Re: [ccp4bb] Difficult data

[Stephen Campbell]

Hi Everybody,

Thanks so much for all the help!  I suppose I should give more info.
Marco, the data is twinned - I forgot about that in my initial posting.  I
can't remember the stats, but it is not a perfect twin.  I thought that
Phaser accounted for twinning, and I haven't got a solution good enough to
worry about the twinning in refinement (so I haven't really been worrying
about that at this point - maybe wrongly?).  I'll send out some diffraction
images tomorrow if you would still like to see.

I'm fairly certain I'm working with the peptide - I have run SDS-PAGE as
well as a Superdex-75 and the purity is perfect, and the size is what I
would expect (3 kDa), so no obvious oligomers in solution.

Dominica, the peptide is synthetic, but CD analysis suggests it is
structured, and helical in nature.  It's is not cyclic,  but likely does
have disulfide bridges keeping it fairly rigid.  The model I am using is an
NMR model (sequence identity is almost 100%), but the ensemble shows very
little flexibility, and any regions that show some sort of flexibility were
trimmed (only two residues at each terminus).  I have also reduced the NMR
model to one chain.  Are there other issues with the NMR models that I
should be aware of?  I don't have much experience using them.

Pete, I did try all of the space groups within the P4 bravais lattice, but
it didn't seem to matter which one I used.  Nico, that's what I thought
too, but unfortunately I've never had such a large unit cell with such a
small peptide, so I'm not sure at which point it gets ridiculous and there
is more likely something wrong with the processing...  Going back to
France, hey?  You're going to miss Edmonton s much!  Especially the
warm winters.

Stephen


On Tue, Apr 16, 2013 at 9:28 PM, Stephen Campbell  wrote:

> Hello,
>
> I am having a few issues with a data set I have been working on recently,
> and was hoping to get some ideas on how to deal with it, if anyone is in
> the mood.
>
> I have been working with a very small bacterocin (about 3 kDa) and set up
> some crystal trays in hope of getting some high diffracting crystals.  I
> failed, but did manage to get a data set of reasonable quality to about 4A
> from crystals that reproduce very poorly.  Now, this sounds horrendous, but
> the MR model I have available is of a similar bacteriocin, whose structure
> is predicted to be essentially identical (different ORF, but almost
> identical sequence).  I was thinking it would be done in a day.  The
> bravais lattice is P4, and 118 x 118 x 165 (which seems HUGE for such a
> small protein...indeed calling it a protein is generous...peptide).  The
> data seems reasonably nice (nice spots, no visible overlap within 4A, but
> is very mosaic, about 1.5 degrees.
>
> The data integrates and scales nicely, with very good chi2, R-factors and
> % rejected reflections.  It is hard to predict the correct space group,
> since all the tetragonal options have the same stats.  The systematic
> absences seem to predict p41212 (as does pointless), but it wouldn't be the
> first time I screwed that up.
>
> I can't get a solution, no matter what I try.  Is this the nature of such
> a small peptide in such a large unit cell (placing the first model is
> difficult for MR since there may be many copies in the AU), or are there
> some tricks?  Is it likely that the unit cell is wrong?  Self Rotation
> functions give 8 peaks, but this is considering a peak fairly generously
> (approx. 15-20% of origin).  Are there some blatantly obvious red flags
> that I may be missing?  Any advice would be great - even if that advice is
> that it may be time to move on.
>
> I should note that I have considered the idea that the peptide may be
> forming some sort of oligomeric structure such as a coiled coil, but it
> fails coiled coil predicting software, and there is no evidence that this
> should be the case.  The homologous structure is very rigid, and I would
> say fairly confidently that mine is likely the same - I just want to
> confirm it.
>
> Thanks so much,
>
> Stephen Campbell
> Post Doctoral Fellow
> Department of Biochemistry
> McGill University
>

[ccp4bb] please see below advert for research technician position

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Re: [ccp4bb] Difficult data

[Edward A. Berry]

Yes- this has happened to us so often recently that I've taken to checking every
new crystal form against the "nearest cell" server before starting heavy atom 
search.
That would account for the poor reproducibility.

Yeast glutamate-cysteine ligase (e.g. 3LVV) crystallizes with nearly identical 
cell
parameters to those given, but nothing else very close.
And i don't suppose one would be expressing a 3 kDa bacterial protein
in Saccharomyces. Peptide synthesis, more likely?


Jim Pflugrath wrote:

Maybe you crystallized something else? Did you look up that unit cell in the 
PDB?


**
*
*
I am having a few issues with a data set I have been working on recently, and 
was hoping to get some ideas on how to
deal with it, if anyone is in the mood.

.

Re: [ccp4bb] Difficult data

[Jim Pflugrath]

Maybe you crystallized something else?  Did you look up that unit cell in the 
PDB?




