Re: [ccp4bb] OT: design of a stable alpha-helical peptide

[Parthasarathy Sampathkumar]

Hi Erik,
A couple of things to consider:
1. Begin the N-terminus from helix "initiating" residues (i.e. one residue
overhang might not be sufficient)
2. If not done already, add the 6-carboxy-fluorescein moiety at C-terminus
of the peptide
Good Luck,
Partha

On Wed, Mar 20, 2024 at 6:03 AM Erik Debler  wrote:

> Hi everyone,
>
> Sorry for the off-topic question, but I thought I may get some
> advice/pointers about the design of a stable alpha-helical peptide. I would
> like to obtain a peptide mimicking a 13-residue alpha-helix that was
> identified in a protein to mediate a protein-protein interaction, which is
> completely abolished by three spatially adjacent point mutations in the
> helix of the protein. I designed a peptide with a 1-residue overhang on
> each side and a 6-carboxy-fluorescein moiety on one end, but the peptide
> did not bind to the interaction partner probed by fluorescence
> polarization. The peptide easily dissolves in water, so solubility does not
> seem to be an issue. Any suggestions or pointers to relevant literature
> would be greatly appreciated.
>
> Cheers!
> Erik
>
> --
>
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Re: [ccp4bb] extra Fo-Fc density in two Cysteines

[Parthasarathy Sampathkumar]

Hi Liliana,
May be it would be good to know if this adduct was formed during
crystallization or protein was modified prior to crystallization.  One
could consider performing a protease digestion, followed by LC-MS/MS to
determine the molecular weight of the adduct and then work backwards to
account for the difference in dalton.

Good Luck,
Partha

On Mon, Dec 18, 2023 at 9:14 AM Liliana Margent <
lmarg...@gradcenter.cuny.edu> wrote:

> Hi there, We’ve been having an issue in trying to clear regions of Fo-Fc
> density from a few cysteines during the refinement process. We were
> wondering if anyone had seen something similar so they could offer some
> insight on the likely chemistry at hand, and a potential refinement
> solution. Attached are two images of the observed extra density at two
> cysteines, 505 and 518. We have modeled acetylated cysteine,
> s-hydroxycysteine, and s-mercaptocysteine but it does not solve the
> density. The protein in question is a Protein Tyrosine Phosphatase known as
> STEP (PTPN5), with data collected to a resolution of 1.79 Å. The crystals
> were grown in bis-tris pH 6.65, 200mM Li2SO4, ~30% PEG3350. Of note, prior
> to data collection the crystal was conserved at room temp for long time
> where it dried, and was subsequently rehydrated with mother liquor. Thank
> you so much.
> --
>
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[ccp4bb] Job Opportunity at Surrozen (SFO Bay Area): Research Associate/Senior Research Associate, Biochemistry and Biophysics

[Parthasarathy Sampathkumar]

Dear All,

I would like to bring to the attention of suitable candidates the following
exciting job opportunities at Surrozen Inc, located in South San Francisco.

Surrozen is a biotechnology company focused on discovering and developing
novel regenerative medicines with a focus on unlocking the powerful
self-renewal properties of the body through specific control of the Wnt
signaling pathway.

Our vision is to engage the body’s own biological tissue repair mechanisms
with our targeted regenerative antibodies, overcoming research and
development hurdles that have been previously intractable, and have held
back drug development efforts in the promising area of Wnt biology.

*Research Associate/Senior Research Associate, Biochemistry and Biophysics:*

https://apply.workable.com/surrozen/j/ACBA0B0D96/

*Scientist, Protein Sciences: *
https://apply.workable.com/surrozen/j/DF4518083F/

*Research Associate, Protein Purification:*
https://apply.workable.com/surrozen/j/7D64A01556/

Many Thanks,
Best Wishes,
Partha Sampathkumar PhD
Principal Scientist
Surrozen Inc
Email: par...@surrozen.com



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Re: [ccp4bb] Help for Twin Refinement in Refmac5 / CCP4i2

[Parthasarathy Sampathkumar]

Dear All,

Please you may ignore my previous email. TWIN refinement in Refmac5 /
CCP4i2 worked well once used "HKLOUT_0-observed_data_asIMEAN.mtz" from the
data-reduction job. I am slowly getting used to the finer-aspects of
CCP4i2. Previously, I used to have both Intensities and Amplitudes in a
single MTZ file :-)

Thanks,
Partha

On Mon, Jan 25, 2021 at 9:47 PM Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear All,
>
> I have not had much experience in refining structure using data from
> twinned crystals and has started using CCP4i2 very recently only.
>
> Here is a background:
> antigen-Fab crystal structure determined by MR first in P4(3)2(1)2 space
> group with 1-complex molecule in the asymmetric unit (ASU). Later, I
> reprocessed the data in P4(3) performed MR to search for the 2nd molecule
> and refined the structure using Refmac5 without the "twin" keyword. With
> the sequence fully modeled for the two complex molecules in the ASU current
> Rcryst = 27.8%and Rfree = 33.2%. Unit cell = "59.9, 59.9, 404.5, 90.0,
> 90.0, 90.0".
>
> Twin fraction estimates from Britton plot = 0.47 and from H-test = 0.43,
> as reported by AIMLESS. Then, attempted TWIN refinement in Refmac5 /
> CCP4i2 by adding the keyword "twin" in the advanced options. However,
> getting following error in the log-file:
>
>  Error==> array size in mtz_refl_read_int
> 
>  Refmac:  Problem with array sizes
>
> Does Refmac5 expects intensities here?!!
> Any help is greatly appreciated.
>
> Best Wishes,
> Partha
>
>
>
>
>



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[ccp4bb] Help for Twin Refinement in Refmac5 / CCP4i2

[Parthasarathy Sampathkumar]

Dear All,

I have not had much experience in refining structure using data from
twinned crystals and has started using CCP4i2 very recently only.

Here is a background:
antigen-Fab crystal structure determined by MR first in P4(3)2(1)2 space
group with 1-complex molecule in the asymmetric unit (ASU). Later, I
reprocessed the data in P4(3) performed MR to search for the 2nd molecule
and refined the structure using Refmac5 without the "twin" keyword. With
the sequence fully modeled for the two complex molecules in the ASU current
Rcryst = 27.8%and Rfree = 33.2%. Unit cell = "59.9, 59.9, 404.5, 90.0,
90.0, 90.0".

Twin fraction estimates from Britton plot = 0.47 and from H-test = 0.43, as
reported by AIMLESS. Then, attempted TWIN refinement in Refmac5 / CCP4i2 by
adding the keyword "twin" in the advanced options. However, getting
following error in the log-file:

 Error==> array size in mtz_refl_read_int

 Refmac:  Problem with array sizes

Does Refmac5 expects intensities here?!!
Any help is greatly appreciated.

Best Wishes,
Partha



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[ccp4bb] Job Posting: RA / SRA - Biophysics / Biochemistry Position at Surrozen

[Parthasarathy Sampathkumar]

Dear All,

Please allow me to post this link for RA / SRA - Biophysics / Biochemistry
Position at Surrozen with focus on Antibody Developability and Structural
Biology.

https://apply.workable.com/surrozen/j/75748FA62D/

I request interested job seekers to apply directly online using the
above link.

Thanks,
Best Wishes,
Partha
__

Parthasarathy Sampathkumar PhD

Senior Scientist, Surrozen Inc.,

171 Oyster Point Blvd., Suite 400

South San Francisco, CA  94080

Email: par...@surrozen.com



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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). 

