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

2023-02-07 Thread kavyashreem 
Dear Patrick, 

I did use microseeding but for a different ligand. Will try with this as
well. 

Thank you 

REgards 

Kavya 

On 2023-02-07 16:20, Patrick Shaw Stewart wrote:

>> As to why previously in a very similar condition you did get your desired 
>> protein plus (other) ligand
> 
> Kavya, did you use microseeding?  That's the way to get consistent results. 
> 
> Since you're changing the ligand I suggest you go back and run a few random 
> screens (with crushed crystals of your protein with the other ligand) - 
> so-called microseed matrix-screening. 
> 
>> https://doi.org/10.1107/S0907444907007652
> 
> Good luck 
> 
> Patrick 
> 
> On Sat, Feb 4, 2023 at 10:32 PM 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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  -- 

 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-07 Thread Patrick Shaw Stewart 
>
> As to why previously in a very similar condition you did get your desired
> protein plus (other) ligand


Kavya, did you use microseeding?  That's the way to get consistent results.

Since you're changing the ligand I suggest you go back and run a few random
screens (with crushed crystals of your protein with the other ligand) -
so-called microseed matrix-screening.

https://doi.org/10.1107/S0907444907007652


Good luck

Patrick






On Sat, Feb 4, 2023 at 10:32 PM 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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-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Patrick Shaw Stewart, Peter Baldock, Stefan Kolek

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36



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

2023-02-06 Thread kavyashreem 
Dear Ben, 

It was a single crystal that was mounted. I did use IZIT dye, did not
trun blue much. Probably its a TCEp crystal. But I did try a negative
control without protein+ligand and rest everything same, the drop was
clear! 

Thank you 

Regards 

Kavya 

On 2023-02-06 22:49, Ben Bax wrote:

> Hi Kavya, More than one crystal?  
> Epitaxial growth (protein crystal growing on salt crystal or vice versa) 
> Did you try IZIT? 
> (https://hamptonresearch.com/product-Izit-Crystal-Dye-33.html). 
> 
> I have seen salt and protein crystals in the same drop before now - but not 
> growing together. 
> Ben
> 
> On 3 Feb 2023, at 22:15, CRAIG A BINGMAN 
> <21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote: 
> 
> Oxidation products of TCEP crystallize in the presence of zinc ions. These 
> crystals fluoresce strongly under UV, which seems like a dirty trick, if your 
> expectation is that only protein crystals can do that. The brightfield white 
> light image of the crystals is also consistent with not-protein crystals, 
> because of the high refractive index contrast between the crystals and the 
> mother liquor. 
> 
> FROM: CCP4 bulletin board  on behalf of kavyashreem 
> 
> DATE: Friday, February 3, 2023 at 2:32 AM
> TO: CCP4BB@JISCMAIL.AC.UK 
> SUBJECT: [ccp4bb] Regarding the diffraction image 
> 
> 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] Regarding the diffraction image

2023-02-06 Thread Ben Bax 
Hi Kavya,
More than one crystal? 
Epitaxial growth (protein crystal growing on salt crystal or vice versa)
Did you try IZIT? 
(https://hamptonresearch.com/product-Izit-Crystal-Dye-33.html).

I have seen salt and protein crystals in the same drop before now - but not 
growing together.
Ben

On 3 Feb 2023, at 22:15, CRAIG A BINGMAN 
<21371e2fba31-dmarc-requ...@jiscmail.ac.uk> wrote:

Oxidation products of TCEP crystallize in the presence of zinc ions. These 
crystals fluoresce strongly under UV, which seems like a dirty trick, if your 
expectation is that only protein crystals can do that. The brightfield white 
light image of the crystals is also consistent with not-protein crystals, 
because of the high refractive index contrast between the crystals and the 
mother liquor.
 
From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of kavyashreem 
mailto:kavyashr...@instem.res.in>>
Date: Friday, February 3, 2023 at 2:32 AM
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Regarding the diffraction image

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

2023-02-05 Thread kavyashreem 
Dear Bernhard Rupp, 

Sure, will look in this direction. 

