Re: [ccp4bb] Crystal structure of an unknown protein

[Kevin Jin]

HI Jiyong,

If you still have protein left, you may try to sequence it. In some cases,
even N-terminal digestion is very helpful. In my previous, I always got a
90KDa protein, which was very close to my target kinase. Based on the
protein sequencing, we identified it was one of those chaperones.

Best,

Kevin

On Sun, Dec 17, 2017 at 11:39 PM, Jiyong Su  wrote:

> Dear Pedro Matias,
>
> Thanks for you advice.
>
> After I manually changed the side chain of the residues, I got a
> "artificial" primary structure. I did a blast by using this primary
> structure.
> Finally, I found the amino acid sequence of this protein. The electron
> density could perfectly match the sequence.
> BTW, this protein is from a lovely weird bacteria which was cultured by a
> student.
>
> Best regards,
>
> Jiyong
>
>
>
>
> --
> Yours Sincerely,
>
> Jiyong Su
>
> The School of Life Sciences
> Northeast Normal University
> Changchun 130024, China
> Email: sujy...@nenu.edu.cn
> Tele: + 0086 13244318851
>
>
>
>
> 在 2017-12-14 21:08:57，Pedro Matias  写道：
> >Hello,
> >
> >Welcome to the club of unexpected results!
> >
> >You don't provide a lot of background, but based on what you wrote you can:
> >
> >1. Do a BLAST search using a known part of your sequence to find whether
> >this sequence has been deposited.
> >
> >2. Assign the different residues based on the chemical environment and
> >electron density and refine the structure.
> >
> >I'm sure you can submit the refined structure to the PDB even from an
> >unknown protein.
> >
> >Regards,
> >
> >Pedro Matias
> >
> >
> >Às 12:08 de 14/12/2017, Jiyong Su escreveu:
> >> Dear CCP4bb,
> >>
> >> In 2014, I collected a high quality data set from a crystal. But I
> >> could not solve the structure of that crystal because this protein is
> >> a contaminate.
> >> Recently, I used StruBE's Contaminer and fortunately got the solution.
> >> Thanks ContaMiner!!!  This protein is a contaminate protein.
> >>
> >> However, I found this protein is an unknown protein (about 180
> >> residues) whose amino acid sequence is not totally same as E.coli.
> >> There are about 20 point mutation sites comparing to the E.coli
> >> protein. This means this protein may be from an unknown bacteria.
> >>
> >> The space group of this crystal is new. There is also a new ligand in
> >> this protein.
> >>
> >> My question is how could I found the primary structure of this protein
> >> and how to deposit this protein in PDB.
> >>
> >> Best regards,
> >>
> >> Jiyong
> >>
> >
> >--
> >
> >Industry and Medicine Applied Crystallography
> >Macromolecular Crystallography Unit
> >___
> >Phones : (351-21) 446-9100 Ext. 1669
> > (351-21) 446-9669 (direct)
> > Fax   : (351-21) 441-1277 or 443-3644
> >
> >email : mat...@itqb.unl.pt
> >
> >http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography
> >http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit
> >
> >Mailing address :
> >Instituto de Tecnologia Quimica e Biologica António Xavier
> >Universidade Nova de Lisboa
> >Av. da República
> >2780-157 Oeiras
> >PORTUGAL
> >
> >ITQB NOVA, a great choice for your PhD
> >https://youtu.be/de6j-aaTWNQ
> >
> >Master Programme in Biochemistry for Health
> >https://youtu.be/UKstDCFjYI8
>
>
>


-- 
Kevin Jin

Sharing knowledge each other is always very joyful..

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

Re: [ccp4bb] Crystal structure of an unknown protein

[Jiyong Su]

Dear Pedro Matias,


Thanks for you advice. 


After I manually changed the side chain of the residues, I got a "artificial" 
primary structure. I did a blast by using this primary structure. 
Finally, I found the amino acid sequence of this protein. The electron density 
could perfectly match the sequence.
BTW, this protein is from a lovely weird bacteria which was cultured by a 
student.  