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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[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] Non crystallography question

[Parthasarathy Sampathkumar]

Hi Vandna,

A typical approach would be to generate different models (experimental or
otherwise, if X-ray structure it could be "completed" further by modeling
missing loops / side-chains) of your sequence to calculate a SAXS profile,
and then compare / fit those to experimental SAXS profile. Some tools are
available at salilab.org

Hope this helps,
Best Wishes
Partha


On Fri, Dec 8, 2017 at 4:50 PM, Vands  wrote:

> Hi All,
>Is there any Web server available where I can input my SAXS
> profile and it will give all closely matched PDB structures from the all
> available PDB.
>
> --
> Vandna Kukshal
> Postdoctral Research Associate
> Dept. Biochemistry and Molecular Biophysics
> Washington University School of Medicine
> 660 S. Euclid
> , Campus
> Box 8231
> St. Louis, MO 63110
>

Re: [ccp4bb] new ContaMiner features

[Parthasarathy Sampathkumar]

Hi All,

To add, on couple of occasions I re-determined the structure of so-called
"contaminants". Once, the structure of Secreted Ferritin (PDB 1Z6O) from
crystals that were supposedly that of TNF ligand:receptor complex (proteins
expressed in Tni cells), and in the second instant re-determined the
structure of bacterial carbonic-anhydrase. On both instants, contaminants
were identified through cell-constant search (at that time no ContaMiner
server available) in PDB, after realizing that standard Molecular
Replacement did not work. Luckily, did not spend too much time on these
cases.

Best Wishes to All for "contaminant" free crystals on this Thanksgiving Day,

Partha



On Thu, Nov 23, 2017 at 6:23 PM, Edward A. Berry  wrote:

> My 2 cents worth:
> I think contaminer is an extremely useful service. I may be a sloppy
> biochemist,
> but I am not the only one. There are multiple structures in the database
> of say
> bacterioferritin or AcrB that were solved from crystals that were supposed
> to
> be something else. I remember in a discussion with the organizer of my
> session
> at a Gordon conference, she excitedly announced that there would be
> preliminary
> crystallographic data on respiratory Complex I. But by the time of the
> conference
> the authors discovered they had crystallized something else. And the
> beautiful crystals
> of Paracoccus Complex II (from Doug Rees's lab?) that graced the catalog of
> Hampton Research (And I believe were part of the basis for the first
> membrane
> protein screen) never saw publication.  The authors of
>   http://www.sciencedirect.com/science/article/pii/S0304416506000894
> certainly feel there is a real problem.  Some proteins crystallize readily
> even when
> present as minor contaminants. And some protein complexes become more
> heterogeneous
> if over-purified due to partial loss of loosely-bound subunits.
> Most of my career I've worked with high-abundance natural-source proteins.
> During a recent foray into the realm of overexpressed proteins, my group
> has
> crystallized (and solved) at least a half dozen wrong proteins from E.
> coli.
> I spent months on one of these (ATCase in Rhomb sg with low-level
> obverse/reverse
> twinning that caused it to sometimes index as P3) Then solved the rest
> rapidly
> by checking the closest several hits with nearest-cell.  All of these
> E.coli proteins
> were already present in the PDB. I wonder how many were from accidental
> crystals.
> And now bacterioferritin (this time from M. smegmatis) keeps coming back
> to haunt us.
>
> I would say any time with a new crystal when a molecular replacement
> unexpectedly fails,
> and even before you start to collect heavy atom or selenomet data, it
> would be worth
> to submit to nearest-cell and contaminer. I would be more likely to
> question the
> utility of an anisotropy correction server, given that modern
> maximum-likelihood
> refinement programs can deal with weak data satisfactorily (speaking from
> ignorance- I'm sure supporting evidence and examples exist, I just haven't
> bothered to look them up. And I know my colleagues here at Upstate have
> used
> anisotropy correction to good effect with a difficult problem- I hope they
> weren't using filled-in maps!)
> eab
>
> On 11/23/2017 03:24 PM, Tristan Croll wrote:
>
>> Dear Radu,
>>
>> I think this is a little harsh. Biology is a fabulously messy thing, and
>> very prone to doing the unexpected. See the excellent paper by
>> Niedzialkowska et al. at https://www.ncbi.nlm.nih.gov/p
>> mc/articles/PMC4815408/#!po=13.6905 for some examples. Sometimes
>> unexpected things (which just happen to have a similar size to your target)
>> carry through all the purification steps - I remember having terrible
>> trouble isolating his-tagged IGF-I (not for crystallization) from Sf9
>> lysates due to a cathepsin-like protease that stuck doggedly to the Ni-NTA
>> column even under 8M urea, yet co-eluted in imidazole. Even if contaminant
>> proteins are barely visible on your SDS-PAGE gel, if they crystallise
>> easily and your target doesn’t...  all these things and many others have
>> happened, and have undoubtedly driven the occasional poor grad student to
>> the brink of giving it all up.
>>
>> I guess in these days of relatively cheap and ubiquitous mass spec it may
>> make sense to sacrifice a crystal to trypsin digest and MS/MS sequencing
>> just for peace of mind, but in the average case I think that’s likely to be
>> overkill. Shooting crystals at a synchrotron is now very routine, so I
>> think it makes perfect sense to provide a computational check for the
>> (hopefully rare) surprise case.
>>
>> Best regards,
>>
>> Tristan
>> Tristan Croll
>> Research Fellow
>> Cambridge Institute for Medical Research
>> University of Cambridge CB2 0XY
>>
>>
>>
>> On 23 Nov 2017, at 19:35, r...@mrc-lmb.cam.ac.uk > r...@mrc-lmb.cam.ac.uk> wrote:
>>
>> Dear Stefan,
>>>
>>> Just a couple of thoughts:
>>>
>>> - first of 

Re: [ccp4bb] Unknown electron density

[Parthasarathy Sampathkumar]

Hi Vijay,

Why there is no 2mFo-DFc (blue) feature on this mFo-DFc (green) map?!! Is
the contour level for blue map set high?!! If there is no 2mFo-DFc density,
should this be consider as noise?!!

Hope this helps,
Best Wishes,
Partha


On Thu, Oct 26, 2017 at 10:23 AM, Vijaykumar Pillalamarri <
vijaypkuma...@gmail.com> wrote:

> Dear ccp4bb,
>
> I am solving the structure of a 1.6 Angstrom data of a metallo protein.
> While everything else is straight forward, this density (see the picture)
> seems unfamiliar to me. I don't see any thing fits in the density. This
> density present just above Histidine-Cobalt. What ever I try to fit, the
> molecule clahes with histidine ( I mean the distance between His and
> molecule is <2 Angstrom).
>
> The crystallization condition is 0.1M Bistris, 19% PEG 3350 and 50%
> glycerol.
>
> Any suggestions?
>
> Thanks,
> Vijaykumar Pillalamarri
> UGC-SRF
> C/O: Dr. Anthony Addlagatta
> Principal Scientist
> CSIR-IICT, Tarnaka
> Hyderabad, India-57
> Mobile: +918886922975 <+91%2088869%2022975>
>

Re: [ccp4bb] His-6 versus His-10 tag

[Parthasarathy Sampathkumar]

Hi Herman,

I worked on a kinase, where moved from 6-His in the literature to 8-His,
and it didn't impact crystallization or diffraction (which around 3Angs).

