Re: [ccp4bb] Resolution limit of index in XDS

[Tim Gruene]

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Hash: SHA1

Dear Niu,

indexing relies on strong reflections only, that is (in very brieft)
why INCLUDE_RESOLUTION_RANGE indeed does not affect the relections
collected in COLSPOT which in turn are used by IDXREF. You can work
around this, however, by making use of TRUSTED_REGION and set it to
e.g. 0.7 or 0.6 (you can use adxv to translate resolution into pixel
and then calculate the fraction you need to set the second number in
TRUSTED_REGION to (or the first if you want to exclude the inner
resolution reflections - I remember one data set where this was
essential for indexing - DNA was involved there)

Best,
Tim

On 03/19/2013 08:53 PM, Niu Tou wrote:
> Dear All,
> 
> Is there any command can set the resolution limit for index step in
> XDS? I only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks
> to be a definition of resolution range after index step as it
> says:
> 
> INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by 
> DEFPIX,INTEGRATE,CORRECT
> 
> Thanks! Niu
> 

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

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Re: [ccp4bb] Philosophical question

[David Schuller]

On 03/19/13 14:41, Jacob Keller wrote:
I don't understand this argument, as it would apply equally to all 
features of the theoretical LUCA (protein and DNA sequences, etc). To 
make it logically sound, I think you have either to include some kind 
of super-high boundary to getting to other possible conventions (you 
probably imply this) or, as I have suggested, it may be a particularly 
good, if not the best, solution (a global minimum, one might say). The 
first hypothesis is similar to the QWERTY keyboard, which is cemented 
in place by many factors, whereas the second is more "survival of the 
fittest."



It would not be a safe idea to assume that LUCA was a single cell with a 
single chromosome (i.e. like a modern bacterium.) It would also not be 
safe to assume that viruses and horizontal gene transfer were not around 
at that time.


As I mentioned privately, I think the relevant slogan would be "winner 
take all" rather than "survival of the fittest."


Another possible explanation for having a recognition tag at the 
beginning of each transcribed gene: to distinguish between host and 
virus. This does not imply that Met has any specific advantage over any 
any other tag which might have been chosen.



--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] Philosophical question

[Ed Pozharski]

Jacob,
So you'd have to explain why the codon convention is so 
intolerant/invariant relative to the other features--it seems to me 
that either it is at an optimum or there is some big barrier holding 
it in place.


Because altering codon convention will result in massive translation errors.

However the original question refers to start codon and its relation to 
methionine.  Notice that AUG is the *only* codon for methionine. If you 
change amino acid specificity of the methionine tRNA synthetase, you'd 
replace every methionine in every protein.  It is very unlikely that an 
organism other than one with a very small genome can survive that.  
Given high fidelity required of tRNA synthetases, changing their 
specificity is also not easy.  Most mutations are likely to incapacitate 
the enzyme rather than switch its specificity, resulting in organism 
that is unable to develop (due to stalled translation), let alone survive.


As for the optimization part - I am also not sure what significant 
benefit you expect from replacing starting methionine with a different 
amino acid.  It is mostly removed anyway.  Why that is? My (uneducated) 
guess is that it is rarely structural and there is benefit in recycling it.


Cheers,

Ed.

Re: [ccp4bb] Philosophical question

[D Bonsor]

You may want to read:

Evolutionary conservation of codon optimality reveals hidden signatures of 
cotranslational folding
Nature structural & molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 237

Here they suggest that the codon bias is such it allows translation to pause 
and folding of the polypeptide. Most proteins probably do not have a problem 
folding so it does not matter which codon is used.

Dan


It is so intolerant to change because reassigning a codon to a different amino 
acid type or stop codon affects thousands of proteins that use that codon 
simultaneously. The probably that none of those mutations are deleterious is 
extremely small.

Genetic code changes are more common in the mitochondrial code. First of all 
the mitochondrial genome is much smaller, ~16kb for vertebrates. Moreover, in 
cases I have looked at the change in codon use seems to happen when first there 
is a case of extreme bias against using a codon. When a codon is (almost) not 
used at all it can be re-purposed without affecting any proteins.

Bart

On Tue, Mar 19, 2013 at 2:05 PM, Jacob Keller  
wrote:

I don't understand this argument, as it would apply equally to all features 
of the theoretical LUCA 

No it won't.  Different features would have different tolerance levels 
to modifications.