I am having a few issues with a data set I have been working on recently, and 
was hoping to get some ideas on how to deal with it, if anyone is in the mood.

.

[ccp4bb] Difficult data

[Stephen Campbell]

Hello,

I am having a few issues with a data set I have been working on recently,
and was hoping to get some ideas on how to deal with it, if anyone is in
the mood.

I have been working with a very small bacterocin (about 3 kDa) and set up
some crystal trays in hope of getting some high diffracting crystals.  I
failed, but did manage to get a data set of reasonable quality to about 4A
from crystals that reproduce very poorly.  Now, this sounds horrendous, but
the MR model I have available is of a similar bacteriocin, whose structure
is predicted to be essentially identical (different ORF, but almost
identical sequence).  I was thinking it would be done in a day.  The
bravais lattice is P4, and 118 x 118 x 165 (which seems HUGE for such a
small protein...indeed calling it a protein is generous...peptide).  The
data seems reasonably nice (nice spots, no visible overlap within 4A, but
is very mosaic, about 1.5 degrees.

The data integrates and scales nicely, with very good chi2, R-factors and %
rejected reflections.  It is hard to predict the correct space group, since
all the tetragonal options have the same stats.  The systematic absences
seem to predict p41212 (as does pointless), but it wouldn't be the first
time I screwed that up.

I can't get a solution, no matter what I try.  Is this the nature of such a
small peptide in such a large unit cell (placing the first model is
difficult for MR since there may be many copies in the AU), or are there
some tricks?  Is it likely that the unit cell is wrong?  Self Rotation
functions give 8 peaks, but this is considering a peak fairly generously
(approx. 15-20% of origin).  Are there some blatantly obvious red flags
that I may be missing?  Any advice would be great - even if that advice is
that it may be time to move on.

I should note that I have considered the idea that the peptide may be
forming some sort of oligomeric structure such as a coiled coil, but it
fails coiled coil predicting software, and there is no evidence that this
should be the case.  The homologous structure is very rigid, and I would
say fairly confidently that mine is likely the same - I just want to
confirm it.

Thanks so much,

Stephen Campbell
Post Doctoral Fellow
Department of Biochemistry
McGill University

[ccp4bb] BioSAXS time available at CHESS: May 8-June 18

[Richard Edward Gillilan]

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Re: [ccp4bb] Puzzling Structure

[MARTYN SYMMONS]

Just as a postscript.

It has been pointed out to me that the space group was correct in the uploaded 
PDB and in the mtz reflection file. And there was no editing of this data 
pre-deposition. 

I am sort of surprised how quickly people are to beat up on the authors in such 
cases since we do not have access to the full data and facts.

Having the uploaded data available would allow better checking. I'm mainly 
ignorant of the details, but isn't this the way sequence data is dealt with? - 
there are the unannotated sequence files which are gradually checked and 
assimilated (but not over-written).   

Cheers 
  Martyn 



 From: Michel Fodje 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Monday, 15 April 2013, 17:19
Subject: Re: [ccp4bb] Puzzling Structure
 

Just to round up this topic, a bug report was submitted to PDBe concerning 
entry 2wlv and the PDB has how responded acknowledging the problem. An updated 
entry will be available in one week.

As pointed out by Savvas, it is very likely the CRYST1 record was manually 
edited prior to deposition resulting in the wrong spacegroup being parsed in by 
AutoDep and subsequent processing moving the waters around in the wrong space 
group. Since there is no logical reason for the authors to do this, it was 
probably inadvertent. I imagine somebody accidentally deleted a space between P 
21 21 2 and 18 and tried to fix it by adding it back, between 1 and 8.  