Good Luck
Partha
On Tue, Sep 19, 2017 at 9:43 AM Oganesyan, Vaheh 
wrote:

> Hi Herman,
>
>
>
> I haven’t done His-6 versus His-10 for the same protein, but have done
> that for different ones with success. However, if in His-6 containing
> protein structure the packing or folding is such that you don’t see His-6
> then it shouldn’t matter it is 6 or 10. Just an opinion.
>
>
>
> *Regards,*
>
>
>
> *Vaheh Oganesyan*
>
> *www.medimmune.com *
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *
> herman.schreu...@sanofi.com
> *Sent:* Tuesday, September 19, 2017 6:11 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] His-6 versus His-10 tag
>
>
>
> Dear BB,
>
>
>
> We are planning the production of a protein for crystallization. From
> literature, we know that the construct with a 6-histidine tag crystallizes.
> However, for other biophysical measurements, we would prefer to have a
> 10-histidine tag.
>
>
>
> Does anyone has experience with His-6 versus His-10 tags in terms of
> crystallization success?
>
>
>
> Thanks for your help!
>
> Herman
>
>
>
>
> To the extent this electronic communication or any of its attachments
> contain information that is not in the public domain, such information is
> considered by MedImmune to be confidential and proprietary. This
> communication is expected to be read and/or used only by the individual(s)
> for whom it is intended. If you have received this electronic communication
> in error, please reply to the sender advising of the error in transmission
> and delete the original message and any accompanying documents from your
> system immediately, without copying, reviewing or otherwise using them for
> any purpose. Thank you for your cooperation.
>

Re: [ccp4bb] Difficult purification with imac columns

[Parthasarathy Sampathkumar]

Hi Narayanan,

This doesn't address your question; may be you could go-around this problem
by using a different purification tag., say like GST?!!

Good Luck,
Partha

On Fri, Sep 15, 2017 at 7:54 AM Narayanan Ramasubbu <
ramas...@sdm.rutgers.edu> wrote:

> Hi. We are working on a periplasmic protein that breaks naked glycans in
> peptidoglycans. There is truncated structure available but our target is
> the full length protein. The difficulty us that it strongly binds to the
> resin with or without his.tag. Changing the resin to acrylamide did not
> help.
> Has anyone come across similar problem and how was it resolved.
> The pdb structure is the catalytic domain and mussing a region that, in my
> opinion, binds to the resin.
> Thank you in advance
> Sent from my iPhone

Re: [ccp4bb] CC(1/2) reference

[Parthasarathy Sampathkumar]

Hi Nicola,

Here are references for CC1/2;

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457925/


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684713/

Best Wishes
Partha
On Tue, Aug 29, 2017 at 10:06 AM Nicola Evans 
wrote:

> Hello all, I have heard at several CCP4 meetings and also at Diamond
> training that a good "cut off" for CC(1/2) is around 0.3 and I/sig(I) is
> 0.2, but I am struggling to find any journal references to say this (other
> than demonstrating the merits of CC(1/2) over Rmeas).
>
> Can anyone point me in the right direction? Thanks all!
>
> Nicola
>

Re: [ccp4bb] Substrate density visible in relatively active mutant

[Parthasarathy Sampathkumar]

Hi Seema,

Couple scenarios plausible (i) mutant is "super slow" compared to WT that
crystallization was setup prior to any significant reaction and / or (ii)
components, as well the pH, of crystallization condition made the reaction
even more slower. Of course, if the reaction has partially occurred, you
are likely to observe occupancy less than 1. These plausible only
scenarios!!

Hope this helps,
Partha
On Fri, May 26, 2017 at 4:59 PM Seema H Irani 
wrote:

> I crystallized a PLP dependent enzyme and wanted to capture the enzyme
> substrate complex, however due high activity of the enzyme I could see no
> density at the active site upon soaking with the substrate. I hence made a
> bunch of mutants of the active site residues and one of them showed density
> consistent with the substrate in some of the molecules in the asymmetric
> unit. Surprisingly however when I measured the activity of this mutant it
> had 30% of the wildtype activity I am wondering why is it still possible
> for me to see substrate density for this mutant?
>
>
>

Re: [ccp4bb] Recommendations for Robotic Crystal Screening Services

[Parthasarathy Sampathkumar]

Hi Elizabeth,
HWI, University of Buffalo, NY offers high-throughout crystallization
screen. See the link for more information:

http://hwi.buffalo.edu/science/high-throughput-crystallization-center/

Good Luck
Partha
On Fri, Apr 14, 2017 at 9:06 AM Elizabeth Diaz 
wrote:

> All,
>
> I am currently attempting to crystallize a peptide/protein complex and am
> wanting to know about robotic screening services that you would recommend.
> We have done some in house conditions with little luck, and want to broaden
> our search, but that would require going externally for screening. Do you
> have any recommendations for robotic crystal screening services, preferably
> in the United States?
>
> Thank you so much,
>
> Elizabeth Diaz
> University of Delaware
>

Re: [ccp4bb] Unknown electron density blob

[Parthasarathy Sampathkumar]

Hi Uma,

It is risky to guess based on one-view of of the density from 2-dimensional
images. What is the contour-level of 2mFo-DFc (blue) map displayed here?!!
If 2mFo-DFc density is continuous from Arg, say at 0.8 sigma, then could it
be an alternate conformation of the Arg side-chain. One could build, and
re-refine and see if negative mFo-DFc features shows up.

Hope this helps,
Partha

On Tue, Jan 24, 2017 at 12:19 PM, Uma Gabale <
0ebb5dcf3eaa-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear all,
> While refining a structure at 2.5 A resolution, we observed a
> semi-circular/crescent shaped electron density blob as shown in the
> attached picture. We have been unable to identify it so far, and would
> appreciate any help in identification.
> The protein was expressed in *E. coli* BL21(DE3), purified on Ni-NTA
> followed by gel filtration. The purification buffers included Tris and NaCl
> (no detergent/ other ingredients except for imidazole for Ni-NTA).
> Crystallization condition had HEPES and PEG3350; no cryoprotectant was used.
> The blob is surrounded by residues Trp, Thr, Gln, Arg, and Phe.
> Thanks and regards,
> Uma.
>
> --
> Uma Gabale, PhD
> Research Associate
> Molecular and Cellular Biochemistry
> Indiana University Bloomington
>

Re: [ccp4bb] Cryo-EM

[Parthasarathy Sampathkumar]

Hi Faisal,

There are some good video introduction available too:
https://www.youtube.com/watch?v=nkGRhYv01ag

HTH,
Partha

On Tue, May 19, 2015 at 3:01 AM, Faisal Tarique faisaltari...@gmail.com
wrote:

 Hi everyone

 A bit off topic..but..I request you to please suggest me some good
 readings related to Cryo-EM..

 Thanx in advance

 --
 Regards

 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] ctruncate error

[Parthasarathy Sampathkumar]

Yes.., I too had similar problem with Ctruncate, and used older truncate to
overcome the issue.

Best Wishes,
Partha


On Thu, Jun 19, 2014 at 2:38 PM, jie liu jl1...@njms.rutgers.edu wrote:

 Hi

 I also encountered the same problem after recent updates (not the most
 recent one, but a couple of updates back). Choosing to run old-truncate
 will get it around.

