Re: [ccp4bb] Renumber residues working PDB file - Applying a sequences numbering to PDB file

[Eleanor Dodson]

Usually you can bully coot into doing it but by bit. Say you need to
renumber A 1-8. I often have to change the chain id to Z say then renumber
Z. And so on . Then go back once you have finished and reset chain id fir
Z1-8 to A. Tedious but possible!

Or just run a few cycles of buccaneer with your structure - buccaneer
should do the job fir you...
Eleanor

On Sat, 4 Feb 2023 at 22:11, Matt McLeod  wrote:

> Hi all,
>
> I have been refining a structure and somehow along the way the residue
> numbers have completely shifted.  For instance, the first section of
> residues are shifted by say 8 numbers, then there is a gap from where the
> resnumbers go from 121, 151, 152, 153...and so on.  Its quite the mess.
>
> Is there a way to take a sequence file with residue numbers correct and
> apply these to the PDB file?  I have tried Renumber Residues in coot but
> this isnt working, it just shifts some of them and does opposite shifts
> elsewhere since its so discontinuous. Align and mutate just shifts them
> incorrectly from the inputted sequence without applying the sequence file
> number to the PDB.
>
> Any suggestions would be appreciated before I go and do this all
> manually...
> Matt
>
> 
>
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Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[kavyashreem]

Dear Mark,  

Thanks for the clarification.  

Regards 

Kavya 

On 2023-02-05 04:02, Mark J. van Raaij wrote:

> PS it's probably not a salt crystal...TCEP is not a salt, your ligand I 
> presume is also not a salt, a small cleaved peptide neither. As to why 
> previously in a very similar condition you did get your desired protein plus 
> (other) ligand crystal, it just means the molecule (TCEP') crystallises in a 
> similar condition to your protein - I don't think you can conclude much more 
> than that (unless there is some other difference like the TCEP being older 
> this time and more oxidised, for example). 
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas, lab 20B
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain 
> tel. +34 91 585 4616 (internal 432092)
> Section Editor Acta Crystallographica F
> https://journals.iucr.org/f/
> 
> On 4 Feb 2023, at 15:48, kavyashreem  wrote: 
> 
> Dear all,  
> 
> Sorry for the confusion created, I did not mean that a protein would have fit 
> in the small unit cell. My question was - 
> 
> 1. Why are there closely spaced spots arising in salt crystal? 
> 
> 2. If TCEP could crystallize in the condition, I have got a protein (same as 
> this)+ligand (different ligand) complex in very close condition. (ligand size 
> is within 500Da). 
> 
> Thank you 
> 
> Kavya 
> 
> On 2023-02-03 14:35, a.perra...@nki.nl wrote: Hi Kavya, 
> 
> Try https://csb.wfu.edu/tools/vmcalc/vm.html  
> 
> This tells you that a 30kD protein simply does not fit the cell. 
> 
> I am pretty sure you crystallised the ligand, or TCEP actually. 
> 
> Also, if you look at the diffractions pattern, its clear the crystal 
> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
> 2.0A. 
> 
> On 3 Feb 2023, at 09:22, kavyashreem  wrote: 
> 
> Dear all, 
> 
> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
> condition 10%PEG3350, 50mM Zinc acetate. 
> 
> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8.  
> 
> Crystal: Crystal:   
> crystal under UV m 
> 
> <8ef9453e.png>
> 
> When we collected the data at an in-house facility, it looked something like 
> this: 
> 
>  
> 
> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
> 
> I have not come across a protein diffraction like this, nor of a salt. When I 
> ran the gel for the incubated protein (protein+ligand), there was no 
> degradation. 
> 
> Although, I was sure there is some problem with this image I tried 
> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
> third. 
> 
> Can anyone please shed some light on this diffraction image? 
> 
> How can it happen?
> 
> Thank you 
> 
> Regards 
> 
> Kavya
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[Mark J. van Raaij]

PS it’s probably not a salt crystal…TCEP is not a salt, your ligand I presume 
is also not a salt, a small cleaved peptide neither. As to why previously in a 
very similar condition you did get your desired protein plus (other) ligand 
crystal, it just means the molecule (TCEP') crystallises in a similar condition 
to your protein - I don’t think you can conclude much more than that (unless 
there is some other difference like the TCEP being older this time and more 
oxidised, for example).