/Michel

>-Original Message-
>From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
>Edward A. Berry
>Sent: April-14-13 9:59 AM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject: Re: [ccp4bb] Puzzling Structure
>
>Robbie Joosten wrote:
>> Hi Martyn,
>>
>>>     I think the question is where the error was made - seeing the
>>> uploaded file would clear this up. But it seems unlikely to me that
>>> the depositor saw a huge R factor discrepancy at the end of refinement
>and just blithely uploaded it.
>>> So scenario 3 :-
>>> PDB : we cannot reproduce your R factor with our programs Depositor :
>>> that's your problem mate - it was fine when it left me...up to you to sort
>it...
>>>     Which seems a sort of reasonable attitude to me.
>
>> Not quite, the depositor has to give, i.e. type, the space group (example
>depositions: https://www.ebi.ac.uk/pdbe- 
>xdep/autodep/AutoDep?param=QovCsvhNv06Mpnr%2BvIkqqfuqeeBd8leFN
>AVymZgS%2Fe7mULyfrCaTMN8jsyaGZUTyUDQyN3gMF3o%3D). Don't ask me
>why, because it is clearly a source of error.
>>
>
>In my experience with RCSB autodep2, assuming the information was in the
>uploaded pdb file, this information is already pre-filled and the depositor 
>just
>glances over to see it is correct. A missing or extra "(1)" might not be 
>noticed.
>So perhaps it is a parsing error, perhaps related to the different ways the
>space group is represented on the CRYST1 card, and the different stringency
>of different programs in interpreting the CRYST1 card.
>
>But the validation report is included with the approval request letter, and
>major discrepancies are noted in the text of the validation letter in case the
>depositor is too busy to actually read the validation report.
>So if the depositor read more than the first line or two of the letter he 
>should
>have known there was a problem.
>
>Then there is the 2-week default release policy:
>
>  "No major issues were raised during data processing. A summary of some of
>the key annotations
>  in your entry is shown below. Please verify that these are correct. If we do
>not hear from
>  you within two weeks, we will consider this entry to have been approved by
>you. The entry
>  will then be released according to the release/hold instructions you have
>provided."
>
>If this 2-week default release is the rule even when there are issues, the
>depositor may have put it aside to find the problem when time was available,
>and waited too long and let the default release kick in.
>
>eab

Re: [ccp4bb] need suggestions

[Bosch, Juergen]

Hi Afshan,

You mention:

The protein prep is same which was using to get the Hits.

So how old is your prep ? Was it stored at 4C or in LN2? Do you know if your 
protein is stable under the conditions you stored it ?

How long after purification did it take you to get those crystals ?

How old is your screen ? MES goes bad as well over time.

Jürgen 
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://lupo.jhsph.edu

On Apr 16, 2013, at 5:21, "Afshan Begum"  wrote:

> The protein prep is same which was using to get the Hits.

Re: [ccp4bb] need suggestions

[Flip GMAIL]

Hi Afshan,

Have look at http://www.douglas.co.uk/Scaling_Up.htm, Patrick has some 
general tips on scaling up. Elsewhere on his site you'll find tips for 
conversion to microbatch, you may want to try that out as well. I assume 
the screen crystals were not big enough to mount?


Flip

Op 4/16/2013 11:21, Afshan Begum schreef:


 Dear All,

I have encountered one problem to optimization the crystallization 
condition manually. Actually i got the good shape and even size 
crystal in a screening plate. The condition are :


20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH 
5.7 contain 50mM NaCl, 1mM EDTA &5mM BME.
When I tried to optimize this on Linbro the drops are become heavily 
precipitate even a mints. I tried so many things to avoid this ppt 
such as start PEG conc from 7%, dilute protein at half conc., add more 
salt in a reservoir solution but no succeeds.

The protein prep is same which was using to get the Hits.

I would be very thankful if you people give me nice suggestions.

Thanks

Best Regards

AFSHAN

Re: [ccp4bb] need suggestions

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Afshan,

- - Can you reproduce the crystals in the Linbro plates with the
identical solution used for the screening plate?
- - Did you compare the composition of the Linbro plate vs. the
screening plate?
- - Did you check e.g. the pH of the original solution?
- - Did you refresh the beta-ME / try a new protein prep? It goes off
after some time, as far as I know.

Regards,
Tim

On 04/16/2013 11:21 AM, Afshan Begum wrote:
> 
> 
> Dear All,
> 
> I have encountered one problem to optimization the crystallization
> condition manually. Actually i got the good shape and even size
> crystal in a screening plate. The condition are :
> 
> 20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH
> 5.7 contain 50mM NaCl, 1mM EDTA &5mM BME. When I tried to optimize
> this on Linbro the drops are become heavily precipitate even a 
> mints. I tried so many things to avoid this ppt such as start PEG
> conc from 7%, dilute protein at half conc., add more salt in a
> reservoir solution but no succeeds. The protein prep is same which
> was using to get the Hits.
> 
> 
> I would be very thankful if you people give me nice suggestions.
> 
> Thanks
> 
> 
> Best Regards
> 
> AFSHAN

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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ySGYCLJmu6yEJ7i94AEjqKQ=
=EJjS
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[ccp4bb] need suggestions

[Afshan Begum]

 Dear All,

I have encountered one problem to optimization the crystallization condition
manually. Actually i got the good shape and even size crystal in a screening 
plate.
The condition are :

20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH 5.7 contain
50mM NaCl, 1mM EDTA &5mM BME.
When I tried to optimize this on Linbro the drops are become heavily 
precipitate even a
mints. I tried so many things to avoid this ppt such as start PEG conc
from 7%, dilute protein at half conc., add more salt in a reservoir solution
but no succeeds.
The protein prep is same which was using to get the Hits.


I would be very thankful if you people give me nice suggestions.

Thanks


Best Regards

AFSHAN