 Best wishes

 Jie

 - Original Message - From: Stephen Carr 
 stephen.c...@rc-harwell.ac.uk
 To: CCP4BB@JISCMAIL.AC.UK
 Sent: Thursday, June 19, 2014 1:11 PM
 Subject: [ccp4bb] ctruncate error



 Dear CCP4bb,

 I am experiencing an unusual error when running truncate.  The program
 appears to be converting I's to F's, but then failing to output them in the
 resulting mtz file, see mtzdump output below:

 Col SortMinMaxNum  % Mean Mean   Resolution   Type
 Column
 num order   Missing complete  abs.   LowHigh label

   1 ASC  0  26  0  100.00 12.6 12.6  79.95   3.00   H H
   2 NONE 0  15  0  100.00  4.3  4.3  79.95   3.00   H K
   3 NONE   -42  42  0  100.00  0.7 16.0  79.95   3.00   H L
   4 NONE0.019.0 0  100.00 9.56 9.56  79.95   3.00   I
 FreeR_flag
   5 BOTH ?   ?  129680.00  ??  -999.00   0.00   F
 F_delta16
   6 BOTH ?   ?  129680.00  ??  -999.00   0.00   Q
 SIGF_delta16
   7 BOTH0.0 0.025   99.81 0.00 0.00  42.22   3.00   D
 DANO_delta16
   8 BOTH0.0 0.0 11253   13.22 0.00 0.00  42.22   3.00   Q
 SIGDANO_delta16
   9 BOTH ?   ?  129680.00  ??  -999.00   0.00   G
 F_delta16(+)
  10 BOTH ?   ?  129680.00  ??  -999.00   0.00   L
 SIGF_delta16(+)
  11 BOTH ?   ?  129680.00  ??  -999.00   0.00   G
 F_delta16(-)
  12 BOTH ?   ?  129680.00  ??  -999.00   0.00   L
 SIGF_delta16(-)
  13 BOTH 0   0 25   99.81  0.0  0.0  42.22   3.00   Y
 ISYM_delta16
  14 NONE   -8.9 72479.225   99.81   198.34   198.44  42.22   3.00   J
 IMEAN_delta16
  15 NONE0.6  3135.725   99.81 5.25 5.25  42.22   3.00   Q
 SIGIMEAN_delta16
  16 NONE  -10.8 72479.225   99.81   198.18   198.37  42.22   3.00   K
 I_delta16(+)
  17 NONE0.0  3135.725   99.81 6.79 6.79  42.22   3.00   M
 SIGI_delta16(+)
  18 NONE   -9.5 72479.225   99.81   198.30   198.47  42.22   3.00   K
 I_delta16(-)
  19 NONE0.0  3135.725   99.81 6.70 6.70  42.22   3.00   M
 SIGI_delta16(-)


 No. of reflections used in FILE STATISTICS12968

 The truncate log file shows no obvious errors apart from the cumulative
 intensity plot which indicates no reflections, all other diagnostic
 indicators for data quality seem to suggest everything is ok.  I get the
 error with both ctruncate and truncate and also automatically as part of
 the scaling/merging pipelines.  The data were processed with imosflm,
 scaled with aimless with neither flagging any errors with the data.  I am
 running CCP4 6.4.0 on a linux box (Centos 6) and have installed the latest
 updates. Any suggestions as to what the fault might be and how to get
 around it would be greatly appreciated.

 best wishes,

 Steve

 Dr Stephen Carr
 Research Complex at Harwell (RCaH)
 Rutherford Appleton Laboratory
 Harwell Oxford
 Didcot
 Oxon OX11 0FA
 United Kingdom
 Email stephen.c...@rc-harwell.ac.uk
 tel 01235 567717
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Re: [ccp4bb] ccp4 v6.4.0 cannot extract mtz

[Parthasarathy Sampathkumar]

Yes Uli. I have the same issue, but not solve it yet :-(

Partha


On Mon, Dec 16, 2013 at 10:52 AM, ulrich.goh...@mdc-berlin.de 
ulrich.goh...@mdc-berlin.de wrote:

  Dear colleagues,

 Trying to run the latest version of ccp4 (6.4.0), say Refmac, I get the
 following error when I fill in the file box for the data file:

 CCP4i encountered an error when trying to extract the data from data.mtz.
 You can view the output from the mtzdump program etc. + crash of the GUI.

 Now, running mtzdump gives me this:

  http://www.hkl-xray.com/data-upload-form-and-bug-report./mtzdump:
 /usr/lib64/libgfortran.so.3: version `GFORTRAN_1.1' not found (required by
 /xprogs/CCP4/CCP4-6.4.0/destination/ccp4-6.4.0/bin/../lib/libccp4f.so.0)

 In /usr/lib64 I have softlinked libgfortran.so.3 - libgfortran.so.3.0.0
 (did so in 2011).

 I am not sure what I need to update here and how. I am running Novell's
 SLES 11 on a 64 bit system with the libgfortran43 runtime library
 installed. I have also checked file permissions (incl. tmp directories),
 and I cannot find anything obvious. I cannot remember having had similar
 problems with v6.3.0 in the beginning.

 Has anyone encountered the same problem and solved it yet?

 My apologies if this has been discussed already; I searched the archive
 and I couldn't find a similar thread.

 Cheers,

  Uli


 ---

 dr ulrich gohlke

 *staff scientist - m**acromolecular structure and interaction*

 max-delbrück-center for molecular medicine (mdc)


 +49 30 9406 - 2725 (w)

 +49 30 9406 - 2548 (fax)

 ulrich.goh...@mdc-berlin.de




 http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/

Re: [ccp4bb] monovalent cation binding sites

[Parthasarathy Sampathkumar]

Hi Ed,

WASP analyse water molecules in high-resolution protein structure to check
if some of those could be metal ions. WASP could be run as a part of STAN
server.
STAN - the STructure ANalysis server from USF (
http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl )

One could also identity potential metal ions within COOT as well.

HTH,
Best Wishes,
Partha





On Fri, Nov 8, 2013 at 9:09 PM, Edward A. Berry ber...@upstate.edu wrote:

 Is there a server or program to predict binding sites for monovalent metal
 ions?
 Ideally should work with just the protein structure, but a program that
 sorts through
 the waters in a high resolution structure and tells which are likely to be
 K+ or Na+
 would also be of interest.

 Ed

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Parthasarathy Sampathkumar]

Hi Andrey,

I am taking a risky guess:

From your slide #2, it looks like a termini (unless this correspond to the
beginning or end of disordered a loop) of the protein chain(s) is (are)
extending toward(s) the solvent channel. Are the density features shown in
slides #3,4, and 5 extend from this terminal residue?!! If this is true,
did you missed to model any uncleaved-tag residues?!! I would also look at
2Fo-Fc at 1.0 sigma.

Hope this helps,
Partha


On Fri, Mar 15, 2013 at 2:39 PM, Andrey Nascimento 
andreynascime...@gmail.com wrote:

 *Dear all,*

 *I have collected a good quality dataset of a protein with 64% of solvent
 in P 2 21 21 space group at 1.7A resolution with good statistical
 parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93%
 Redun.=2.4, the overall values are better than last shell). The structure
 solution with molecular replacement goes well, the map quality at the
 protein chain is very good, but in the final of refinement, after addition
 of a lot of waters and other solvent molecules, TLS refinement, etc. ...
 the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree=
 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower
 symmetry space group (P21), but I got the same problem, and I tried all
 possible space groups for P222, but with other screw axis I can not even
 solve the structure.*

 *A strange thing in the structure are the large solvent channels with a
 lot of electron density positive peaks!? I usually did not see too many
 peaks in the solvent channel like this. This peaks are the only reason for
 these high R's in refinement that I can find. But, why are there too many
 peaks in the solvent channel???*

 *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map
 figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf*

 *
 *

 *Do someone have an explanation or solution for this?*

 * *

 *Cheers,*

 *Andrey*

Re: [ccp4bb] software to generate protein topology diagram like those available at PDBsum webserver

[Parthasarathy Sampathkumar]

Dear All,

Guillaume Ponchel referred me to ProOrigami (which can be installed
locally) to generate protein topology diagrams:

http://munk.csse.unimelb.edu.au/pro-origami/


Thanks again.

Best wishes,
Partha


On Mon, Apr 16, 2012 at 12:12 AM, Parthasarathy Sampathkumar 
spart...@gmail.com wrote:

 Dear All,

 Thanks to Herb, John, Simanshu, and Anu for link:

 http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html

 to generate PDBsum like topology diagrams.

