Re: [ccp4bb] I/sigmaI of >3.0 rule

[Graeme Winter]

Hi James,

May I just offer a short counter-argument to your case for not including
weak reflections in the merging residuals?

Unlike many people I rather like Rmerge, not because it tells you how good
the data are, but because it gives you a clue as to how well the unmerged
measurements agree with one another. It's already been mentioned on this
thread that Rmerge is ~ 0.8 /  which means that the inverse is also
true - an Rmerge of 0.8 indicates that the average measurement in the shell
has an I/sigma of ~ 1 (presuming there are sufficient multiple measurements
- if the multiplicity is < 3 or so this can be nonsense)

This does not depend on the error model or the multiplicity. It just talks
about the average. Now, if we exclude all measurements with an I/sigma of
less than three we have no idea of how strong the reflections in the shell
are on average. We're just top-slicing the good reflections and asking how
well they agree. Well, with an I/sigma > 3 I would hope they agree rather
well if your error model is reasonable. It would suddenly become rare to see
an Rmerge > 0.3 in the outer shell.

I like Rpim. It tells you how good the average measurement should be
provided you have not too much radiation damage. However, without Rmerge I
can't get a real handle on how well the measurements agree.

Personally, what I would like to see is the full contents of the Scala log
file available as graphs along with Rd from xdsstat and some other choice
statistics so you can get a relatively complete picture, however I
appreciate that this is unrealistic :o)

Just my 2c.

Cheerio,

Graeme

On 8 March 2011 20:07, James Holton  wrote:

> Although George does not mention anything about data reduction programs, I
> take from his description that common small-molecule data processing
> packages (SAINT, others?), have also been modernized to record all data (no
> I/sigmaI > 2 or 3 cutoff).  I agree with him that this is a good thing!  And
> it is also a good thing that small-molecule refinement programs use all
> data.  I just don't think it is a good idea to use all data in R factor
> calculations.
>
> Like Ron, I will probably be dating myself when I say that when I first got
> into the macromolecular crystallography business, it was still commonplace
> to use a 2-3 sigma spot intensity cutoff.  In fact, this is the reason why
> the PDB wants to know your "completeness" in the outermost resolution shell
> (in those days, the outer resolution was defined by where completeness drops
> to ~80% after the 3 sigma spot cutoff).  My experience with this, however,
> was brief, as the maximum-likelihood revolution was just starting to take
> hold, and the denzo manual specifically stated that only bad people use
> sigma cutoffs > -3.0.  Nevertheless, like many crystallographers from this
> era, I have fond memories of the REALLY low R factors you can get by using
> this arcane and now reviled practice.  Rsym values of 1-2% were common.
>
> It was only recently that I learned enough about statistics to understand
> the wisdom of my ancestors and that a 3-sigma cutoff is actually the "right
> thing to do" if you want to measure a fractional error (like an R factor).
>  That is all I'm saying.
>
> -James Holton
> MAD Scientist
>
>
> On 3/6/2011 2:50 PM, Ronald E Stenkamp wrote:
>
>> My small molecule experience is old enough (maybe 20 years) that I doubt
>> if it's even close to representing current practices (best or otherwise).
>>  Given George's comments, I suspect (and hope) that less-than cutoffs are
>> historical artifacts at this point, kept around in software for making
>> comparisons with older structure determinations.  But a bit of scanning of
>> Acta papers and others might be necessary to confirm that.  Ron
>>
>>
>> On Sun, 6 Mar 2011, James Holton wrote:
>>
>>
>>> Yes, I would classify anything with I/sigmaI < 3 as "weak".  And yes, of
>>> course it is possible to get "weak" spots from small molecule crystals.
>>> After all, there is no spot so "strong" that it cannot be defeated by a
>>> sufficient amount of background!  I just meant that, relatively speaking,
>>> the intensities diffracted from a small molecule crystal are orders of
>>> magnitude brighter than those from a macromolecular crystal of the same
>>> size, and even the same quality (the 1/Vcell^2 term in Darwin's formula).
>>>
>>> I find it interesting that you point out the use of a 2 sigma(I)
>>> intensity cutoff for small molecule data sets!  Is this still common
>>> practice?  I am not a card-carrying "small molecule crystallographer", so
>>> I'm not sure. However, if that is the case, then by definition there are no
>>> "weak" intensities in the data set.  And this is exactly the kind of data
>>> you want for least-squares refinement targets and computing "% error"
>>> quality metrics like R factors.  For likelihood targets, however, the "weak"
>>> data are actually a powerful restraint.
>>>
>>> -James Holton
>>> MAD Scientist
>>>
>>> On 3/6/2011 11