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 4 Feb 2023, at 15:48, kavyashreem  wrote:
> 
> Dear all, 
> 
> Sorry for the confusion created, I did not mean that a protein would have fit 
> in the small unit cell. My question was -
> 
> 1. Why are there closely spaced spots arising in salt crystal?
> 
> 2. If TCEP could crystallize in the condition, I have got a protein (same as 
> this)+ligand (different ligand) complex in very close condition. (ligand size 
> is within 500Da).
> 
> Thank you
> 
> Kavya
> 
> 
> 
> On 2023-02-03 14:35, a.perra...@nki.nl wrote:
> 
>> Hi Kavya,
>>  
>> Try https://csb.wfu.edu/tools/vmcalc/vm.html 
>>  
>>  
>> This tells you that a 30kD protein simply does not fit the cell.
>>  
>> I am pretty sure you crystallised the ligand, or TCEP actually.
>>  
>> Also, if you look at the diffractions pattern, its clear the crystal 
>> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
>> 2.0A.
>>  
>>  
>> 
>>> On 3 Feb 2023, at 09:22, kavyashreem >> > wrote:
>>> Dear all,
>>> 
>>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>>> condition 10%PEG3350, 50mM Zinc acetate.
>>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 
>>> 8. 
>>> Crystal: Crystal:   
>>> crystal under UV m
>>> <8ef9453e.png>
>>> When we collected the data at an in-house facility, it looked something 
>>> like this:
>>> 
>>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>>> I have not come across a protein diffraction like this, nor of a salt. When 
>>> I ran the gel for the incubated protein (protein+ligand), there was no 
>>> degradation.
>>> Although, I was sure there is some problem with this image I tried 
>>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not 
>>> the third.
>>> Can anyone please shed some light on this diffraction image?
>>> How can it happen?
>>>  
>>> Thank you
>>> Regards
>>> Kavya
>>>  
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>>> 
>> To unsubscribe from the CCP4BB list, click the following link:
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> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[Mark J. van Raaij]

the unit cell in the c direction is quite long, 49 Å, this gives the relatively 
close spots in one direction.

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 4 Feb 2023, at 15:48, kavyashreem  wrote:
> 
> Dear all, 
> 
> Sorry for the confusion created, I did not mean that a protein would have fit 
> in the small unit cell. My question was -
> 
> 1. Why are there closely spaced spots arising in salt crystal?
> 
> 2. If TCEP could crystallize in the condition, I have got a protein (same as 
> this)+ligand (different ligand) complex in very close condition. (ligand size 
> is within 500Da).
> 
> Thank you
> 
> Kavya
> 
> 
> 
> On 2023-02-03 14:35, a.perra...@nki.nl wrote:
> 
>> Hi Kavya,
>>  
>> Try https://csb.wfu.edu/tools/vmcalc/vm.html 
>>  
>>  
>> This tells you that a 30kD protein simply does not fit the cell.
>>  
>> I am pretty sure you crystallised the ligand, or TCEP actually.
>>  
>> Also, if you look at the diffractions pattern, its clear the crystal 
>> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
>> 2.0A.
>>  
>>  
>> 
>>> On 3 Feb 2023, at 09:22, kavyashreem >> > wrote:
>>> Dear all,
>>> 
>>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>>> condition 10%PEG3350, 50mM Zinc acetate.
>>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 
>>> 8. 
>>> Crystal: Crystal:   
>>> crystal under UV m
>>> <8ef9453e.png>
>>> When we collected the data at an in-house facility, it looked something 
>>> like this:
>>> 
>>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>>> I have not come across a protein diffraction like this, nor of a salt. When 
>>> I ran the gel for the incubated protein (protein+ligand), there was no 
>>> degradation.
>>> Although, I was sure there is some problem with this image I tried 
>>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not 
>>> the third.
>>> Can anyone please shed some light on this diffraction image?
>>> How can it happen?
>>>  
>>> Thank you
>>> Regards
>>> Kavya
>>>  
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>>> 
>> To unsubscribe from the CCP4BB list, click the following link:
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>> 
> 
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[ccp4bb] Renumber residues working PDB file - Applying a sequences numbering to PDB file

[Matt McLeod]

Hi all,

I have been refining a structure and somehow along the way the residue numbers 
have completely shifted.  For instance, the first section of residues are 
shifted by say 8 numbers, then there is a gap from where the resnumbers go from 
121, 151, 152, 153...and so on.  Its quite the mess.

Is there a way to take a sequence file with residue numbers correct and apply 
these to the PDB file?  I have tried Renumber Residues in coot but this isnt 
working, it just shifts some of them and does opposite shifts elsewhere since 
its so discontinuous. Align and mutate just shifts them incorrectly from the 
inputted sequence without applying the sequence file number to the PDB.

Any suggestions would be appreciated before I go and do this all manually...
Matt



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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[kavyashreem]

Dear all,  

Sorry for the confusion created, I did not mean that a protein would
have fit in the small unit cell. My question was - 

1. Why are there closely spaced spots arising in salt crystal? 

2. If TCEP could crystallize in the condition, I have got a protein
(same as this)+ligand (different ligand) complex in very close
condition. (ligand size is within 500Da). 

