a different buffer before diffraction data collection, what
does the electron density map look like?

Jim

Re: [ccp4bb] on unit cell and asymmetric unit

[adarsh kumar]

Dear John

A unit cell can be produced by the symmetry operations on an asymmetric
unit. So, in your summary, if you just include number of copies in the
asymmetric unit along with the space group, it should do the job.

On Sat, Feb 4, 2017 at 4:37 PM,  <
0ef8f1cfe4cc-dmarc-requ...@jiscmail.ac.uk> wrote:

>  Dear All,
>
> Some papers write in the way that how many copies of proteins exist in the
> crystal unit cell (for example, in each unit cell there was monomer or
> dimer or 3 proteins), some write in the way that how many copies of
> proteins exist in the crystal assymetric unit.
>
> I am writing a paragraph to sumarize how many copies of proteins exist in
> the crystal unit cell or in the  crystal assymetric unit, based on
> published papers. In this situation can I treat unit cell and assymetric
> unit as same, especially if I need to prepare a table to indicate the
> copies of proteins in the unit cell or asymmetric unit?
>
> I am looking forward to getting a reply from you.
>
> Best regards.
>
> John
>



-- 

*Regards.*



*Adarsh Kumar*

*Senior Research Fellow (SPMF)*

*Dr. S. Karthikeyan's Group (Protein Crystallography Unit)*

*Dept. of Protein Science and Engineering*

*Institute of Microbial Technology (CSIR)*

*Sector 39A, Chandigarh - 160036*

*India*

[ccp4bb] on unit cell and asymmetric unit

 Dear All,
Some papers write in the way that how many copies of proteins exist in the 
crystal unit cell (for example, in each unit cell there was monomer or dimer or 
3 proteins), some write in the way that how many copies of proteins exist in 
the crystal assymetric unit.
I am writing a paragraph to sumarize how many copies of proteins exist in the 
crystal unit cell or in the  crystal assymetric unit, based on published 
papers. In this situation can I treat unit cell and assymetric unit as same, 
especially if I need to prepare a table to indicate the copies of proteins in 
the unit cell or asymmetric unit? 
I am looking forward to getting a reply from you.
Best regards.
John