Best regards,


Jiyong




--
Yours Sincerely,


Jiyong Su


The School of Life Sciences
Northeast Normal University
Changchun 130024, China
Email: sujy...@nenu.edu.cn
Tele: + 0086 13244318851
 




在 2017-12-14 21:08:57，Pedro Matias  写道：
>Hello,
>
>Welcome to the club of unexpected results!
>
>You don't provide a lot of background, but based on what you wrote you can:
>
>1. Do a BLAST search using a known part of your sequence to find whether
>this sequence has been deposited.
>
>2. Assign the different residues based on the chemical environment and
>electron density and refine the structure.
>
>I'm sure you can submit the refined structure to the PDB even from an
>unknown protein.
>
>Regards,
>
>Pedro Matias
>
>
>Às 12:08 de 14/12/2017, Jiyong Su escreveu:
>> Dear CCP4bb,
>>
>> In 2014, I collected a high quality data set from a crystal. But I
>> could not solve the structure of that crystal because this protein is
>> a contaminate. 
>> Recently, I used StruBE's Contaminer and fortunately got the solution.
>> Thanks ContaMiner!!!  This protein is a contaminate protein. 
>>
>> However, I found this protein is an unknown protein (about 180
>> residues) whose amino acid sequence is not totally same as E.coli.
>> There are about 20 point mutation sites comparing to the E.coli
>> protein. This means this protein may be from an unknown bacteria.  
>>
>> The space group of this crystal is new. There is also a new ligand in
>> this protein. 
>>
>> My question is how could I found the primary structure of this protein
>> and how to deposit this protein in PDB. 
>>
>> Best regards,
>>
>> Jiyong
>>
>
>-- 
>
>Industry and Medicine Applied Crystallography
>Macromolecular Crystallography Unit
>___
>Phones : (351-21) 446-9100 Ext. 1669
> (351-21) 446-9669 (direct)
> Fax   : (351-21) 441-1277 or 443-3644
>
>email : mat...@itqb.unl.pt
>
>http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography
>http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit
>
>Mailing address :
>Instituto de Tecnologia Quimica e Biologica António Xavier
>Universidade Nova de Lisboa
>Av. da República
>2780-157 Oeiras
>PORTUGAL
>
>ITQB NOVA, a great choice for your PhD
>https://youtu.be/de6j-aaTWNQ
>
>Master Programme in Biochemistry for Health
>https://youtu.be/UKstDCFjYI8

Re: [ccp4bb] Crystal structure of an unknown protein

[Petr Leiman]

To keep this thread on topic - there is no Blue Stone on the Galveston island. 
Lots of white sand beaches, lots of black birds, lots of new BMW cars in the 
port (they unload the ship here), a few oil rigs, an occasional cruise ship, 
but no Blue Stone…

Petr


Petr Leiman
University of Texas Medical Branch
Department of BMB
Basic Sciences Building 6.600D
301 University Blvd
Galveston, TX 77555-0647
Cell: 832 908 6635
Office: 409 747 2078
 
https://scsb.utmb.edu/faculty/Leiman.asp


> On Dec 16, 2017, at 4:42 PM, Anastassis Perrakis  wrote:
> 
> 
> I wonder why you assume know there are "about 20 point mutation sites” if 
> "this protein is an unknown protein”.
> It looks like you are comparing the sequence of a protein you do not know 
> what it is to the sequence of a protein you dont really know what it is (1).
> 
> I would consider it more likely, it is the E. coli protein and the electron 
> density is ambigious so some side chains might have been erronously assigned 
> to a false identity.
> 
> A.
> 
> (1)
> Percy: You know, they do say that the Infanta's eyes are more beautiful than 
> the famous Stone of Galveston. 
> Edmund: Mm! ... What?
> Percy: The famous Stone of Galveston, My Lord.
> Edmund: And what's that, exactly?
> Percy: Well, it's a famous blue stone, and it comes ... from Galveston.
> Edmund: I see. And what about it?
> Percy: Well, My Lord, the Infanta's eyes are bluer than it, for a start.
> Edmund: I see. And have you ever seen this stone?
> Percy: (nods) No, not as such, My Lord, but I know a couple of people who 
> have, and they say it's very very blue indeed. 
> Edmund: And have these people seen the Infanta's eyes?
> Percy: No, I shouldn't think so, My Lord.
> Edmund: And neither have you, presumably.
> Percy: No, My Lord.
> Edmund: So, what you're telling me, Percy, is that something you have never 
> seen is slightly less blue than something else you have never seen.
> 
> 
>>> Dear CCP4bb,
>>> In 2014, I collected a high quality data set from a crystal. But I could 
>>> not solve the structure of that crystal because this protein is a 
>>> contaminate.
>>> Recently, I used StruBE's Contaminer and fortunately got the solution. 
>>> Thanks ContaMiner!!!  This protein is a contaminate protein.
>>> However, I found this protein is an unknown protein (about 180 residues) 
>>> whose amino acid sequence is not totally same as E.coli. There are about 20 
>>> point mutation sites comparing to the E.coli protein. This means this 
>>> protein may be from an unknown bacteria.
>>> The space group of this crystal is new. There is also a new ligand in this 
>>> protein.
>>> My question is how could I found the primary structure of this protein and 
>>> how to deposit this protein in PDB.
>>> Best regards,
>>> Jiyong
>> 
>> 
> 

Re: [ccp4bb] Crystal structure of an unknown protein

[Anastassis Perrakis]

I wonder why you assume know there are "about 20 point mutation sites” if "this 
protein is an unknown protein”.
It looks like you are comparing the sequence of a protein you do not know what 
it is to the sequence of a protein you dont really know what it is (1).