 Partha
 On Sun, Apr 15, 2012 at 8:08 PM, Parthasarathy Sampathkumar 
 spart...@gmail.com wrote:

 Dear All,

 I would like to generate a topology diagram similar to the ones available
 for deposited entries at the PDBsum webpage (attached one such diagram here
 for reference) for a new structure. Is this program is available for
 download?

 I am aware of TopDraw. However, I am trying to analyze a ~850 a.a.
 structure. So, some automation would be helpful.

 Thanks in advance for your help.

 Best wishes,
 Partha

Re: [ccp4bb] software to generate protein topology diagram like those available at PDBsum webserver

[Parthasarathy Sampathkumar]

Dear All,

Thanks to Herb, John, Simanshu, and Anu for link:

http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html

to generate PDBsum like topology diagrams.

Partha
On Sun, Apr 15, 2012 at 8:08 PM, Parthasarathy Sampathkumar 
spart...@gmail.com wrote:

 Dear All,

 I would like to generate a topology diagram similar to the ones available
 for deposited entries at the PDBsum webpage (attached one such diagram here
 for reference) for a new structure. Is this program is available for
 download?

 I am aware of TopDraw. However, I am trying to analyze a ~850 a.a.
 structure. So, some automation would be helpful.

 Thanks in advance for your help.

 Best wishes,
 Partha

Re: [ccp4bb] contaminant when overexpressing a GST tagged protein

[Parthasarathy Sampathkumar]

Hi Maria,

As mentioned by Tony, it could be a chaperonin. Having little of ATP (0.5mM
or less) and Mg2+ (1mM) in lysis buffer might help.

Good Luck,
Partha

2012/3/22 SANCHEZ BARRENA, MARIA JOSE xmj...@iqfr.csic.es

 Dear all,

 I am trying to express a eukatiotic protein (E. coli codon optimized
 sequence) with a GST tag at the N-terminus. I always get my overexpressed
 protein and a contaminant around 60kDa. This contaminant is not washed out
 of the column when washing glutathione beads with 1M NaCl-buffer. However,
 during o/n incubation with proteases that cleave the GST off (thrombin or
 TEV), the contaminant is in the soluble fraction.

 Has someone had this experience? I know about contaminants that bind to
 Ni2+ when overexpressing a His-tagged protein, but this is the first time I
 get such thing with a GST-tagged protein.

 One could think that that contaminant could be a protein that binds to
 my overexpressed protein, but I do not think so, cause I always get a huge
 band of the contaminant, independently on the amount of the protein of
 interest
 Many thanks in advance for all your suggestions and sorry for asking about
 non-crystallographic topics.
 Regards,

 Maria


  -**

 María José Sánchez-Barrena, PhD**

 Departamento de Cristalografía y Biología Estructural.**

 Instituto de Química Física Rocasolano. CSIC**

 Serrano 119. 28006 Madrid (Spain)**

Re: [ccp4bb] Water

[Parthasarathy Sampathkumar]

Dear Uma,

The water pictured in W12-1.jpg: could this be a potential metal ion? If
you flip the side chain on Asn at 3.08Angstrom, then this has 3 or 4
coordination with oxygen atoms. So, provided your crystallization condition
or buffer contains metal ion(s), you could attempt to see if it fits better
with a refinement cycle.

May be a similar situation with the water described in W11-1.jpg as well?
Difficult to say from these figures.

COOT within the validate wizard has an option to search for
hihgly-coordinated waters like the one you have pictured.

Hope this helps,
Partha

On Wed, Mar 7, 2012 at 4:21 PM, Uma Ratu rosiso2...@gmail.com wrote:

 Dear Roger:

 Thank you very much for your comments. I use them as guideline and remove
 many 'false waters.

 Still, I am not clear of some of these 'waters' are real or not. I have
 the pic attached.

 In Pic-W11-1, the 'water' is connected to the adjust residues with 4
 contacts, which are 'N' or 'O' atoms. I would consider this 'water' is
 false. My question is: if these 4 contacts include C from residues, will
 it be a polar contact or not?

 In Pic-W12-1, the 'water' is connected to the adjust residues with 3
 contacts. The 4th is to another 'water'.
 Will this 'water' is true or not? Similar case is seen in Pic-W190-1

 In Pic-W109-1, some 'waters' are connected to adjust residues, some not.
 Are these 'water' true or not?

 Further more,
  and the b-factors are not way out of line,

 I am not clear on how to define out of line.
 How to find b-factor of individual residue in Coot? I search the web, but
 find no answer.

 Thank you for advice

 Uma

 On Wed, Mar 7, 2012 at 11:44 AM, Roger Rowlett rrowl...@colgate.eduwrote:

 Uma,

 Remember that your structure, ultimately, is a model. A model is your
 best judgment of the true representation of the protein structure in your
 crystal. Your model should make chemical sense. Coot is pretty good at
 placing waters, but it cannot substitute entirely for the experimentalist.
 Coot will miss some waters, and mis-assign others into weak, unmodeled or
 alternate side- or main-chain density, or into density that might be
 attributable to cations and anions or other crystallization materials. Your
 waters should be subjected to inspection and verification. It is really
 helpful to turn on environment distances in Coot when you do this. Even in
 a large protein model, it is possible to inspect all waters for
 reasonableness pretty quickly. If you have no significant positive or
 negative difference density, and the b-factors are not way out of line, and
 hydrogen bonding partners are reasonable, then modeling a water is probably
 a good call.

 Waters should have hydrogen bonding partners with side chains or
 main-chain polar atoms, within reasonable distances, or be withing hydrogen
 bonding distance of other waters that are (chains of waters). If a water
 has strong electron density and more than 4 polar contacts, you might
 consider anion or cation occupancy. Most anions and cations will have
 higher electron density, and appropriately different types of polar
 contacts. (e.g. you might find sulfates near a cluster of basic residues).
 Low occupancy anions can often look a lot like water. PEGs can create ugly
 snakes of variable density that may be challenging to model. Modeling
 non-protein structural bits is endlessly entertaining for the protein
 crystallographer. ;)

 Cheers,

 ___
 Roger S. Rowlett
 Gordon  Dorothy Kline Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346

 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu


 On 3/7/2012 11:20 AM, Uma Ratu wrote:

 Dear All:

 I try to add water to my model.

 Here is how I did:
 Coot: Find Wates
  Map: FWT PHWT;  1.8 rmsd; Distances to protein atoms:
 2.4 min/3.2 max

 Coot found 270 water molecules.

 I then examed these waters. Most of them had ball shape. Some had two or
 more balls together. Some had irregular shape (not glabol shape).

 I run Water Check. The program did not find any mis-matched water.

 Here is my question: how could I tell the waters are real? Or something
 else?

 Thank you for advice

 Ros

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Parthasarathy Sampathkumar]

Hi Mike,

Often, I generate independent freeR set (especially in cases where soak
dataset is of different resolution (usually worse) compared to the native
dataset and do following two things to get rid-off the bias: 1. add a noise
to the coordinates (this can be done using PDBSET). 2. set the Bvalues to
the wilson B of the soak dataset (within Refmac5 before rigidbody
refinement).