Thank you 

Regards 

Kavya 

On 2023-02-06 00:41, Bernhard Rupp wrote:

> TCEP is known to form transition metal complexes. With the molecule alone 
> already about 10A across, a ~11x11x46 cell is not unreasonable, and there 
> might be alternatives to P3 (can't tell from the single image). Would be 
> interesting to collect and, as mentioned, toss into Direct methods assisted 
> by anomalous P (or Zn?) signal... 
> 
> Have fun, BR 
> 
> FROM: CCP4 bulletin board  ON BEHALF OF Mark J. van 
> Raaij
> SENT: Saturday, February 4, 2023 14:32
> TO: CCP4BB@JISCMAIL.AC.UK
> SUBJECT: Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image 
> 
> 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 
> 
> -
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-05 Thread Bernhard Rupp 
TCEP is known to form transition metal complexes. With the molecule alone 
already about 10A across, a ~11x11x46 cell is not unreasonable, and there might 
be alternatives to P3 (can’t tell from the single image). Would be interesting 
to collect and, as mentioned, toss into Direct methods assisted by anomalous P 
(or Zn?) signal... 

 

Have fun, BR

 

From: CCP4 bulletin board  On Behalf Of Mark J. van Raaij
Sent: Saturday, February 4, 2023 14:32
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

 

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 mailto:kavyashr...@instem.res.in> > 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 <mailto: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 mailto:kavyashr...@instem.res.in> > 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

 

 


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

2023-02-04 Thread 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
> 
> -
> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-04 Thread 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 
>>> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-04 Thread 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 
>>> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-04 Thread 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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> 
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-04 Thread 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:
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Re: [ccp4bb] [EXT] Re: [ccp4bb] Regarding the diffraction image

2023-02-04 Thread 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

2023-02-04 Thread 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
> 102 Avenue Gaston Roussel 
> 93230 Romainville 
> France
> T: +33 7 81 26 95 70
> www.glpg.com [3] 
> 
> 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]
> phone: +49-451-3101-3105
> Ratzeburger Allee 160
> 23562 Lübeck
> Germany
> -- 
> 
>> 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
>> 
>> -
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Regarding the diffraction image

2023-02-03 Thread Jessica Bruhn 
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] Regarding the diffraction image

2023-02-03 Thread Artem Evdokimov 
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] Regarding the diffraction image

2023-02-03 Thread Mark J. van Raaij 
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] Regarding the diffraction image

2023-02-03 Thread Thomas Flower 
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)<https://www.ccdc.cam.ac.uk/structures/WebCSD/UnitCell>

Best,
Thomas



Thomas Flower, PhD
Senior Scientist, Protein Science

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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<mailto:mest...@biochem.uni-luebeck.de>. Learn 
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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<https://eur05.safelinks.protection.outlook.com/?url=https%3A%2F%2Forcid.org%2F-0001-8532-6699=05%7C01%7CThomas.flower%40GLPG.COM%7C44212ddb46284392893a08db05c40c61%7C627f3c33bccc48bba033c0a6521f7642%7C1%7C0%7C638110111905504664%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C=BCI5EW%2B3Ha6ZJkPsy1sOX2ePH1Sa21Nm5iXg7%2F%2B64ys%3D=0>
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Am 03.02.2023 um 09:22 schrieb kavyashreem 
mailto:kavyashr...@instem.res.in>>:

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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Re: [ccp4bb] Regarding the diffraction image

2023-02-03 Thread a . perrakis 
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 
mailto:kavyashr...@instem.res.in>> 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





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Re: [ccp4bb] Regarding the diffraction image

2023-02-03 Thread mesters@biochem 
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



University of Lübeck
Institute of Biochemistry
https://www.biochem.uni-luebeck.de
phone: +49-451-3101-3105
Ratzeburger Allee 160
23562 Lübeck
Germany
--

> 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
>  
> 
> 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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