Re: [ccp4bb] data processing with split/bad crystals

[graeme.win...@diamond.ac.uk]

Dear Andreas, all,


With DIALS, correct treatment of overlapping reflections is current work in 
progress - yes, you can index and refine two or more independent lattices, but 
running dials integration on this will probably not work as well as we'd like 
at the moment. It should do something sensible-ish but... Split lattices are 
tricky: at low angle the spots tend to overlap / merge, at higher angles become 
resolved, so treating them as independent lattices as Andreas describes may not 
be successful.


My experience of chemical crystallography at a synchrotron is that a large 
fraction of samples come with satellites etc, so developing robust handling for 
this is very much on our to-do and in current active development.


Of course, this is one of those data processing problems which is perhaps best 
fixed with a better sample  but that's not a very helpful answer!


Best wishes Graeme



From: CCP4 bulletin board  on behalf of Andreas Förster 

Sent: 16 July 2018 22:14:49
To: ccp4bb
Subject: Re: [ccp4bb] data processing with split/bad crystals

Dear Tommi,

DIALS is good with multiple lattices.  It might not have given you best
results as part of the Diamond pipeline, but give it a try with the
max_lattices=2 parameter during dials.index and see where it takes you.

That said, you'll end up with worse statistics if you have two lattices.
  Don't expect magic from your processing programs.

All best.


Andreas



On 16/07/2018 10:55, Kajander, Tommi A wrote:
> Dear All,
>
> I was wondering what would be the best software nowadays to try to process 
> data from crystal that clearly is split or
> has a secondary set of lattice points (close, poor data) in the raw data - 
> data can be processed with XDS (2.9-2.8 Å) but Rmerge tends to be
> bit high at low resolution (close to 7-8% depending on processing) - using 
> XSCALE helps with the radiation damage correction some what.
>
> Data looks like primitive orthorombic but not quite sure (also seems like it 
> has one screw axis e.g. P2212 - but oddly phaser finds
> solutions in P22121 also or even preferably…..). I am wondering a bit if it 
> isn’t actually monoclinic.
>
> Based on automated processing by Diamond pipeline XDS seems most robust - but 
> any hints on such cases would
> be welcome. Of course we will try to get better crystal but so far no luck.
>
> Thanks for comments,
>
> Best
> Tommi
>
>
>
> ---
>
> Tommi Kajander, Ph.D.
> Structural Biology and Biophysics
> Institute of Biotechnology
> University of Helsinki
> Viikinkaari 1 (P.O. Box 65)
> 00014 Helsinki, Finland
>
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Hi Radu,

Many Thanks for your clarifications, and reference for background
materials. I will implement all suggestions in my next expression batch.

Best Wishes,
Partha

On Mon, Jul 16, 2018 at 1:53 PM,  wrote:

> Hi Partha,
>
> In this case (un-treated HEK293), you cannot use EndoH with already
> purified
> proteins (as per your reply to Artem's email). The N-glycans will be
> fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background
> infos). You can still use PNGase. But, as Artem suggested, this often
> causes
> protein precipitation when it can access the sites on folded proteins.
> Apart
> from shaving the glycan shield, PNGase causes unwanted mutations at the
> N-linked sites: Asn -> Asp, which is where most problems stem from.
>
> Nevertheless, since your fully glycosylated protein doesn't crystallize,
> there's not much else you can do with the current prep Except for
> example
> masking your glycans with a nice lectin such as griffithsin, which would be
> very elegant for crystallography. Or indeed trying cryo-EM, where wild-type
> N-linked glycans are extremely helpful! Why would anyone crystallize
> proteins
> these days anyway :-)
>
> Best wishes,
>
> Radu
> --
> Radu Aricescu
> MRC Laboratory of Molecular Biology
> Francis Crick Avenue
> Cambridge Biomedical Campus
> Cambridge CB2 0QH, U.K.
> tel: +44-(0)1223-267049
> fax: +44-(0)1223-268305
> www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu
>
> > Hi Savvas,
> > Many Thanks for your inputs and references. This cell line used is not
> HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
> cell-line
> stably expressing (created using lenti-methods by a
> > former colleague) the protein of interest. In future, I will plan to do
> expression in the presence of Kifunensine, followed by EndoH treatment
> before complexation and crystallization.
> > Best Wishes,
> > Partha
> > On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides <
> savvas.savvi...@ugent.be>
> wrote:
> >> Dear Partha
> >> you do not specify which HEK293 cell line you have used, but if it so
> happens that it is the very handy HEK293S *MGAT1-/- *cell line
> >> (previously known as HEK293S *GnTI-/- ) *which produces
> >> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH
> (e.g.
> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
> immuni.2017.12.008).
> >> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
> quite well for protein expression in HEK293T and renders N-linked glycans
> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et al.
> doi:10.1038/nsmb.2367).
> >> If resources and protein material allow, you might also want to consider
> the permutation exercise of subjecting the complex to deglycosylation, or
> the individual components followed by complex formation/purification, or
> just one of the two components followed by complex formation/purification,
> or even one of the two components followed by further deglycosylation of
> the complex. We are becoming more and more apprehensive of the possible
> role of glycans in complex formation.
> >> And then there is of course the option to apply mutagenesis, e.g. via
> N—>Q, to eliminate certain N-linked glycans either as a standalone
> approach
> >> or in combination with enzymatic glycan digestions as described above.
> Best
> wishes
> >> Savvas
> >> *---*
> >> *Savvas Savvides*
> >> VIB Center for Inflammation Research
> >> Dept. Biochemistry & Microbiology, Ghent University
> >> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
> >> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
> savvas.savvides_skype
> >> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
> On
> 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
> wrote:
> >> Dear All,
> >> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
> >> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
> >> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing
> Endo F1, F2, F3 be sufficient or should this be tried in combination with
> PNGase (which requires
> >> desaturating conditions)?!!
> >> Many Thanks in advance for your suggestions, and reference.
> >> 

Re: [ccp4bb] data processing with split/bad crystals

[Rangana Warshamanage]

CrysAlisPro would be my best bet. It has an option for multicystals and
twinning. Also it offers a very good visual tools to see any relationships
between multiple lattices, if they have.

Rangana

On Mon, 16 Jul 2018 at 22:16 Andreas Förster  wrote:

> Dear Tommi,
>
> DIALS is good with multiple lattices.  It might not have given you best
> results as part of the Diamond pipeline, but give it a try with the
> max_lattices=2 parameter during dials.index and see where it takes you.
>
> That said, you'll end up with worse statistics if you have two lattices.
>   Don't expect magic from your processing programs.
>
> All best.
>
>
> Andreas
>
>
>
> On 16/07/2018 10:55, Kajander, Tommi A wrote:
> > Dear All,
> >
> > I was wondering what would be the best software nowadays to try to
> process data from crystal that clearly is split or
> > has a secondary set of lattice points (close, poor data) in the raw data
> - data can be processed with XDS (2.9-2.8 Å) but Rmerge tends to be
> > bit high at low resolution (close to 7-8% depending on processing) -
> using XSCALE helps with the radiation damage correction some what.
> >
> > Data looks like primitive orthorombic but not quite sure (also seems
> like it has one screw axis e.g. P2212 - but oddly phaser finds
> > solutions in P22121 also or even preferably…..). I am wondering a bit if
> it isn’t actually monoclinic.
> >
> > Based on automated processing by Diamond pipeline XDS seems most robust
> - but any hints on such cases would
> > be welcome. Of course we will try to get better crystal but so far no
> luck.
> >
> > Thanks for comments,
> >
> > Best
> > Tommi
> >
> >
> >
> > ---
> >
> > Tommi Kajander, Ph.D.
> > Structural Biology and Biophysics
> > Institute of Biot
> echnology
> > University of Helsinki
> > Viikinkaari 1 (P.O. Box 65)
> > 00014 Helsinki, Finland
> >
> >
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> >
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] data processing with split/bad crystals

[Andreas Förster]

Dear Tommi,

DIALS is good with multiple lattices.  It might not have given you best 
results as part of the Diamond pipeline, but give it a try with the 
max_lattices=2 parameter during dials.index and see where it takes you.


That said, you'll end up with worse statistics if you have two lattices. 
 Don't expect magic from your processing programs.


All best.