Re: [ccp4bb] How to crystallize a helicase

[Savvas Savvides]

I would certainly try cocrystallizing with adp, amppnp, or ATPgS
Many helicases have been crystallized in this way.   Papers by Sawaya et al and 
Singleton et al come readily to mind. 

Best of luck
Savvas

On 09 Mar 2011, at 15:52, Ed Pozharski  wrote:

> On Wed, 2011-03-09 at 10:52 +, Art wrote:
>> No protein crystal was found after some typical conditions (kit I, kit
>> II, and Index) were repeatly screened for more than three times.
> 
> See Einstein's definition of insanity :)
> 
>> Protein concentration gradient was considered, but it can not work.
> 
> Not sure what you mean by this, but you can also try diluting reservoir
> solutions.  Also, given your protein buffer, you may be more or less
> locking yourself into a narrow pH range - try 10mM MES instead.
> 
> -- 
> "I'd jump in myself, if I weren't so good at whistling."
>   Julian, King of Lemurs

Re: [ccp4bb] Supplementary density

[Jacob Keller]

I would think that any peptide column used in an ESI-MS setup would be
good for this. Are you doing ESI or MALDI?

JPK

On Wed, Mar 9, 2011 at 8:55 AM, Ed Pozharski  wrote:
> On Wed, 2011-03-09 at 14:14 +0100, Fabien Bergeret wrote:
>> I would be much grateful for any advice on peptide separation
>> methods?
>
> How about gel filtration in denaturing conditions?  If your protein is
> his-tagged, nickel column will do too, again in denaturing conditions.
>
> --
> "I'd jump in myself, if I weren't so good at whistling."
>                               Julian, King of Lemurs
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Supplementary density

[Daniel Bonsor]

If the protein is His-tagged, load the purified protein on to a Nickel column 
and place the column in a water bath above the protein's melting temperature. 
Recycle 5mls of water through the column to collect the peptide and then dry 
freeze or speed vac the sample to concentrate the peptide. 

I have seen this done for peptidoglycan fragments:
Peptidoglycan Recognition by Pal, an Outer Membrane Lipoprotein Biochemistry, 
2006, 45 (7), pp 2122–2128

I cannot see why it should not work for peptides. Don't forget if you are going 
to run a gel of the peptide to use tricine gels.


Dan

Re: [ccp4bb] Supplementary density

[Ed Pozharski]

On Wed, 2011-03-09 at 14:14 +0100, Fabien Bergeret wrote:
> I would be much grateful for any advice on peptide separation
> methods? 

How about gel filtration in denaturing conditions?  If your protein is
his-tagged, nickel column will do too, again in denaturing conditions.

-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] How to crystallize a helicase

[Ed Pozharski]

On Wed, 2011-03-09 at 10:52 +, Art wrote:
> No protein crystal was found after some typical conditions (kit I, kit
> II, and Index) were repeatly screened for more than three times.

See Einstein's definition of insanity :)

> Protein concentration gradient was considered, but it can not work.

Not sure what you mean by this, but you can also try diluting reservoir
solutions.  Also, given your protein buffer, you may be more or less
locking yourself into a narrow pH range - try 10mM MES instead.

-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] question about composite omit map

[Ed Pozharski]

On Wed, 2011-03-09 at 02:25 -0500, Kyuwon Baek wrote:
> Is there any resolution limit for the composite omit map?

Probably not and I would think that it is more useful at low resolution
where model bias is stronger.  But shouldn't you be talking about
simulated annealing as a bias removal tool rather than composite omit
map?  As far as maps go, solvent flattening may be even better option.

As a general comment, outcome of almost any refinement trick may vary
from dataset to dataset.  There are general tendencies, but the best way
to answer your questions is perhaps to calculate the map and look at the
result.