Yes, this "tolerance" is the second (hidden or implicit) principle I 
referred to. So you'd have to explain why the codon convention is so 
intolerant/invariant relative to the other features--it seems to me that either 
it is at an optimum or there is some big barrier holding it in place. And you'd 
have to explain this without invoking interchange of DNA, viruses, etc, as 
we're talking about a LUCA here, right? And you'll have to make sure that 
whatever reason you invoke cannot be applied to other features of this LUCA 
which are indeed seen to be variable.

JPK

Re: [ccp4bb] Philosophical question

[Edward A. Berry]

> Why use a big expensive amino acid instead of choosing one of the glycine 
codons?
>
> I can't quickly track anything down in the literature to back this up, but 
"expensive" could be part of it. The cell
> doesn't want to start translation if there isn't ample resources to finish 
the job. Perhaps Met concentration is a proxy
> for anabolic potential of the cell? Or at least was primordially and "QWERTY'd 
in"?

That makes sense.
Also, It occurred to me after posting that the Met is not necessarily wasted
if it gets cleaved off by N-peptidase. It can get loaded onto another tRNA and
go through the cycle again. presumably the cost in ATP for loading Met-tRNA
and gly-tRNA is the same.
Maybe Met makes a better "handle" for some step in initiation.


Shane Caldwell wrote:

why doesn't initiation occur also at methionines in the middle of proteins?


It can and does. I can show you expression gels where I make full-length 
protein and a fragment from an internal
initiation.

Why use a big expensive amino acid instead of choosing one of the glycine 
codons?


I can't quickly track anything down in the literature to back this up, but 
"expensive" could be part of it. The cell
doesn't want to start translation if there isn't ample resources to finish the 
job. Perhaps Met concentration is a proxy
for anabolic potential of the cell? Or at least was primordially and "QWERTY'd 
in"?

/wild speculation

Shane Caldwell
McGill University



On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry mailto:ber...@upstate.edu>> wrote:

Opher Gileadi wrote:

Hi Theresa,

To add to Anat's comments: Although the AUG codon for the first 
methionine and all other methionines in a
protein coding sequence look the same, they are read in a very 
different way by the ribosomal machinery. The
first AUG is recognized by the initiation complex, which includes the 
separate small ribosomal subunit (40s), a
special tRNA-methionine, and initiation factors (proteins) including 
eIF2. This leads to assembly of a complete
ribosome and initiation of protein synthesis. Subsequently, in the 
process of elongation, AUG codons are read by
a different tRNA, which is brought to the 80s ribosome bound to a 
protein called elongation factor 1a. This is
an oversimplification, of course, but the point is that the initiation 
codon (=the first amino acid to be
incorporated to the protein) is read by a special tRNA, hence the 
universal use of methionine.

Opher

Yes, but why methionine? Half the time it has to be removed by N-terminal 
peptidase to give a small first residue,
or by leader sequence processing. Why use a big expensive amino acid 
instead of choosing one of the glycine codons?
Is there an obvious reason, or just "it had to be something, and Met happened to 
get selected"?

And why sometimes alternate start codons can be used? and why doesn't 
initiation occur also at methionines in the
middle of proteins? I'm guessing it has to do with 5' untranslated region 
and ribosome binding sites. So could the
start codon actually be anything you want, provided there is a strong 
ribosome binding site there?

Just being philosophical, and not afraid to display my ignorance,
eab

Re: [ccp4bb] Philosophical question

[Bart Hazes]

It is so intolerant to change because reassigning a codon to a different
amino acid type or stop codon affects thousands of proteins that use that
codon simultaneously. The probably that none of those mutations are
deleterious is extremely small.

Genetic code changes are more common in the mitochondrial code. First of
all the mitochondrial genome is much smaller, ~16kb for vertebrates.
Moreover, in cases I have looked at the change in codon use seems to happen
when first there is a case of extreme bias against using a codon. When a
codon is (almost) not used at all it can be re-purposed without affecting
any proteins.

Bart

On Tue, Mar 19, 2013 at 2:05 PM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:

> I don't understand this argument, as it would apply equally to all
> features of the theoretical LUCA
>
>> No it won't.  Different features would have different tolerance levels to
>> modifications.
>
>
> Yes, this "tolerance" is the second (hidden or implicit) principle I
> referred to. So you'd have to explain why the codon convention is so
> intolerant/invariant relative to the other features--it seems to me that
> either it is at an optimum or there is some big barrier holding it in
> place. And you'd have to explain this without invoking interchange of DNA,
> viruses, etc, as we're talking about a LUCA here, right? And you'll have to
> make sure that whatever reason you invoke cannot be applied to other
> features of this LUCA which are indeed seen to be variable.
>
> JPK
>
>
> ***
>
> Jacob Pearson Keller, PhD
>
> Looger Lab/HHMI Janelia Farms Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> email: kell...@janelia.hhmi.org
>
> ***
>



-- 

Bart Hazes
Associate Professor
Dept. of Medical Microbiology & Immunology
University of Alberta

Re: [ccp4bb] Philosophical question

[Jacob Keller]

I don't understand this argument, as it would apply equally to all features
of the theoretical LUCA

> No it won't.  Different features would have different tolerance levels to
> modifications.