Andreas



On 16/07/2018 10:55, Kajander, Tommi A wrote:

Dear All,

I was wondering what would be the best software nowadays to try to process data 
from crystal that clearly is split or
has a secondary set of lattice points (close, poor data) in the raw data - data 
can be processed with XDS (2.9-2.8 Å) but Rmerge tends to be
bit high at low resolution (close to 7-8% depending on processing) - using 
XSCALE helps with the radiation damage correction some what.

Data looks like primitive orthorombic but not quite sure (also seems like it 
has one screw axis e.g. P2212 - but oddly phaser finds
solutions in P22121 also or even preferably…..). I am wondering a bit if it 
isn’t actually monoclinic.

Based on automated processing by Diamond pipeline XDS seems most robust - but 
any hints on such cases would
be welcome. Of course we will try to get better crystal but so far no luck.

Thanks for comments,

Best
Tommi



---

Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1 (P.O. Box 65)
00014 Helsinki, Finland





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1





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Re: [ccp4bb] data processing with split/bad crystals

[Tim Gruene]

Dear Tommi,

saint (Part of the Bruker APEX/Proteum suite) can integrate with, I think, up 
to five orientation matrices at a time. It integrates smoothly with twinabs. 
Saint seems quite sophisticated in modelling the reflection profiles from the 
different lattices.
EvalCCD (Eval15?) may be able to to similar things.

Other packages can integrate different lattices one after the other (XDS can 
do this with little effort), but they don't take into account that spots may 
be the result of overlapping reflections from different lattices.

You can check your next Chemistry department. Chances they have an APEX 
installation are quite high.

Best,
Tim

On Monday, July 16, 2018 10:55:15 PM CEST Kajander, Tommi A wrote:
> Dear All, 
> 
> I was wondering what would be the best software nowadays to try to process
> data from crystal that clearly is split or
 has a secondary set of lattice
> points (close, poor data) in the raw data - data can be processed with XDS
> (2.9-2.8 Å) but Rmerge tends to be bit high at low resolution (close to
> 7-8% depending on processing) - using XSCALE helps with the radiation
> damage correction some what. 
> Data looks like primitive orthorombic but not quite sure (also seems like it
> has one screw axis e.g. P2212 - but oddly phaser finds 
 solutions in
> P22121 also or even preferably…..). I am wondering a bit if it isn’t
> actually monoclinic. 
> Based on automated processing by Diamond pipeline XDS seems most robust -
> but any hints on such cases would
 be welcome. Of course we will try to get
> better crystal but so far no luck. 
> Thanks for comments,
> 
> Best
> Tommi
> 
> 
> 
> ---
> 
> Tommi Kajander, Ph.D.
> Structural Biology and Biophysics
> Institute of Biotechnology
> University of Helsinki
> Viikinkaari 1 (P.O. Box 65)
> 00014 Helsinki, Finland
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

-- 
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OSUA/204
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[radu]

Oops,

I made a mistake in my previous email, apologies! Griffithsin would also
require high-Man glycans, so kifunensine treatment or GnTI-/- cells So
yes, it's either PNGase or cryo-EM...