-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] PDB data mining

[Tom Oldfield]

 Robert Immormino

Does this service answer you question.  It is limited to 10,000 return
results for the statistical analysis so it is possible to ask general 
questions

where the statistics are truncated.

http://www.ebi.ac.uk/pdbe-as/pdbestatistics/PDBeStatisticsAtom.jsp

Tom Oldfield


Hi Cale,
My advice would be to find someone local with experience in scripting
possibly in python or perl.  This is a tractable problem with a bit of
scripting and if you aren't already experienced this may be a good
opportunity to delve into a bit of programing.
Good Luck,
-bob

On Tue, Mar 8, 2011 at 7:20 PM, Cale Dakwar  wrote:

Thanks for the quick reply.  This is a very good resource but not quite what
I need.  As far as I can tell, this resource is based on a dataset from
10-08-1998 and then only included x-ray crystal structure with better than 2
angstrom data.  I also can't in this database a mention of the PDB
structures in which e.g. the shortest His-His distances have been observed.
- C

On Tue, Mar 8, 2011 at 6:19 PM, Ed Pozharski  wrote:

On Tue, 2011-03-08 at 17:44 -0500, Cale Dakwar wrote:

Amongst other things, I want e.g. generate a histogram from this table
and determine e.g. the shortest HisND1 pair distance observed and the
structure in which this happens.

For this specific question this can be useful

http://www.biochem.ucl.ac.uk/bsm/sidechains/index.html



--
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] Supplementary density

[Fabien Bergeret]

 Hello

A while ago, I reported the structure determination of a protein in two 
different space groups. In both structures, the electron density maps 
revealed a well defined supplementary electron density. We built an 
oligopeptide and a putative sequence has been assigned. To confirm the 
sequence, we have set up several MS experiments and Edman sequencing 
from purified protein batches and dissolved crystals. For some reasons, 
the peptide could not be easily characterized using these approaches. As 
a last resort, we are planning to separate the peptide from the protein 
on gel and in solution before mass spectrometry.
I would be much grateful for any advice on peptide separation methods? 
For instance, what kind of gel or extraction protocols would you suggest?

Thank you
Fabien

-


Hello

We’re resolving a structure of a soluble protein and in the electronic 
density map (maximum resolution at 2.2Å), we observe a supplementary 
density that does not belong to the protein. This density is present 
in two different crystalline forms obtained in different 
crystallization conditions.
This density could be represented by an oligopeptide ~10 residues long 
for which there is no ambiguity about its polarity. Furthermore, side 
chains are quite easily visible and a sequence can therefore be assigned.
The deduced sequence doesn’t belong to the sequence of the protein of 
interest, meaning that the oligopeptide has been co-purified and 
co-crystallized.


Has somebody met a similar situation? Could you please give us some 
advices in terms of refinement, validation, etc.?


Thanks in advance

Best regards


Fabien
PhD student
fabien.berge...@ipbs.fr 

Re: [ccp4bb] How to crystallize a helicase

[Poul Nissen]

Dear Art,

It seems twisted, indeed.

First of all you need to verify that your protein is at all in a good
state (you seem to imply that it is not with your remark on protein
concentration). Basic biophyscial characterisation for stability and
structural homogeniety is required to reach proper buffer conditions for
your protein, and you may consider a  complex with relevant ligands

Poul

> Dear all!
> We've been trying to crystallize a helicase. No protein crystal was found
> after some typical conditions (kit I, kit II, and Index) were repeatly
> screened for more than three times. 50 mM MES (pH 6.0), 5% glycerol, and
> 1mM DTT is used as the final storage buffer. The most significant problem
> of this protein is its precipitation in most of the screening conditions.
> Protein concentration gradient was considered, but it can not work.
> I was wondering if there is any way to work aroud this problem. Look
> forward to your suggestions!
> Thanks!
> Art
>

[ccp4bb] How to crystallize a helicase

[Art]