Yes, this "tolerance" is the second (hidden or implicit) principle I
referred to. So you'd have to explain why the codon convention is so
intolerant/invariant relative to the other features--it seems to me that
either it is at an optimum or there is some big barrier holding it in
place. And you'd have to explain this without invoking interchange of DNA,
viruses, etc, as we're talking about a LUCA here, right? And you'll have to
make sure that whatever reason you invoke cannot be applied to other
features of this LUCA which are indeed seen to be variable.

JPK


***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***

[ccp4bb] Resolution limit of index in XDS

[Niu Tou]

Dear All,

Is there any command can set the resolution limit for index step in XDS? I
only found a keyword INCLUDE_RESOLUTION_RANGE, but it looks to be a
definition of resolution range after index step
as it says:

INCLUDE_RESOLUTION_RANGE=20.0 0.0 !Angstroem; used by
DEFPIX,INTEGRATE,CORRECT

Thanks!
Niu

Re: [ccp4bb] Philosophical question

[Ed Pozharski]

On 03/19/2013 02:41 PM, Jacob Keller wrote:
I don't understand this argument, as it would apply equally to all 
features of the theoretical LUCA 
No it won't.  Different features would have different tolerance levels 
to modifications.


Philosophically, one is wrong to expect that living organisms will 
evolve in a fashion that we find optimal.  Whenever I feel that a 
protein behaves in a way I find stupid, I simply say "giraffe laryngeal 
nerve" and all comes back to normal.

Re: [ccp4bb] Philosophical question

[Shane Caldwell]

> why doesn't initiation occur also at methionines in the middle of proteins?


It can and does. I can show you expression gels where I make full-length
protein and a fragment from an internal initiation.



> Why use a big expensive amino acid instead of choosing one of the glycine
> codons?
>

I can't quickly track anything down in the literature to back this up, but
"expensive" could be part of it. The cell doesn't want to start translation
if there isn't ample resources to finish the job. Perhaps Met concentration
is a proxy for anabolic potential of the cell? Or at least was primordially
and "QWERTY'd in"?

/wild speculation

Shane Caldwell
McGill University



On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry  wrote:

> Opher Gileadi wrote:
>
>> Hi Theresa,
>>
>> To add to Anat's comments: Although the AUG codon for the first
>> methionine and all other methionines in a protein coding sequence look the
>> same, they are read in a very different way by the ribosomal machinery. The
>> first AUG is recognized by the initiation complex, which includes the
>> separate small ribosomal subunit (40s), a special tRNA-methionine, and
>> initiation factors (proteins) including eIF2. This leads to assembly of a
>> complete ribosome and initiation of protein synthesis. Subsequently, in the
>> process of elongation, AUG codons are read by a different tRNA, which is
>> brought to the 80s ribosome bound to a protein called elongation factor 1a.
>> This is an oversimplification, of course, but the point is that the
>> initiation codon (=the first amino acid to be incorporated to the protein)
>> is read by a special tRNA, hence the universal use of methionine.
>>
>> Opher
>>
>>  Yes, but why methionine? Half the time it has to be removed by
> N-terminal peptidase to give a small first residue, or by leader sequence
> processing. Why use a big expensive amino acid instead of choosing one of
> the glycine codons? Is there an obvious reason, or just "it had to be
> something, and Met happened to get selected"?
>
> And why sometimes alternate start codons can be used? and why doesn't
> initiation occur also at methionines in the middle of proteins? I'm
> guessing it has to do with 5' untranslated region and ribosome binding
> sites. So could the start codon actually be anything you want, provided
> there is a strong ribosome binding site there?
>
> Just being philosophical, and not afraid to display my ignorance,
> eab
>

Re: [ccp4bb] Philosophical question

[Jacob Keller]

I don't understand this argument, as it would apply equally to all features
of the theoretical LUCA (protein and DNA sequences, etc). To make it
logically sound, I think you have either to include some kind of super-high
boundary to getting to other possible conventions (you probably imply this)
or, as I have suggested, it may be a particularly good, if not the best,
solution (a global minimum, one might say). The first hypothesis is similar
to the QWERTY keyboard, which is cemented in place by many factors, whereas
the second is more "survival of the fittest."