Best wishes,

Radu



> Hi Savvas,
>
> Many Thanks for your inputs and references. This cell line used is not
> HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
> cell-line stably expressing (created using lenti-methods by a
> former colleague) the protein of interest. In future, I will plan to do
> expression in the presence of Kifunensine, followed by EndoH treatment
> before complexation and crystallization.
>
> Best Wishes,
> Partha
>
>
> On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
> wrote:
>
>> Dear Partha
>> you do not specify which HEK293 cell line you have used, but if it so
>> happens that it is the very handy HEK293S *MGAT1-/- *cell line
>> (previously known as HEK293S *GnTI-/- ) *which produces
>> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
>> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
>> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
>> immuni.2017.12.008).
>> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
>> quite well for protein expression in HEK293T and renders N-linked glycans
>> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
>> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et
>> al. doi:10.1038/nsmb.2367).
>>
>> If resources and protein material allow, you might also want to consider
>> the permutation exercise of subjecting the complex to deglycosylation, or
>> the individual components followed by complex formation/purification, or
>> just one of the two components followed by complex formation/purification,
>> or even one of the two components followed by further deglycosylation of
>> the complex. We are becoming more and more apprehensive of the possible
>> role of glycans in complex formation.
>> And then there is of course the option to apply mutagenesis, e.g. via
>> N—>Q, to eliminate certain N-linked glycans either as a standalone
>> approach
>> or in combination with enzymatic glycan digestions as described above.
>>
>> Best wishes
>> Savvas
>>
>>
>> *---*
>> *Savvas Savvides*
>> VIB Center for Inflammation Research
>> Dept. Biochemistry & Microbiology, Ghent University
>> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
>> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
>> savvas.savvides_skype
>> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
>>
>> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
>> wrote:
>>
>> Dear All,
>>
>> I am in a situation, almost for the first time within my limited
>> experience, that deglycosylation might be necessary to obtain crystal. So,
>> I thought of tapping to vast experience of CCP4BBers, while I am searching
>> literature.
>>
>> I have protein that has been expressed in HEK293 cells, secreted into
>> media, purified over IMAC and SEC columns. Crystallization-screens with its
>> binding partners (they form good complexes based on analytical SEC) have
>> not produced any useful hits (whereas complexes with related proteins
>> worked well). So, I plan to re-try complex formation and \crystallization
>> screen after deglysosylation.
>>
>> My question is: In practice, Does a kit (for example here: https://www.
>> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
>> containing Endo F1, F2, F3 be sufficient or should this be tried in
>> combination with PNGase (which requires
>> desaturating conditions)?!!
>>
>> Many Thanks in advance for your suggestions, and reference.
>>
>> Best Wishes,
>> Partha
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>>
>>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu



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[ccp4bb] data processing with split/bad crystals

[Kajander, Tommi A]

Dear All, 

I was wondering what would be the best software nowadays to try to process data 
from crystal that clearly is split or
has a secondary set of lattice points (close, poor data) in the raw data - data 
can be processed with XDS (2.9-2.8 Å) but Rmerge tends to be
bit high at low resolution (close to 7-8% depending on processing) - using 
XSCALE helps with the radiation damage correction some what. 

Data looks like primitive orthorombic but not quite sure (also seems like it 
has one screw axis e.g. P2212 - but oddly phaser finds 
solutions in P22121 also or even preferably…..). I am wondering a bit if it 
isn’t actually monoclinic. 

Based on automated processing by Diamond pipeline XDS seems most robust - but 
any hints on such cases would
be welcome. Of course we will try to get better crystal but so far no luck.

Thanks for comments,

Best
Tommi



---

Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1 (P.O. Box 65)
00014 Helsinki, Finland





To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[radu]

Hi Partha,

In this case (un-treated HEK293), you cannot use EndoH with already purified
proteins (as per your reply to Artem's email). The N-glycans will be
fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background
infos). You can still use PNGase. But, as Artem suggested, this often causes
protein precipitation when it can access the sites on folded proteins. Apart
from shaving the glycan shield, PNGase causes unwanted mutations at the
N-linked sites: Asn -> Asp, which is where most problems stem from.

Nevertheless, since your fully glycosylated protein doesn't crystallize,
there's not much else you can do with the current prep Except for example
masking your glycans with a nice lectin such as griffithsin, which would be
very elegant for crystallography. Or indeed trying cryo-EM, where wild-type
N-linked glycans are extremely helpful! Why would anyone crystallize proteins
these days anyway :-)