Thank you 

Kavya 

On 2023-02-03 14:35, a.perra...@nki.nl wrote:

> Hi Kavya, 
> 
> Try https://csb.wfu.edu/tools/vmcalc/vm.html  
> 
> This tells you that a 30kD protein simply does not fit the cell. 
> 
> I am pretty sure you crystallised the ligand, or TCEP actually. 
> 
> Also, if you look at the diffractions pattern, its clear the crystal 
> diffracts beyond 1.0A, diffraction spots are really very very very strong at 
> 2.0A. 
> 
>> On 3 Feb 2023, at 09:22, kavyashreem  wrote: 
>> 
>> Dear all, 
>> 
>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>> condition 10%PEG3350, 50mM Zinc acetate. 
>> 
>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8. 
>>  
>> 
>> Crystal: Crystal:   
>> crystal under UV m 
>> 
>> <8ef9453e.png>
>> 
>> When we collected the data at an in-house facility, it looked something like 
>> this: 
>> 
>>  
>> 
>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>> 
>> I have not come across a protein diffraction like this, nor of a salt. When 
>> I ran the gel for the incubated protein (protein+ligand), there was no 
>> degradation. 
>> 
>> Although, I was sure there is some problem with this image I tried 
>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
>> third. 
>> 
>> Can anyone please shed some light on this diffraction image? 
>> 
>> How can it happen?
>> 
>> Thank you 
>> 
>> Regards 
>> 
>> Kavya
>> 
>> -
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
> -
> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[kavyashreem]

Dear Mark,  

I did think it was salt, so I checked the same batch of protein which
went for crystallization by running a gel, it was intact, no cleavage.
My doubts arose because of two things 

1. I crystallized the same protein with another ligand, in very similar
condition (10% PEG3350, 50mM Zn acetate, 0.25M Sod citrate) which
diffracted and ligand density was visible in the protein structure 

2. When can salt crystals give closely spaced spots which is seen in the
image that was attached 

Thank you 

Regards 

Kavya 

On 2023-02-03 16:50, Mark J. van Raaij wrote:

> like others mentioned, looks like something in between a salt and a protein, 
> perhaps TCEP, the ligand, a peptide cleaved from your protein by trace 
> protease. 
> If possible, I would move the detector closer, collect an atomic resolution 
> dataset and try to solve the structure by direct methods. You never know, it 
> could be something interesting. 
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas, lab 20B
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. +34 91 585 4616 (internal 432092)
> Section Editor Acta Crystallographica F
> https://journals.iucr.org/f/ 
> https://namedrop.io/markvanraaij 
> 
>> On 3 Feb 2023, at 09:22, kavyashreem  wrote: 
>> 
>> Dear all, 
>> 
>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>> condition 10%PEG3350, 50mM Zinc acetate. 
>> 
>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8. 
>>  
>> 
>> Crystal: Crystal:   
>> crystal under UV m 
>> 
>> <8ef9453e.png>
>> 
>> When we collected the data at an in-house facility, it looked something like 
>> this: 
>> 
>>  
>> 
>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>> 
>> I have not come across a protein diffraction like this, nor of a salt. When 
>> I ran the gel for the incubated protein (protein+ligand), there was no 
>> degradation. 
>> 
>> Although, I was sure there is some problem with this image I tried 
>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
>> third. 
>> 
>> Can anyone please shed some light on this diffraction image? 
>> 
>> How can it happen?
>> 
>> Thank you 
>> 
>> Regards 
>> 
>> Kavya
>> 
>> -
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[kavyashreem]

Hi Jessica,  

I am quite sure the protein cannot be fit in this unitcell. I was just
curious why the diffraction has closely spaced spots.  

Thanks 

On 2023-02-04 00:02, Jessica Bruhn wrote:

> Hi Kavya, 
> 
> As others have mentioned, the unit cell is too small to contain your protein. 
> With a volume of ~4820 Ang^3, the unit cell can contain at most ~268 atoms, 
> excluding hydrogens (divide the volume by 18 to get this number). If the 
> symmetry is P3, then the asymmetric unit can only contain ~89 atoms (divide 
> the number of atoms in the unit cell by 3), which is not a lot. It is most 
> likely something organic from your buffers (the ligand, TCEP, protein 
> fragment, other buffer components, etc).  
> 
> Searching the CCDC (https://www.ccdc.cam.ac.uk/structures/?) or the COD 
> (https://nanocrystallography.org/search.html) databases may be helpful. The 
> CCDC also has a unit cell searcher tool (CellCheckCSD) that you can download 
> and use without a license 
> (https://www.ccdc.cam.ac.uk/support-and-resources/downloads/).   
> Collecting higher resolution data (<1 Ang) and trying to solve this with 
> SHELXT would likely get to the bottom of things if you really want to know. 
> 
> Best of luck! 
> 
> Kind regards, 
> Jessica
> 
> On Fri, Feb 3, 2023 at 7:37 AM Artem Evdokimov  
> wrote: 
> With 50 mM Zn++ in solution, whatever it is will probably have a Zn salt in 
> it. So if you wanted to solve it by direct methods or via SAD - that should 
> do well. Sadly (hur hur) it's probably quite small, whatever it is.
> 
> Artem 
> 
> - Cosmic Cats approve of this message 
> 
> On Fri, Feb 3, 2023 at 6:20 AM Mark J. van Raaij  
> wrote: 
> like others mentioned, looks like something in between a salt and a protein, 
> perhaps TCEP, the ligand, a peptide cleaved from your protein by trace 
> protease. 
> If possible, I would move the detector closer, collect an atomic resolution 
> dataset and try to solve the structure by direct methods. You never know, it 
> could be something interesting. 
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas, lab 20B
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. +34 91 585 4616 (internal 432092)
> Section Editor Acta Crystallographica F
> https://journals.iucr.org/f/ 
> https://namedrop.io/markvanraaij 
> 
> On 3 Feb 2023, at 09:22, kavyashreem  wrote: 
> 
> Dear all, 
> 
> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
> condition 10%PEG3350, 50mM Zinc acetate. 
> 
> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8.  
> 
> Crystal: Crystal:   
> crystal under UV m 
> 
> <8ef9453e.png>
> 
> When we collected the data at an in-house facility, it looked something like 
> this: 
> 
>  
> 
> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
> 
> I have not come across a protein diffraction like this, nor of a salt. When I 
> ran the gel for the incubated protein (protein+ligand), there was no 
> degradation. 
> 
> Although, I was sure there is some problem with this image I tried 
> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
> third. 
> 
> Can anyone please shed some light on this diffraction image? 
> 
> How can it happen?
> 
> Thank you 
> 
> Regards 
> 
> Kavya
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 
> -
> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

[kavyashreem]

Dear Thomas,  

Interestingly, I had crystallized the same protein with another ligand
before with the same condition except that it had sodium citrate in
addition. I was able to collect the data for this and it was a
protein-ligand complex, which could be seen in the density. So I was
speculating about this data especially because of the closely spaced
diffraction spots, and if the crystal has some peculiar defect.  

Thank you 

Regards 

Kavya 

On 2023-02-03 14:27, Thomas Flower wrote:

> Dear Kavya, 
> 
> 4 mM is quite a high concentration of TCEP, perhaps they are TCEP crystals. 
> 
> You could try a Unit Cell Search of the CCDC to see if you find a match: Unit 
> Cell Search - WebCSD (cam.ac.uk) [2] 
> 
> Best, 
> 
> Thomas 
> 
> Thomas Flower, PhD
> Senior Scientist, Protein Science
> 
> Galapagos
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> 
> FROM: CCP4 bulletin board  ON BEHALF OF mesters@biochem
> SENT: vendredi 3 février 2023 9:53 AM
> TO: CCP4BB@JISCMAIL.AC.UK
> SUBJECT: Re: [ccp4bb] Regarding the diffraction image 
> 
> You don't often get email from mest...@biochem.uni-luebeck.de. Learn why this 
> is important [4]  
> 
> A and B unit cell dimensions are hardly bigger than twice the uni cell of 
> cubic NaCl and will probably not accommodate a 30 kDa protein. 
> 
> Make a SDS gel of washed & dissolved crystals to be sure these are not alt or 
> inhibitor crystals. You can stain the crystals with IzIt... 
> 
> J. 
> 
> --
> Dr. math. et dis. nat. Jeroen R. Mesters
> https://orcid.org/-0001-8532-6699 [5] 
> 
> University of Lübeck
> Institute of Biochemistry
> https://www.biochem.uni-luebeck.de [6]
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> 
>> Am 03.02.2023 um 09:22 schrieb kavyashreem : 
>> 
>> Dear all, 
>> 
>> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the 
>> condition 10%PEG3350, 50mM Zinc acetate. 
>> 
>> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH 8. 
>>  
>> 
>> Crystal: Crystal:   
>> crystal under UV m 
>> 
>> <8ef9453e.png> 
>> 
>> When we collected the data at an in-house facility, it looked something like 
>> this: 
>> 
>>  
>> 
>> The minimum resolution spot is around 9Ang and maximum ~2.2Ang. 
>> 
>> I have not come across a protein diffraction like this, nor of a salt. When 
>> I ran the gel for the incubated protein (protein+ligand), there was no 
>> degradation. 
>> 
>> Although, I was sure there is some problem with this image I tried 
>> processing, which could not be, But indexing showed a unit cell  of 11Ang, 
>> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not the 
>> third. 
>> 
>> Can anyone please shed some light on this diffraction image? 
>> 
>> How can it happen?
>> 
>> Thank you 
>> 
>> Regards 
>> 
>> Kavya
>> 
>> -
>> 
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