It should perhaps be noted parenthetically that prima facie the "accident
of history" QWERTY hypothesis is at variance with radical Darwinism.

JPK


On Tue, Mar 19, 2013 at 1:56 PM, David Waterman wrote:

> I believe that the reason all organisms share the convention (more or
> less) is that it dates back to LUCA - the Last Universal Common Ancestor of
> all extant life. LUCA must have had the basic transcription and translation
> machinery that we now see somewhat divergently-evolved versions of in all
> cells. This does not answer why that particular convention was "chosen",
> but it does count against the idea that it is the best possible system, or
> indeed should continue to be selected for (except that mutations to this
> machinery tend to be very much deleterious).
>
> -- David
>
>
> On 19 March 2013 14:34, Jacob Keller wrote:
>
>> Never one to shrink from philosophizing, I wonder generally why the codon
>> conventions are the way they are? Is it like the QWERTY keyboard--basically
>> an historical accident--or is there some more beautiful reason? One might
>> argue that since basically all organisms share the convention (are there
>> exceptions, even?), that it must be the "best of all possible" conventions.
>> I have often wondered whether maybe this particular convention allows for
>> the most effective pathways between proteins of significant function, e.g.,
>> through the fewest mutations perhaps? One certainly cannot maintain that
>> every possible protein sequence has been made at some time or another in
>> the history of the biological world (go quantitate!) so there must be a way
>> to ensure that mostly the "best" ones got made. On the other hand, since
>> many organisms share DNA, maybe they had to "agree" on a system (I think
>> this is the dogma?). Was there a "United Organisms" convention at some
>> point, reminiscent of "Les Immortels" of the French language or POSIX or
>> something, to ensure compliance? What was the penalty for non-compliance?
>>
>> Anyway, I like the question about the methionines,
>>
>> Jacob
>>
>>
>> On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry wrote:
>>
>>> Opher Gileadi wrote:
>>>
 Hi Theresa,

 To add to Anat's comments: Although the AUG codon for the first
 methionine and all other methionines in a protein coding sequence look the
 same, they are read in a very different way by the ribosomal machinery. The
 first AUG is recognized by the initiation complex, which includes the
 separate small ribosomal subunit (40s), a special tRNA-methionine, and
 initiation factors (proteins) including eIF2. This leads to assembly of a
 complete ribosome and initiation of protein synthesis. Subsequently, in the
 process of elongation, AUG codons are read by a different tRNA, which is
 brought to the 80s ribosome bound to a protein called elongation factor 1a.
 This is an oversimplification, of course, but the point is that the
 initiation codon (=the first amino acid to be incorporated to the protein)
 is read by a special tRNA, hence the universal use of methionine.

 Opher

  Yes, but why methionine? Half the time it has to be removed by
>>> N-terminal peptidase to give a small first residue, or by leader sequence
>>> processing. Why use a big expensive amino acid instead of choosing one of
>>> the glycine codons? Is there an obvious reason, or just "it had to be
>>> something, and Met happened to get selected"?
>>>
>>> And why sometimes alternate start codons can be used? and why doesn't
>>> initiation occur also at methionines in the middle of proteins? I'm
>>> guessing it has to do with 5' untranslated region and ribosome binding
>>> sites. So could the start codon actually be anything you want, provided
>>> there is a strong ribosome binding site there?
>>>
>>> Just being philosophical, and not afraid to display my ignorance,
>>> eab
>>>
>>
>>
>>
>> --
>> ***
>>
>> Jacob Pearson Keller, PhD
>>
>> Looger Lab/HHMI Janelia Farms Research Campus
>>
>> 19700 Helix Dr, Ashburn, VA 20147
>>
>> email: kell...@janelia.hhmi.org
>>
>> ***
>>
>
>


-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

*

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

[Edward A. Berry]

Maybe this thread still needs some more pedagogy/explanation for those newbies and for biologist/ wanna-be 
crystallographers like me. My original reaction was- if the true space group is P21 you wouldn't want to expand from 
data reduced in higher symmetry, because you would be enforcing that higher symmetry.


But if it were simply a case of P21 symmetry, with three molecules in the AU, that happened to have a beta angle of 90, 
merging statistics would have prevented reducing the data in p222 in the first place.