Best wishes,

Radu
-- 
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu

> Hi Savvas,
> Many Thanks for your inputs and references. This cell line used is not
HEK293S *MGAT1-/-. *This particular batch was purified from HEK293 cell-line
stably expressing (created using lenti-methods by a
> former colleague) the protein of interest. In future, I will plan to do
expression in the presence of Kifunensine, followed by EndoH treatment
before complexation and crystallization.
> Best Wishes,
> Partha
> On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
wrote:
>> Dear Partha
>> you do not specify which HEK293 cell line you have used, but if it so
happens that it is the very handy HEK293S *MGAT1-/- *cell line
>> (previously known as HEK293S *GnTI-/- ) *which produces
>> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
immuni.2017.12.008).
>> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
quite well for protein expression in HEK293T and renders N-linked glycans
digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et al.
doi:10.1038/nsmb.2367).
>> If resources and protein material allow, you might also want to consider
the permutation exercise of subjecting the complex to deglycosylation, or
the individual components followed by complex formation/purification, or
just one of the two components followed by complex formation/purification,
or even one of the two components followed by further deglycosylation of
the complex. We are becoming more and more apprehensive of the possible
role of glycans in complex formation.
>> And then there is of course the option to apply mutagenesis, e.g. via
N—>Q, to eliminate certain N-linked glycans either as a standalone
approach
>> or in combination with enzymatic glycan digestions as described above. Best
wishes
>> Savvas
>> *---*
>> *Savvas Savvides*
>> VIB Center for Inflammation Research
>> Dept. Biochemistry & Microbiology, Ghent University
>> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
>> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
savvas.savvides_skype
>> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx On
16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
wrote:
>> Dear All,
>> I am in a situation, almost for the first time within my limited
experience, that deglycosylation might be necessary to obtain crystal. So,
I thought of tapping to vast experience of CCP4BBers, while I am searching
literature.
>> I have protein that has been expressed in HEK293 cells, secreted into
media, purified over IMAC and SEC columns. Crystallization-screens with its
binding partners (they form good complexes based on analytical SEC) have
not produced any useful hits (whereas complexes with related proteins
worked well). So, I plan to re-try complex formation and \crystallization
screen after deglysosylation.
>> My question is: In practice, Does a kit (for example here: https://www.
sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US) containing
Endo F1, F2, F3 be sufficient or should this be tried in combination with
PNGase (which requires
>> desaturating conditions)?!!
>> Many Thanks in advance for your suggestions, and reference.
>> Best Wishes,
>> Partha
>> --
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Hi Savvas,

Many Thanks for your inputs and references. This cell line used is not
HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
cell-line stably expressing (created using lenti-methods by a
former colleague) the protein of interest. In future, I will plan to do
expression in the presence of Kifunensine, followed by EndoH treatment
before complexation and crystallization.

Best Wishes,
Partha


On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides 
wrote:

> Dear Partha
> you do not specify which HEK293 cell line you have used, but if it so
> happens that it is the very handy HEK293S *MGAT1-/- *cell line
> (previously known as HEK293S *GnTI-/- ) *which produces
> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
> immuni.2017.12.008).
> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
> quite well for protein expression in HEK293T and renders N-linked glycans
> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et
> al. doi:10.1038/nsmb.2367).
>
> If resources and protein material allow, you might also want to consider
> the permutation exercise of subjecting the complex to deglycosylation, or
> the individual components followed by complex formation/purification, or
> just one of the two components followed by complex formation/purification,
> or even one of the two components followed by further deglycosylation of
> the complex. We are becoming more and more apprehensive of the possible
> role of glycans in complex formation.
> And then there is of course the option to apply mutagenesis, e.g. via
> N—>Q, to eliminate certain N-linked glycans either as a standalone approach
> or in combination with enzymatic glycan digestions as described above.
>
> Best wishes
> Savvas
>
>
> *---*
> *Savvas Savvides*
> VIB Center for Inflammation Research
> Dept. Biochemistry & Microbiology, Ghent University
> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
> savvas.savvides_skype
> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
>
> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar 
> wrote:
>
> Dear All,
>
> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
>
> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
>
> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing Endo F1, F2, F3 be sufficient or should this be tried in
> combination with PNGase (which requires
> desaturating conditions)?!!
>
> Many Thanks in advance for your suggestions, and reference.
>
> Best Wishes,
> Partha
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Many Thanks Artem. I will use Kifunensine in culture for next batch
expression, and will use EndoH with already purified proteins.

Best Wishes,
Partha

On Mon, Jul 16, 2018 at 12:26 PM, Artem Evdokimov  wrote:

> Dear Partha
>
> Treat your culture with Kifunensine prior to transfection (or throughout
> growth if you're using stables) and then treat purified protein with EndoH.
> Pretty cheap and effective.
>
> PNGase F does not *require* denaturing conditions. It just likes the
> protein to be 'loose' - in my experience about half the time the accessible
> sites will fall off even under native conditons. However, PNGase F is
> somewhat expensive and after treatment certain proteins have fallen out of
> solution (presumably because not a single carbohydrate remained, as opposed
> to 'shielding' provided by the single remaining sugar left behind by
> EndoH). Caveat emptor.
>
> Artem
>
> - Cosmic Cats approve of this message
>
> On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar <
> spart...@gmail.com> wrote:
>
>> Dear All,
>>
>> I am in a situation, almost for the first time within my limited
>> experience, that deglycosylation might be necessary to obtain crystal. So,
>> I thought of tapping to vast experience of CCP4BBers, while I am searching
>> literature.
>>
>> I have protein that has been expressed in HEK293 cells, secreted into
>> media, purified over IMAC and SEC columns. Crystallization-screens with its
>> binding partners (they form good complexes based on analytical SEC) have
>> not produced any useful hits (whereas complexes with related proteins
>> worked well). So, I plan to re-try complex formation and \crystallization
>> screen after deglysosylation.
>>
>> My question is: In practice, Does a kit (for example here:
>> https://www.sigmaaldrich.com/catalog/product/SIGMA/
>> NDEGLY?lang=en=US) containing Endo F1, F2, F3 be sufficient or
>> should this be tried in combination with PNGase (which requires
>> desaturating conditions)?!!
>>
>> Many Thanks in advance for your suggestions, and reference.
>>
>> Best Wishes,
>> Partha
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>
>



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Savvas Savvides]

Dear Partha
you do not specify which HEK293 cell line you have used, but if it so happens 
that it is the very handy HEK293S MGAT1-/- cell line (previously known as 
HEK293S GnTI-/- ) which produces N-linked Man5GlcNAc2 glycans you might want to 
consider using EndoH (e.g. see Verstraete et al. DOI: 10.1038/ncomms14937) or 
even Jack-bean alpha-mannosidase (e.g. see Bloch et al. 
https://doi.org/10.1016/j.immuni.2017.12.008 
).
Kifunensine, an inhibitor of N-glycosylation processing, tends to work quite 
well for protein expression in HEK293T and renders N-linked glycans digestible 
by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix et al. 
http://dx.doi.org/10.1016/j.str.2015.06.019 
 ; Elegheert et al. 
doi:10.1038/nsmb.2367).

If resources and protein material allow, you might also want to consider the 
permutation exercise of subjecting the complex to deglycosylation, or the 
individual components followed by complex formation/purification, or just one 
of the two components followed by complex formation/purification, or even one 
of the two components followed by further deglycosylation of the complex. We 
are becoming more and more apprehensive of the possible role of glycans in 
complex formation.
And then there is of course the option to apply mutagenesis, e.g. via N—>Q, to 
eliminate certain N-linked glycans either as a standalone approach or in 
combination with enzymatic glycan digestions as described above.

Best wishes
Savvas


---
Savvas Savvides
VIB Center for Inflammation Research
Dept. Biochemistry & Microbiology, Ghent University
Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 


> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar  
> wrote:
> 
> Dear All, 
> 
> I am in a situation, almost for the first time within my limited experience, 
> that deglycosylation might be necessary to obtain crystal. So, I thought of 
> tapping to vast experience of CCP4BBers, while I am searching literature. 
> 
> I have protein that has been expressed in HEK293 cells, secreted into media, 
> purified over IMAC and SEC columns. Crystallization-screens with its binding 
> partners (they form good complexes based on analytical SEC) have not produced 
> any useful hits (whereas complexes with related proteins worked well). So, I 
> plan to re-try complex formation and \crystallization screen after 
> deglysosylation. 
> 
> My question is: In practice, Does a kit (for example here: 
> https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US 
> )
>  containing Endo F1, F2, F3 be sufficient or should this be tried in 
> combination with PNGase (which requires
> desaturating conditions)?!! 
> 
> Many Thanks in advance for your suggestions, and reference. 
> 
> Best Wishes,
> Partha 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Artem Evdokimov]

Dear Partha

Treat your culture with Kifunensine prior to transfection (or throughout
growth if you're using stables) and then treat purified protein with EndoH.
Pretty cheap and effective.

PNGase F does not *require* denaturing conditions. It just likes the
protein to be 'loose' - in my experience about half the time the accessible
sites will fall off even under native conditons. However, PNGase F is
somewhat expensive and after treatment certain proteins have fallen out of
solution (presumably because not a single carbohydrate remained, as opposed
to 'shielding' provided by the single remaining sugar left behind by
EndoH). Caveat emptor.