I would consider it more likely, it is the E. coli protein and the electron 
density is ambigious so some side chains might have been erronously assigned to 
a false identity.

A.

(1)
Percy: You know, they do say that the Infanta's eyes are more beautiful than 
the famous Stone of Galveston. 
Edmund: Mm! ... What?
Percy: The famous Stone of Galveston, My Lord.
Edmund: And what's that, exactly?
Percy: Well, it's a famous blue stone, and it comes ... from Galveston.
Edmund: I see. And what about it?
Percy: Well, My Lord, the Infanta's eyes are bluer than it, for a start.
Edmund: I see. And have you ever seen this stone?
Percy: (nods) No, not as such, My Lord, but I know a couple of people who have, 
and they say it's very very blue indeed. 
Edmund: And have these people seen the Infanta's eyes?
Percy: No, I shouldn't think so, My Lord.
Edmund: And neither have you, presumably.
Percy: No, My Lord.
Edmund: So, what you're telling me, Percy, is that something you have never 
seen is slightly less blue than something else you have never seen.


>> Dear CCP4bb,
>> In 2014, I collected a high quality data set from a crystal. But I could not 
>> solve the structure of that crystal because this protein is a contaminate.
>> Recently, I used StruBE's Contaminer and fortunately got the solution. 
>> Thanks ContaMiner!!!  This protein is a contaminate protein.
>> However, I found this protein is an unknown protein (about 180 residues) 
>> whose amino acid sequence is not totally same as E.coli. There are about 20 
>> point mutation sites comparing to the E.coli protein. This means this 
>> protein may be from an unknown bacteria.
>> The space group of this crystal is new. There is also a new ligand in this 
>> protein.
>> My question is how could I found the primary structure of this protein and 
>> how to deposit this protein in PDB.
>> Best regards,
>> Jiyong
> 
> 

Re: [ccp4bb] Crystal structure of an unknown protein

[Ivan Shabalin]

Hi Jiyong,

Try running the FITMUNK server in the sequencing mode:

http://witold.med.virginia.edu/fitmunk/server/


More information on using FITMUNK to identify your protein:

https://www.ncbi.nlm.nih.gov/pubmed/26894674

And here:
https://www.ncbi.nlm.nih.gov/pubmed/26660914

Ivan




With best regards,
Ivan Shabalin, Ph.D.
Research Scientist,
Department of Molecular Physiology and Biological Physics,
University of Virginia,
1340 Jefferson Park Avenue, Pinn Hall,Room 4223,
Charlottesville, VA 22908

On 12/14/2017 07:08 AM, Jiyong Su wrote:

Dear CCP4bb,

In 2014, I collected a high quality data set from a crystal. But I could 
not solve the structure of that crystal because this protein is a 
contaminate.
Recently, I used StruBE's Contaminer and fortunately got the solution. 
Thanks ContaMiner!!!  This protein is a contaminate protein.


However, I found this protein is an unknown protein (about 180 residues) 
whose amino acid sequence is not totally same as E.coli. There are about 
20 point mutation sites comparing to the E.coli protein. This means this 
protein may be from an unknown bacteria.


The space group of this crystal is new. There is also a new ligand in 
this protein.


My question is how could I found the primary structure of this protein 
and how to deposit this protein in PDB.


Best regards,

Jiyong

Re: [ccp4bb] Crystal structure of an unknown protein

[Edward A. Berry]

In this case you know the protein is closely relaed to whatefer contaminer 
found, and you have good sequence data, so I agree the next step is blast. 
Maybe it is an isozyme of the structure used.

In cases where you solve an unknown by heavy atom derivatives and have no idea 
what it is; and the resolution is too low to get a good read on the sequence, 
but you can trace the chain; the DALI server is the way to go. That is how we 
identified 4HEI.