Does order/disorder mean that the third molecule is actually present in two different orientations with equal occupancy, 
so that on the average it does obey the higher symmetry? Like our "heme on a special position" in the bacterioferritin 
paper?
And structure factors add because the two orientations are present in the same domain, whereas with twinning the two 
orientations are present in different domains that diffract like separate crystals, and the resulting intensities add on 
the "film"?



herman.schreu...@sanofi.com wrote:

If it is crystal packing disorder (F's added instead of I's), the switches 
between the alternative conformations need to be very frequent, to be within 
coherent range, so I would asume that the alternative conformations will be 
present in equal proportions. Still the alternatives need to be modeled somehow 
and if this can be conveniently done in a lower symmetry spacegroup this would 
not artificially lower the free R-factors. As Phoebe mentioned, ignoring the 
higher symmetry relations and repicking the free Rflags at lower symmetry would 
lead free reflections to be linked to the working set, leading to too low Rfree 
values. However, with perfect packing disorder, no extra information would be 
gained by reprocessing in lower symmetry (in contrast to cases with pseudo 
symmetry).

My 2 cents,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. 
Rice
Sent: Tuesday, March 19, 2013 4:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
   If the issue is perfect twinning, I agree - good point!
   But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry&  Molecular Biology The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is that 
data can be merged in the higher-symmetry lattice, while the number of 
molecules in the asymmetric unit (3 in P21) is not divisible by the higher 
symmetry factor (2, by going from P21 to P21212).

From my experience, this is more likely a case of order-disorder than 
merohedral twinning. The difference between these two is that structure factors 
are added for the alternative conformations in the case of order-disorder, 
while intensities (structure factors squared) are added in the case of 
merohedral twinning.


Now an important comment on how to proceed in the cases where data can be 
merged in a higher symmetry, but the structure needs to be solved in a lower 
symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag, and 
THEN expanded to the lower symmetry. Reprocessing the data in a lower sy

Re: [ccp4bb] Philosophical question

[David Waterman]

I believe that the reason all organisms share the convention (more or
less) is that it dates back to LUCA - the Last Universal Common Ancestor of
all extant life. LUCA must have had the basic transcription and translation
machinery that we now see somewhat divergently-evolved versions of in all
cells. This does not answer why that particular convention was "chosen",
but it does count against the idea that it is the best possible system, or
indeed should continue to be selected for (except that mutations to this
machinery tend to be very much deleterious).

-- David


On 19 March 2013 14:34, Jacob Keller  wrote:

> Never one to shrink from philosophizing, I wonder generally why the codon
> conventions are the way they are? Is it like the QWERTY keyboard--basically
> an historical accident--or is there some more beautiful reason? One might
> argue that since basically all organisms share the convention (are there
> exceptions, even?), that it must be the "best of all possible" conventions.
> I have often wondered whether maybe this particular convention allows for
> the most effective pathways between proteins of significant function, e.g.,
> through the fewest mutations perhaps? One certainly cannot maintain that
> every possible protein sequence has been made at some time or another in
> the history of the biological world (go quantitate!) so there must be a way
> to ensure that mostly the "best" ones got made. On the other hand, since
> many organisms share DNA, maybe they had to "agree" on a system (I think
> this is the dogma?). Was there a "United Organisms" convention at some
> point, reminiscent of "Les Immortels" of the French language or POSIX or
> something, to ensure compliance? What was the penalty for non-compliance?
>
> Anyway, I like the question about the methionines,
>
> Jacob
>
>
> On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry wrote:
>
>> Opher Gileadi wrote:
>>
>>> Hi Theresa,
>>>
>>> To add to Anat's comments: Although the AUG codon for the first
>>> methionine and all other methionines in a protein coding sequence look the
>>> same, they are read in a very different way by the ribosomal machinery. The
>>> first AUG is recognized by the initiation complex, which includes the
>>> separate small ribosomal subunit (40s), a special tRNA-methionine, and
>>> initiation factors (proteins) including eIF2. This leads to assembly of a
>>> complete ribosome and initiation of protein synthesis. Subsequently, in the
>>> process of elongation, AUG codons are read by a different tRNA, which is
>>> brought to the 80s ribosome bound to a protein called elongation factor 1a.
>>> This is an oversimplification, of course, but the point is that the
>>> initiation codon (=the first amino acid to be incorporated to the protein)
>>> is read by a special tRNA, hence the universal use of methionine.
>>>
>>> Opher
>>>
>>>  Yes, but why methionine? Half the time it has to be removed by
>> N-terminal peptidase to give a small first residue, or by leader sequence
>> processing. Why use a big expensive amino acid instead of choosing one of
>> the glycine codons? Is there an obvious reason, or just "it had to be
>> something, and Met happened to get selected"?
>>
>> And why sometimes alternate start codons can be used? and why doesn't
>> initiation occur also at methionines in the middle of proteins? I'm
>> guessing it has to do with 5' untranslated region and ribosome binding
>> sites. So could the start codon actually be anything you want, provided
>> there is a strong ribosome binding site there?
>>
>> Just being philosophical, and not afraid to display my ignorance,
>> eab
>>
>
>
>
> --
> ***
>
> Jacob Pearson Keller, PhD
>
> Looger Lab/HHMI Janelia Farms Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> email: kell...@janelia.hhmi.org
>
> ***
>