HTH,
Partha


On Mon, Nov 21, 2011 at 5:47 PM, Michael Thompson mi...@chem.ucla.eduwrote:

 - Forwarded Message -
 From: Michael Thompson mi...@chem.ucla.edu
 To: e dodson e.dod...@ysbl.york.ac.uk
 Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
 Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

 A question regarding the plea for less MR (which I support):

 There have been several recent instances in which I have used the solution
 of an isomorphous structure to do rigid body refinement for a new crystal
 (as described by Eleanor). It has always produced good results. My question
 is about how to best handle the free set of reflections when doing this? I
 have heard a number of differing opinions about whether or not it is
 important to carry the freeR flags from the original structure over to the
 new data set. I have heard equally convincing arguments from both sides, so
 my young and impressionable mind does not know who to believe. I was hoping
 I could get an opinion from the advocates for less MR.

 Sorry for hijacking this thread, but hopefully it will provide some
 insight that is relevant to the original post.

 Thanks!

 Mike




 - Original Message -
 From: Eleanor Dodson c...@ysbl.york.ac.uk
 To: CCP4BB@JISCMAIL.AC.UK
 Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific
 Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

 Just a plea for less molecular replacement.

 If you get a new crystal of a known protein with the  same cell
 dimension as youur old crystal, the most likely scenario is that it has
 the same group, and you really should not try MR - use the previous
 solution as input to do rigid body refinement, and then
  a) the R factor will tell you if this is a reasonable hypothesis (it
 usually is..) and
 b) you dont have this awful problem of not being able to compare the
 solutions..

  Eleanor

 On 11/20/2011 03:57 PM, Napoleão Valadares wrote:
  Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
  Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.
 
  I think I understand it now, I always thought the one ring to rule them
  all translated in the crystallography realms to one origin to rule
  them all. That probably means I have a long road in front of me.
 
  I'm still half confused, I definitely need to read more, as much as I
  read about symmetry and space groups I never seem to improve or get a
  better understanding, but I'll keep trying.
 
  About the same origin:
  The pdbs of both Solution-1 and Solution-2 present the same space group
  and cell, as observed opening the pdbs as text files or in pymol. When I
  open both maps on coot they are not superposed but present the same cell
  and origin.
 
  If I open both solutions on pymol they clash. If I generate the symmetry
  mates of both solutions none of them are superposed, instead they clash.
  But I think they are related as you all pointed, I'll check it out.
 
  Thank you all for your kind answers and your patience with a beginner.
  Regards from a sunny Brazil,
  Napo
 
 
  On 11/20/2011 2:58 AM, Felix Frolow wrote:
  Napoleao,
  It is so called alternative origins play a game with you. You do not
  change your structure by shifting 1/2 translation (or even combination
  of these translations)
  into directions of the main axes of your unit cell. Structure factors
  after this operation stay the same, however phases change
  systematically, producing however the same
  map features.
  Would I be a begin crystallographer now, I would read a bit more old
  fashioned books
  on crystallography such as probably Jensen and Stout…
  FF
  Dr Felix Frolow
  Professor of Structural Biology and Biotechnology
  Department of Molecular Microbiology
  and Biotechnology
  Tel Aviv University 69978, Israel
 
  Acta Crystallographica F, co-editor
 
  e-mail: mbfro...@post.tau.ac.il mailto:mbfro...@post.tau.ac.il
  Tel: ++972-3640-8723
  Fax: ++972-3640-9407
  Cellular: 0547 459 608
 
  On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:
 
  Hello,
  I'm observing a very strange phenomena (at least to me, I'm a
  beginner). It is related to symmetry (I think).
 
  I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas  35% in
  the last shell) and a partially refined solution with R/Rfree 22/24,
  166 aminoacids observed and around 30 solvent molecules. I'll call
  this Solution-1. The refinement was smooth, the densities were very
  clearly asking for the correct missing side chains and the map
  looks good.
 
  The space group I'm using 

Re: [ccp4bb] (off-topic) Measurement of channel pore dimensions

[Parthasarathy Sampathkumar]

Dear Bert,

You  could try the program Hole from
http://www.ncbi.nlm.nih.gov/pubmed/9195488

J Mol Graph. http://www.ncbi.nlm.nih.gov/pubmed/9195488 1996
Dec;14(6):354-60, 376.
HOLE: a program for the analysis of the pore dimensions of ion channel
structural models.

Smart OShttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Smart%20OS%22%5BAuthor%5D
, Neduvelil 
JGhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Neduvelil%20JG%22%5BAuthor%5D
, Wang Xhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Wang%20X%22%5BAuthor%5D
, Wallace 
BAhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Wallace%20BA%22%5BAuthor%5D
, Sansom 
MShttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Sansom%20MS%22%5BAuthor%5D
.


-Partha


On Wed, Mar 23, 2011 at 9:02 AM, Van Den Berg, Bert 
lambertus.vandenb...@umassmed.edu wrote:

  Hello all,

 Does anyone know how to get values for pore sizes of membrane channels? I’m
 not interested in A x B angstrom values measured between atom centers and
 assuming a regular pore shape, but a “real-life” value of either surface
 area at the narrowest point or the volume of a block centered on the
 narrowest point of the pore.

 Thanks, Bert

[ccp4bb] Blotting paper for plasmid DNA

[Parthasarathy Sampathkumar]

Dear All,

Apologies for non-CCP4 / non-Crystallography question.

I planning to spot plasmid DNA on a blotting paper for transport. I do not
have any idea what type of blotting paper is used for this purpose.

1. what type of blotting/filter paper should be used? will any type of
Whatman paper is good for this purpose? where (which supplier / catalog
number) could I purchase that blogging paper?

2. what is the typical lowest volume for spotting (will 5 micro lit.
sufficient?)

3. Are these spotted DNA are good option for longtime storage (for about 6
months)?

With thanks in advance,
-Partha

[ccp4bb] Summary of answers to question on “Blotting paper for plasmid DNA”.

[Parthasarathy Sampathkumar]

08 December 2010

Dear All,



Below is a summary of answers I obtained for my questions on “Blotting paper
for plasmid DNA”.

Thank you all very much for your quick response and thanks to CCP4BB.
-Partha
__
Hideaki Moriyama:
How about FTA filter papers (Whatman).

https://dgrc.cgb.indiana.edu/vectors/

http://www.whatman.com/FTAElute.aspx


Dima Klenchin :
1. what type of blotting/filter paper should be used? will any type of
Whatman paper is good for this purpose? where (which supplier / catalog
number) could I purchase that blogging paper?


Just about any will do. Whatman #1 works fine, Whatman 3MM is
nice because it is thicker and takes up more solution.

2. what is the typical lowest volume for spotting (will 5 micro lit.
sufficient?)

Any piece that can be conveniently fit in the eppendorf
eventually - enough to make it wet. 5 ul is plenty for about 0.3 cm2 of
Whatman #1.

3. Are these spotted DNA are good option for longtime storage (for about 6
months)?


Yes as long as there was EDTA in the DNA solution (e.g., TE).
Kept in the dark, of course.

___

Patrick J. Loll:
Whatman #1 is fine. We frequently use ca. 2 uL of a maxi-prep solution (so,
say, maybe 100-200 ng). I haven't conducted any analysis of long term
stability, but I wouldn't suggest storing it any longer than necessary (say,
a week or so). If you need to store longer, there are better ways to do it.


__

Konstantin v. Korotkov:
I planning to spot plasmid DNA on a blotting paper for transport. I do not
have any idea what type of blotting paper is used for this purpose.

1. what type of blotting/filter paper should be used? will any type of
Whatman paper is good for this purpose? where (which supplier /
catalog  number)
could I purchase that blogging paper?

Any filter paper will do. I fact, any paper can be used as
well. If you are worried about contamination, you could use sterile strips.

2. what is the typical lowest volume for spotting (will 5 micro lit.
sufficient?)

Even less will be sufficient. 1-2ul of a miniprep of plasmid DNA
is plenty.