On 12/14/2017 07:08 AM, Jiyong Su wrote:

Dear CCP4bb,

In 2014, I collected a high quality data set from a crystal. But I could not 
solve the structure of that crystal because this protein is a contaminate.
Recently, I used StruBE's Contaminer and fortunately got the solution. Thanks 
ContaMiner!!!  This protein is a contaminate protein.

However, I found this protein is an unknown protein (about 180 residues) whose 
amino acid sequence is not totally same as E.coli. There are about 20 point 
mutation sites comparing to the E.coli protein. This means this protein may be 
from an unknown bacteria.

The space group of this crystal is new. There is also a new ligand in this 
protein.

My question is how could I found the primary structure of this protein and how 
to deposit this protein in PDB.

Best regards,

Jiyong

Re: [ccp4bb] Crystal structure of an unknown protein

[Keller, Jacob]

Wow, pretty cool—you must have solved it to very high resolution to know the 
sequence from the structure. I cannot imagine, however, how you got this 
contaminant—maybe phage infection of your bacterial culture? Anyway, I agree 
with BLAST-ing the sequence, seeing what you get that is closest.

JPK


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jiyong Su
Sent: Thursday, December 14, 2017 7:09 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystal structure of an unknown protein

Dear CCP4bb,

In 2014, I collected a high quality data set from a crystal. But I could not 
solve the structure of that crystal because this protein is a contaminate.
Recently, I used StruBE's Contaminer and fortunately got the solution. Thanks 
ContaMiner!!!  This protein is a contaminate protein.

However, I found this protein is an unknown protein (about 180 residues) whose 
amino acid sequence is not totally same as E.coli. There are about 20 point 
mutation sites comparing to the E.coli protein. This means this protein may be 
from an unknown bacteria.

The space group of this crystal is new. There is also a new ligand in this 
protein.

My question is how could I found the primary structure of this protein and how 
to deposit this protein in PDB.

Best regards,

Jiyong

Re: [ccp4bb] Crystal structure of an unknown protein

[Pedro Matias]

Hello,

Welcome to the club of unexpected results!

You don't provide a lot of background, but based on what you wrote you can:

1. Do a BLAST search using a known part of your sequence to find whether
this sequence has been deposited.

2. Assign the different residues based on the chemical environment and
electron density and refine the structure.

I'm sure you can submit the refined structure to the PDB even from an
unknown protein.

Regards,

Pedro Matias


Às 12:08 de 14/12/2017, Jiyong Su escreveu:
> Dear CCP4bb,
>
> In 2014, I collected a high quality data set from a crystal. But I
> could not solve the structure of that crystal because this protein is
> a contaminate. 
> Recently, I used StruBE's Contaminer and fortunately got the solution.
> Thanks ContaMiner!!!  This protein is a contaminate protein. 
>
> However, I found this protein is an unknown protein (about 180
> residues) whose amino acid sequence is not totally same as E.coli.
> There are about 20 point mutation sites comparing to the E.coli
> protein. This means this protein may be from an unknown bacteria.  
>
> The space group of this crystal is new. There is also a new ligand in
> this protein. 
>
> My question is how could I found the primary structure of this protein
> and how to deposit this protein in PDB. 
>
> Best regards,
>
> Jiyong
>

-- 

Industry and Medicine Applied Crystallography
Macromolecular Crystallography Unit
___
Phones : (351-21) 446-9100 Ext. 1669
 (351-21) 446-9669 (direct)
 Fax   : (351-21) 441-1277 or 443-3644

email : mat...@itqb.unl.pt

http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography
http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit

Mailing address :
Instituto de Tecnologia Quimica e Biologica António Xavier
Universidade Nova de Lisboa
Av. da República
2780-157 Oeiras
PORTUGAL

ITQB NOVA, a great choice for your PhD
https://youtu.be/de6j-aaTWNQ

Master Programme in Biochemistry for Health
https://youtu.be/UKstDCFjYI8

[ccp4bb] Crystal structure of an unknown protein

[Jiyong Su]

Dear CCP4bb,


In 2014, I collected a high quality data set from a crystal. But I could not 
solve the structure of that crystal because this protein is a contaminate. 
Recently, I used StruBE's Contaminer and fortunately got the solution. Thanks 
ContaMiner!!!  This protein is a contaminate protein. 


However, I found this protein is an unknown protein (about 180 residues) whose 
amino acid sequence is not totally same as E.coli. There are about 20 point 
mutation sites comparing to the E.coli protein. This means this protein may be 
from an unknown bacteria.  


The space group of this crystal is new. There is also a new ligand in this 
protein. 


My question is how could I found the primary structure of this protein and how 
to deposit this protein in PDB. 


Best regards,


Jiyong