Artem

- Cosmic Cats approve of this message

On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear All,
>
> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
>
> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
>
> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing Endo F1, F2, F3 be sufficient or should this be tried in
> combination with PNGase (which requires
> desaturating conditions)?!!
>
> Many Thanks in advance for your suggestions, and reference.
>
> Best Wishes,
> Partha
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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[ccp4bb] Suggestions for deglycosylaiton enzymes

[Parthasarathy Sampathkumar]

Dear All,

I am in a situation, almost for the first time within my limited
experience, that deglycosylation might be necessary to obtain crystal. So,
I thought of tapping to vast experience of CCP4BBers, while I am searching
literature.

I have protein that has been expressed in HEK293 cells, secreted into
media, purified over IMAC and SEC columns. Crystallization-screens with its
binding partners (they form good complexes based on analytical SEC) have
not produced any useful hits (whereas complexes with related proteins
worked well). So, I plan to re-try complex formation and \crystallization
screen after deglysosylation.

My question is: In practice, Does a kit (for example here:
https://www.sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
containing Endo F1, F2, F3 be sufficient or should this be tried in
combination with PNGase (which requires
desaturating conditions)?!!

Many Thanks in advance for your suggestions, and reference.

Best Wishes,
Partha



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Re: [ccp4bb] [ccpem] Creating map volume of custom size

[Edward A. Berry]

Hi Melanie,
Mapmask was suggested. The XYZLIM command of mapmask takes values in grid 
points or fractional coordinates.
You could make a 300A cube by giving fractional coordinates of 300/(current 
cell edge),
But since that doesn't change the grid it won't change the thickness of your 
slices.
I don't think mapmask (old name "extend") can change the grid.

Maprot was also mentioned, and I think this should work. You need to generate a 
300x300x300 target map (WORKIN) and perhaps a mask (could be your entire 
current cell). Use the identity rot-trans operator, or with some non-zero 
translation if necessary to get your model entirely in one cell.

But it might be much easier to just recalculate your map from structure factors 
with equal grid spacings using fft.
So if you want half-angstrom slices, set the grid values (nx,ny,nz) to twice 
the cell dimensions in Angstroms.
The FFT has some restrictions on the grid so you may not be able to choose 
exactly what you want, but could get pretty close to even spacing. (In fact 
programs that write maps tend to make approximately equal spacing, like 1/2 or 
1/3 of the resolution, by default- so it would be surprising if your current 
map has drastically different spacing along the different axes).  If you expand 
your data to P1 before fft, then the only restrictions are that nz be an even 
number (and none of the nx,ny,nz have prime factors greater than 19). You 
should be able to get very nearly equal spacing with that.

I wonder if there is any program that uses conventional FT and allows arbitrary 
grid choices?
eab


On 07/16/2018 11:28 AM, melanie.voll...@diamond.ac.uk wrote:

Thanks everyone who replied to my post. I got a wealth of ideas.


Melanie

Hello everyone,


I have a question about manipulating map files.


I want to use my map derived from X-ray diffraction and create a standard 
volume for it, say 300A3.

This is to be able to create slices of equal dimensions along all three 
directions of the electron density stored in the map file. Currently my slices 
differ in dimensions as the default volume is defined by the unit cell.


I don't think there is anything in the map tools from the X-ray field I know. I 
had a play with Chimera and I think there is a tool in there to use some form 
of reference cell created from coordinates. But I don't want to make use of a 
coordinates file.


I just want to inflate my unit cell to a standard volume.


Any ideas would be very welcome.


Thanks


Melanie

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please notify us of receipt by returning the e-mail and do not use, copy, 
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Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments 
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Re: [ccp4bb] [ccpem] Creating map volume of custom size

[melanie.voll...@diamond.ac.uk]

Thanks everyone who replied to my post. I got a wealth of ideas.


Melanie

Hello everyone,


I have a question about manipulating map files.


I want to use my map derived from X-ray diffraction and create a standard 
volume for it, say 300A3.

This is to be able to create slices of equal dimensions along all three 
directions of the electron density stored in the map file. Currently my slices 
differ in dimensions as the default volume is defined by the unit cell.


I don't think there is anything in the map tools from the X-ray field I know. I 
had a play with Chimera and I think there is a tool in there to use some form 
of reference cell created from coordinates. But I don't want to make use of a 
coordinates file.