3. Are these spotted DNA are good option for longtime storage (for about 6
months)?

I do not have long-term experience myself, but I would guess it
should be quite stable as long as it's dry.

_

Dhirendra K Simanshu:


I can suggest at least what works for me.

1. I feel any clean Whatman filter/blotting paper would be fine. I have used
1mm for my purpose. Main purpose is to absorb all the liquid.

2. It will depend on DNA concentration. But I think 5-6 ul should be more
than sufficient.

3. I am sure they will be good for six months in dried form.

Generally I cut small pieces of Whatman Filter paper and then write the name
of the construct using pencil in the corner and then drop 5-6 ul in the
center and then draw a circle around the drop. Because once the drop is
dried,  this will help in locating the position. Generally I cover the paper
with thin plastic wrap for transfer purposes. For extracting, I just cut the
circled part of paper and then put it in few ul of Tris buffer at 37 C and
then I have enough to transform and make large quantity of it.

Re: [ccp4bb] An alternative to glycerol in keeping protein soluble?

[Parthasarathy Sampathkumar]

Dear Serah,

It is likely that glycerol stabilized your protein via binding as it was
available in plenty during purification. Therefore, you could attempt to
purify the mutant enzymes in the presence of substrate or ligands. I am not
sure if this will work, but worth a try since you have mentioned that
interferes with the binding of the substrates to my protein.

Good Luck,
-Partha

On Tue, Feb 23, 2010 at 3:07 AM, SERAH KIMANI serah.kim...@uct.ac.zawrote:

 Dear all,

 Does anyone have an idea of something else that I can use instead glycerol
 to maintain solubility of my protein? I have been having 10% glycerol in my
 protein solution and this has helped me get crystals in like three different
 conditions. However, I would want to get crystals of mutants-substrate
 complexes, but unfortunately glycerol interferes with the binding of the
 substrates to my protein. So, for the complexes to form, glycerol has to be
 out (I have tested this using the wildtype enzyme both in the presence and
 absence of glycerol). Now, in the absence of glycerol, I get a lot of
 precipitation, and no crystals even after optimizing around the known
 conditions with different protein concentrations. I have tried re-screening
 for new conditions in the absence of glycerol, but I haven't found any
 condition that yields crystals in the absence of glycerol.

 I was wondering if anyone might know of something else that I could use in
 place of glycerol in my protein solution??

 Regards,

 Serah
 University of Cape Town

 __


 UNIVERSITY OF CAPE TOWN

 This e-mail is subject to the UCT ICT policies and e-mail disclaimer
 published on our website at
 http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from
 +27 21 650 4500. This e-mail is intended only for the person(s) to whom it
 is addressed. If the e-mail has reached you in error, please notify the
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 _

Re: [ccp4bb] crystal contacts

[Parthasarathy Sampathkumar]

Dear Amit,

You might want to take a look at NOXclass webserver:
http://noxclass.bioinf.mpi-inf.mpg.de/help.php

Relevant paper is available at:
http://www.biomedcentral.com/1471-2105/7/27

Goold Luck,
Partha Sampathkumar
NYSGXRC

On Mon, Feb 22, 2010 at 6:49 AM, amit sharma 3112a...@gmail.com wrote:

 Dear All,

 Apologies for a non-CCP4question. I have a structure of a dimeric molecule,
 where the interfaces between monomers is held by a couple of tyrosine
 residues (per monomer)  juxtaposed with each other. The molecule exists as a
 dimer in solution. Are there ways/programs to show that the interaction
 between the tyrosine residues is not a consequence of crystal contacts. I
 guess the fact that the molecule occurs as a dimer in solution strongly
 suggests so. Also, any directions towards literature showing similar cases
 would be of great help.

 Many thanks in advance
 --
 Amit Sharma
 Postdoctoral Fellow,
 Department of Biophysics,
 Johns Hopkins University,
 Baltimore,
 MD21218

Re: [ccp4bb] FW: [ccp4]: TDS upon flashcooling

[Parthasarathy Sampathkumar]

Hi Rafael,

If it has not been already suggested: try DMSO (20% to 40%).

In my limited experience I found that often DMSO works well for
crystallization conditions with high-salt or high buffer component
(like 1M D,L,-Malic acid).

HTH,
-Partha

On Thu, Dec 17, 2009 at 1:39 PM, Meitian Wang meitian.w...@psi.ch wrote:

  good point!  recently we managed to collect very good room temperature
 data with PILATUS detector at SLS.  if your crystals are large enough, say
 100 microns or so, you have chance.  regards, meitian


  On Dec 15, 2009, at 1:42 PM, mjvanraaij wrote:

  why not stay with room temp?
 many structures have been solved at RT...


 Mark J. van Raaij
 Dpto de Bioquimica, Facultad de Farmacia
 Universidad de Santiago
 15782 Santiago de Compostela
 Spain
 http://web.usc.es/~vanraaij/
 researcherID: B-3678-2009






 On 15 Dec 2009, at 13:20, Natalie Zhao wrote:

 -Original Message-

 From: owner-c...@dl.ac.uk [mailto:owner-c...@dl.ac.uk] On Behalf Of Rafael
 Couñago

 Sent: 14 December 2009 20:22

 To: c...@ccp4.ac.uk

 Subject: [ccp4]: TDS upon flashcooling


 Dear all,


 I got these beautiful looking crystals that grow in high salt (1.8M) and

 diffract under 2.0A at room temp.  My attempts so far to cryo protect

 them have resulted in a loss of resolution (2.5A tops) and increased

 anisotropy.


 I have tried some of the usual suspects; no cryo, ethylene glycol,

 glycerol (even 5% makes my crystal crack), sucrose, glucose, paratone-n

 (no diffraction at all).  I have tried both dipping the crystal straight

 into liquid nitrogen and flash cooling it in the cryostream.


 An interesting observation is that the diffraction pattern following

 freezing has a substantial amount of thermal diffuse scattering (but no

 ice rings).  If I remove the crystal from the cryostream and re-anneal

 it at room temp (in air or in mother liquor or mother liquor + cryo)

 most of the TDS goes away, but the max resolution is still around 2.5A

 and the higher anisotropy is still there.  Extending re-annealing times

 lead to cracking of the crystal.


 My two questions would be:


 - any thoughts on cryo solutions?

 - does the result from the re-annealing experiment  ring any bells?

 Would this be an indication that I need the cooling to be faster or slower?


 Cheers,


 Rafael.


 --

 Rafael Couñago

 Research Fellow

 Department of Biochemistry

 University of Otago


 710 Cumberland St

 Dunedin, New Zealand

 ph: (03) 479 5148


 --

 Scanned by iCritical.


__
 Meitian Wang
 Swiss Light Source at Paul Scherrer Institut
 CH-5232 Villigen PSI - http://sls.web.psi.ch
 Phone: +41 56 310 4175
 Fax:  +41 56 310 5292

[ccp4bb] Unit of electrostatic potential in PyMol

[Parthasarathy Sampathkumar]

Dear All,

I used PyMol to generate a qualitative Vacum Electrostatic surface (without
APBS calculations).
Positve and negative potentials are displayed in the range 56.3 to -56.3.
What is the units of these numbers?

Thank you,
-Partha

[ccp4bb] Fwd: Unit of electrostatic potential in PyMol

[Parthasarathy Sampathkumar]

Oops.., I meant to ask a different quesition. Let me try again:

I used PyMol to generate a qualitative Vacum Electrostatic surface (without
APBS calculations). The potential is displayed in the range -56.3 to +56.3.

Is it possible to change this scale in PyMol, if so how?