Dear all!
We've been trying to crystallize a helicase. No protein crystal was found after 
some typical conditions (kit I, kit II, and Index) were repeatly screened for 
more than three times. 50 mM MES (pH 6.0), 5% glycerol, and 1mM DTT is used as 
the final storage buffer. The most significant problem of this protein is its 
precipitation in most of the screening conditions. Protein concentration 
gradient was considered, but it can not work.
I was wondering if there is any way to work aroud this problem. Look forward to 
your suggestions!
Thanks!
Art

Re: [ccp4bb] PDB data mining

[Christian Fufezan]

Hi Cale,

this could be done equally with a python module called p3d.
A script would look like this

#!/usr/bin/env python3.1
'''Calculate ND1pairwise distances by Ch. Fufezan 2011

usage: ND1_pairs.py 

see online documentation http://p3d.fufezan.net 
'''
import sys
import p3d
import itertools

if (__name__ == '__main__'):
if (len(sys.argv) != 2):
print (__doc__)
sys.exit(1)

pdb  = p3d.protein.Protein(sys.argv[1])
ND1s = pdb.query("protein and resname HIS and atom type ND1")
for atom1,atom2 in itertools.combinations(ND1s,2):
print("{atom1_info} {atom2_info} {distance}".format(
atom1_info = atom1.info(lvl='min'),
atom2_info = atom2.info(lvl='min'),
distance   = atom1.distanceTo(atom2)
))
 
Cheers :)

[ccp4bb] SUMMARY -until now- [OT] which column to use in SLS/MALS instruments

[Sebastiano Pasqualato]

Dear all,
thanks for the wealth of replies.
I'll try to summarize and give some answers and clarification.
Here the original post:

Dear all,
I was wondering if somebody could help me out by suggesting the "best" column 
to be used in a Static Light Scattering (I guess it would be the same for a 
Multi Angle Light Scattering) instrument.
We were suggested using a silica-based column, with very high separation 
properties, but it seems that these columns are highly sensitive to (even 
slightly) basic pH's. Even running the column in PBS, it looks like injecting 
samples at pH 8.0 ruins the column resin, making it unusable.
On the other hand, GE Healthcare columns would require a huge amount of 
material to be loaded.
What are you guys using in your instruments? 
Thanks a lot in advance for the feedback,
best,
ciao
Sebastiano

Here's replies and comments:

Laurent Terradot wrote:

> Dea Sebastiano,
> I encountered a similar problem and lost one of these columns. (We use 
> Dawn-Treos/OptiReX in my lab with an akata purifier)
> I am now using GE superdex columns (S200 and S75) and load around 100ul of 
> protein at 10mg/ml. That' s a lot but I fractionate and recover it. The 
> results are good.
> 
> If money is not a problem, Wyatt make their own columns, which might be 
> better for you. I have heard good things about it.
> I 'll be happy to have info if you have some feed back later.
> Best regards,
> 
> Laurent


Well that's one of our problems. We are using a Viscotek SLS instrument on 
their own HPLC machine, and don't have a fractionation unit in line, since we 
thought we could be using analytical amounts, without recovering.
In this respect, 100 ul at 10 mg/ml are way to much.
However, protein amount can be much lower, it seems:

Nikos Pinotsis wrote:

> Hi Sebastiano,
> 
> we have used both silica based and superdex columns and we found the second 
> as a better option for general usage.
> Most of our proteins are on basic buffer and about 50% of them stick on the 
> silica based column. On the other side a superdex 200 10/30 from GE performed 
> quite well, we got quite accurate results for both a ~250kDa hexameric enzyme 
> as also for a 0.3 mg/ml injection of an insulin sample.
> Eventually I think depends on the applications you prefer to have.
> 
> cheers
> Nikos


 Engin Özkan wrote:

> On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote:
>> On the other hand, GE Healthcare columns would require a huge amount of 
>> material to be loaded.
>> 
> What do you mean by a huge amount of material? You would not be using a 16/60 
> column (125 ml column volume) for an analytical experiment. How about a 
> Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have 
> column volumes at 25 ml and 3 ml, respectively, have great resolution, and 
> probably already compatible with your proteins and buffers.
> 
> We used to use HPLC columns, but some proteins would never elute from these 
> columns. Then we switched to good old Superdex 200 10/300, and it works like 
> a charm every time. We inject <100 ul material at concentrations around 1 
> mg/ml (depending on the molecular weight of the protein in question). The 
> only issue is we have to run these columns at 0.35 ml/min flow rates (instead 
> of the default 0.5 ml/min), since our HPLC has a lot of back pressure for 
> FPLC columns.
> 
> Best,
> Engin

Engin Özkan wrote:

> And as others have said shedding of dextran is a problem with GE columns 
> (this was confirmed to me by GE people), but after extensive system 
> equilibration, we do not see a problem significant enough to ever hurt our 
> light scattering measurements.