I just want to inflate my unit cell to a standard volume.


Any ideas would be very welcome.


Thanks


Melanie

--
This e-mail and any attachments may contain confidential, copyright and or 
privileged material, and are for the use of the intended addressee only. If you 
are not the intended addressee or an authorised recipient of the addressee 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not 
necessarily of Diamond Light Source Ltd.
Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments 
are free from viruses and we cannot accept liability for any damage which you 
may sustain as a result of software viruses which may be transmitted in or with 
the message.
Diamond Light Source Limited (company no. 4375679). Registered in England and 
Wales with its registered office at Diamond House, Harwell Science and 
Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom







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Re: [ccp4bb] salt bridges etc..

[Eleanor Dodson]

Thank you Gert - very clear explanation..
Eleanor

On 16 July 2018 at 13:31, Gert Vriend  wrote:

> Dear Eleanor,
>
> Salt bridges are a compromise between entropy and enthalpy. If, say, an
> Asp and an Arg side chain are a bit restricted in their freedom and the
> charges are close, enthalpy wins, and if they are very exposed, and not
> close at all, entropy wins. The enthalpic gain upon protein folding from
> obtaining one salt bridge has been given many values in the literature, but
> in practice boils down to about 1 kCal/Mole. The enthalpic contribution is
> a bit higher than 1kcal/Mole when the positive and negative charges are
> very close to each other (in which case you loose entropy upon folding).
> Most salt bridges are at the surface where they continuously compromise
> between entropy and enthalpy. So, they move around, but most of the time
> the charges are close together, and that is why you can see them with Xray.
> When the two charged groups come close to each other there are always
> certain local conformations that are preferred over others. Those
> (sometimes multiple) locally preferred conformations we see in Xray if we
> have good crystals. It does not matter, however, how many local
> conformations we observe. It is just one salt bridge, and its energetic
> contribution to protein folding remains (very roughly, and this is
> practical experience for which no good theory exists) about 1kCal/Mole.
>
> Gert
>
> On 11-7-2018 16:52, Eleanor Dodson wrote:
>
> How do people decide on what is a salt bridge within a molecule and how to
> count them for those Tables?
>
> I have been looking at 2z2f - paper claims some score..-
>
> But there are several residues in alternate conformation
>
> with NZ A  to OE1Aand NZ A to OE1B  and NZ B to OE1B etc
>
> Is that one salt bridge   or 3 salt bridges
>
> PISA lists salt bridges between molecules but not within a molecule I dont
> think?
>
> Suggestions gratefully received.
> Eleanor
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
>
>



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Re: [ccp4bb] salt bridges etc..

[Gert Vriend]

Dear Eleanor,

Salt bridges are a compromise between entropy and enthalpy. If, say, an 
Asp and an Arg side chain are a bit restricted in their freedom and the 
charges are close, enthalpy wins, and if they are very exposed, and not 
close at all, entropy wins. The enthalpic gain upon protein folding from 
obtaining one salt bridge has been given many values in the literature, 
but in practice boils down to about 1 kCal/Mole. The enthalpic 
contribution is a bit higher than 1kcal/Mole when the positive and 
negative charges are very close to each other (in which case you loose 
entropy upon folding). Most salt bridges are at the surface where they 
continuously compromise between entropy and enthalpy. So, they move 
around, but most of the time the charges are close together, and that is 
why you can see them with Xray. When the two charged groups come close 
to each other there are always certain local conformations that are 
preferred over others. Those (sometimes multiple) locally preferred 
conformations we see in Xray if we have good crystals. It does not 
matter, however, how many local conformations we observe. It is just one 
salt bridge, and its energetic contribution to protein folding remains 
(very roughly, and this is practical experience for which no good theory 
exists) about 1kCal/Mole.


Gert


On 11-7-2018 16:52, Eleanor Dodson wrote:
How do people decide on what is a salt bridge within a molecule and 
how to count them for those Tables?


I have been looking at 2z2f - paper claims some score..-

But there are several residues in alternate conformation

with NZ A  to OE1A    and NZ A to OE1B  and NZ B to OE1B etc

Is that one salt bridge   or 3 salt bridges

PISA lists salt bridges between molecules but not within a molecule I 
dont think?


Suggestions gratefully received.
Eleanor







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