Thank you,
-Partha

-- Forwarded message --
From: Parthasarathy Sampathkumar spart...@gmail.com
Date: Mon, Nov 2, 2009 at 3:41 PM
Subject: Unit of electrostatic potential in PyMol
To: CCP4BB@jiscmail.ac.uk


Dear All,

I used PyMol to generate a qualitative Vacum Electrostatic surface (without
APBS calculations).
Positve and negative potentials are displayed in the range 56.3 to -56.3.
What is the units of these numbers?

Thank you,
-Partha

Re: [ccp4bb] Colored proteins :)

[Parthasarathy Sampathkumar]

ThyX (also known as Flavin Dependent Thymidyalte Synthase, FDTS) is yellow
due to bound FAD.
-Partha

On Tue, Oct 20, 2009 at 5:25 PM, Artem Evdokimov ar...@xtals.org wrote:

 Hello CCP4 folks!

 I have a quick question - could you suggest a few naturally intensely
 colored proteins? Colors based on small molecule co-factors (i.e. metal
 ions, flavonoids, etc.) are perfectly fine for my needs :)

 I already looked into GFP and its relatives, (bacterio)rodopsin,
 azurins/pseudoazurins, and hemoglobins - but I would appreciate more
 examples.

 I am sure there's a nice review out there somewhere but so far I've not
 found it.

 Thank you,

 Artem

Re: [ccp4bb] To set a crystal tray with protein and Trypsin protease.

[Parthasarathy Sampathkumar]

Hi Jing,

Methods to perform In site proteolysis are available in the following
publications:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005094


http://www.nature.com/nmeth/journal/v4/n12/abs/nmeth1118.html

Good Luck,
-Partha


On Sun, Oct 11, 2009 at 5:13 PM, jwangliang jwangli...@gmail.com wrote:

  Hello,

 I am sorry to put this question which didn’t related to the CCP4 software.
 I couldn’t get the crystals from the protein only. So I want to try the
 different way to work it out. I hear that it is possible to set a crystal
 tray of protein with Trypsin protease. I will be very appreciated about who
 will provide some detail of this method about how to do this., e.g.  How
 much trypsin shall I add into the protein, How long shall I mix protease
 with the proteins before I set the tray.   Thanks for all.
  I will also put on the summary of all the answers.

 Another question is about protein express in insect cell.  10% of my
 protein was degradated after  1st purification. Is anyway that I can
 prohibit this degradation?



 Thanks


 Jing

Re: [ccp4bb] To set a crystal tray with protein and Trypsin protease.

[Parthasarathy Sampathkumar]

It should be In situ and not 'In site'. Sorry for the typo error.
-Partha

On Sun, Oct 11, 2009 at 9:59 PM, Parthasarathy Sampathkumar 
spart...@gmail.com wrote:

 Hi Jing,

 Methods to perform In site proteolysis are available in the following
 publications:

 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0005094


 http://www.nature.com/nmeth/journal/v4/n12/abs/nmeth1118.html

 Good Luck,
 -Partha



 On Sun, Oct 11, 2009 at 5:13 PM, jwangliang jwangli...@gmail.com wrote:

  Hello,

 I am sorry to put this question which didn’t related to the CCP4 software.

 I couldn’t get the crystals from the protein only. So I want to try the
 different way to work it out. I hear that it is possible to set a crystal
 tray of protein with Trypsin protease. I will be very appreciated about who
 will provide some detail of this method about how to do this., e.g.  How
 much trypsin shall I add into the protein, How long shall I mix protease
 with the proteins before I set the tray.   Thanks for all.
  I will also put on the summary of all the answers.

 Another question is about protein express in insect cell.  10% of my
 protein was degradated after  1st purification. Is anyway that I can
 prohibit this degradation?



 Thanks


 Jing

Re: [ccp4bb] Active aggregates?

[Parthasarathy Sampathkumar]

Hi Toyoyuki,

If your protein bind to metal ions you could try low concentration of
chelating agents in the purification and storage buffer. Take a look at the
following reference:

Chelating Agents Stabilize the Monomeric State of the Zinc Binding Human
Papillomavirus 16 E6 Oncoprotein
Degenkolbe et al., Biochemistry, 2003, 42 (13), pp 3868–3873

Few years back, based on the above mentioned paper, keeping low amount of
EGTA (note: NOT eDta) and DTT helped me to stabilize a Zinc binding RING
domain as dimer instead of soluble aggregate. Of course, every protein is
different but above reference might help you.

If you lucky enough to identify a chelating agent that prevent aggregation
of your protein, you might also try to include that chemical in the
expression media.

Good luck and all the best,
-Partha
Partha Sampathkumar
NYSGXRC,
Lilly Biotechnology Center
San Diego CA 92121

PS: At that time I purified this particular RING domain over NiNTA column.
Since both EGTA and DTT are not good for the NiNTA resin I kept required
higher concentration of both waiting for the protein in the 1.7ml eppendroff
tubes that were used to collection elution fractions.


On Sat, Aug 29, 2009 at 7:52 PM, Xuan Yang pattisy...@gmail.com wrote:

 Dear James,

 Could you provide the reference of your success story? My protein also
 formed large soluble aggregates and I am desperate for such successful
 stories!

 By the way, have your performed DLS to your protein? What about the
 polydispensity? Is it lower than 20%?

 Thanks in advance!

 Sincerely,

 Xuan Yang
 2009/8/27 James Stroud xtald...@gmail.com

 Just try crystallizing it. What is a crystal but a massive aggregate?
 That they are still soluble and active is great news.

 As a grad student, I had a similar phenomenon with an early project. I
 showed a gel in group meeting where both activity and aggregation were
 obvious, said the aggregate was no problem, got ridiculed when I said I was
 going to throw it in trays despite what anyone said, had giant crystals
 after a few trays, and solved the structure with miras.

 Get the structure and then worry about why it's aggregating. The structure
 will probably provide you with the clues you need.


 On Aug 26, 2009, at 5:55 PM, ose toyoyuki wrote:


 Dear all,

 This is a question on how to cope with the protein that seems to form
 massive aggregates in solution but enzymatically active.

 I'm working on a protein whose molecular weight is around 70kDa and can
 be
 divided into two domains (say A and B domains). We expressed this protein
 in
 E.coli fused with GST and purified using some chromatography. The GST
 affinity chromatography works well and proteinase digestion to remove the
 tag does wonders too. The purified protein was confirmed to be active
 enough, we can detect both activities from these two domains. But the
 retention time from the gel filtration clearly shows it is awfully
 aggregated (comes out at the void region). DLS measurement indicates the
 averaged diameter is around 45 nm, which I feel is a bit too long.
 Analytical ultracentrifuge result implies that the distribution of the
 molecular species is wide, some portion got precipitated with 1K rcf
 (means
 the molecular weight is more than 5MDa) and the rest is ranging from 1MD
 to
 5MDa with a peak at 1MDa.
 I made new two constructs covering the A and B domains respectively, both
 of
 them are active again, but only the A domain has got the same symptom as
 the
 intact protein. The B domain seems to exist as a monomer in solution.

 Here come my questions, (I) How can I interpret this phenomenon? (II) Is
 there anything we can try to change the situation? (III) Does it make
 sense
 to try crystallization? (probably not).(IV) Has anyone got such
 experience?

 I tried the methylation on lysine side chains, I also tried the buffer
 with
 0.2M arginine or 10% glycerol but the all the results just seem hopeless.
 The protein before the proteinase treatment also comes out at the void
 region from the gel filtration.


 cheers

 toyoyuki

 --?
 Toyoyuki Ose
 o...@sci.hokudai.ac.jp

 Graduate School of Life Science
 Hokkaido University
 N21W11 Kita-ku, Sapporo
 001-0021 Japan