With respect to the choice of which GE Column to use, it looks there's a 
consensus for Superdex/Superose 10/300 columns. Some have suggested Superdex 
5/150 columns, which would allow much less material to be loaded (they have a 
diameter of ~5 mm, as the suggested silica column), but others have pointed out 
that the resolution power is way less than those of the 30 cm columns, and this 
is indeed our experience, too, and the reason we dropped those columns.

Sally Pham Thanh Van wrote:

> But I doubt if GE 5/150 column has the same resolution with
> the 10/30 one. When I had mixture of several species, the 10/30 always
> gave better resolution for me, and this is crucial in obtaining
> accurate information about oligomerization state and/or absolute
> molecular weight of the protein. So I think if choosing GE columns for
> light scattering experiment, I would go for 10/30.
> 
> Best wishes,
> Sally.

M T wrote:

> Dear Sebastiano,
> 
> In our case we don't have magical column... We are using small silica columns 
> from shodex or wyatt and for proteins that cannot be happy in pH<7.5 we have 
> a 25 mL superdex 200 from GE. Maybe you can try to use a very small superdex 
> 200 from GE (ref : 17-1089-01) but I think that the maximum flow rate of 
> these columns is too low to be suitable with your system. The peoples of

Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments

[Sally Pham Thanh Van]

Regarding to amount of material loaded, I think it may also depend on
the buffer. When I did light scattering experiment with membrane
protein at angle of 90 degree, I got much better base line with buffer
containing DDM than one containing OG. This maybe due to high
concentration of OG required I guess. When I increased concentration
of protein in OG buffer, it helped increase signal-noise ratio.

In this issue, I don't know if Wyatt column can help reduce the amount
of sample. But I doubt if GE 5/150 column has the same resolution with
the 10/30 one. When I had mixture of several species, the 10/30 always
gave better resolution for me, and this is crucial in obtaining
accurate information about oligomerization state and/or absolute
molecular weight of the protein. So I think if choosing GE columns for
light scattering experiment, I would go for 10/30.

Best wishes,
Sally.

On Tue, Mar 8, 2011 at 7:44 PM, Engin Özkan  wrote:
> And as others have said shedding of dextran is a problem with GE columns
> (this was confirmed to me by GE people), but after extensive system
> equilibration, we do not see a problem significant enough to ever hurt our
> light scattering measurements.
>
> Engin
>
> On 3/8/11 10:38 AM, Engin Özkan wrote:
>>
>> On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote:
>>>
>>> On the other hand, GE Healthcare columns would require a huge amount of
>>> material to be loaded.
>>>
>> What do you mean by a huge amount of material? You would not be using a
>> 16/60 column (125 ml column volume) for an analytical experiment. How about
>> a Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have
>> column volumes at 25 ml and 3 ml, respectively, have great resolution, and
>> probably already compatible with your proteins and buffers.
>>
>> We used to use HPLC columns, but some proteins would never elute from
>> these columns. Then we switched to good old Superdex 200 10/300, and it
>> works like a charm every time. We inject <100 ul material at concentrations
>> around 1 mg/ml (depending on the molecular weight of the protein in
>> question). The only issue is we have to run these columns at 0.35 ml/min
>> flow rates (instead of the default 0.5 ml/min), since our HPLC has a lot of
>> back pressure for FPLC columns.
>>
>> Best,
>> Engin
>>
>
>
> --
> Engin Özkan
> Post-doctoral Scholar
> Howard Hughes Medical Institute
> Dept of Molecular and Cellular Physiology
> 279 Campus Drive, Beckman Center B173
> Stanford School of Medicine
> Stanford, CA 94305
> ph: (650)-498-7111
>