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

[Herman . Schreuder]

If it is crystal packing disorder (F's added instead of I's), the switches 
between the alternative conformations need to be very frequent, to be within 
coherent range, so I would asume that the alternative conformations will be 
present in equal proportions. Still the alternatives need to be modeled somehow 
and if this can be conveniently done in a lower symmetry spacegroup this would 
not artificially lower the free R-factors. As Phoebe mentioned, ignoring the 
higher symmetry relations and repicking the free Rflags at lower symmetry would 
lead free reflections to be linked to the working set, leading to too low Rfree 
values. However, with perfect packing disorder, no extra information would be 
gained by reprocessing in lower symmetry (in contrast to cases with pseudo 
symmetry).

My 2 cents,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phoebe A. 
Rice
Sent: Tuesday, March 19, 2013 4:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is that 
data can be merged in the higher-symmetry lattice, while the number of 
molecules in the asymmetric unit (3 in P21) is not divisible by the higher 
symmetry factor (2, by going from P21 to P21212).
>From my experience, this is more likely a case of order-disorder than 
>merohedral twinning. The difference between these two is that structure 
>factors are added for the alternative conformations in the case of 
>order-disorder, while intensities (structure factors squared) are added in the 
>case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be 
merged in a higher symmetry, but the structure needs to be solved in a lower 
symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag, and 
THEN expanded to the lower symmetry. Reprocessing the data in a lower symmetry 
is an absolutely wrong procedure and it will artificially reduce R-free, as the 
new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and these 
are infrequent, reprocessing the data in a lower symmetry seems to be 
frequently abused, essentially in order to reduce R-free. Generally, when data 
CAN be merged in a higher symmetry, the only proper procedure in going to a 
lower-symmetry structure is by expanding these higher-symmetry data to a lower 
symmetry, and not by rescaling and merging the data in a lower symmetry.

Zbyszek Otwinowski

> Dear all,
> We have solved the problem. Data processing in P1 looks better (six 
> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three 
> molecules in ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first 
> round of refinement (without put waters, ligands, etc.).
>
> Indeed, there were one more molecule in ASU, but the over-merged data 
> in an orthorhombic lattice hid the correct solution.
>
> Thank you very much for all y

Re: [ccp4bb] Philosophical question

[Katherine Sippel]

On Tue, Mar 19, 2013 at 9:34 AM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:

> One might argue that since basically all organisms share the convention
> (are there exceptions, even?), that it must be the "best of all possible"
> conventions.
>

There are actually lots of exceptions. For example the UGA stop codon in E.
coli codes for Trp in Mycoplasma species. A fairly comprehensive list of
the codon variations as annotated by the NCBI can be found here
http://www.bioinformatics.org/JaMBW/2/3/TranslationTables.html#SG4

Cheers,
Katherine

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

[Phoebe A. Rice]

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that "perfect twinning" 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is
that data can be merged in the higher-symmetry lattice, while the number
of molecules in the asymmetric unit (3 in P21) is not divisible by the
higher symmetry factor (2, by going from P21 to P21212).
>From my experience, this is more likely a case of order-disorder than
merohedral twinning. The difference between these two is that structure
factors are added for the alternative conformations in the case of
order-disorder, while intensities (structure factors squared) are added in
the case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be
merged in a higher symmetry, but the structure needs to be solved in a
lower symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag,
and THEN expanded to the lower symmetry. Reprocessing the data in a lower
symmetry is an absolutely wrong procedure and it will artificially reduce
R-free, as the new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and
these are infrequent, reprocessing the data in a lower symmetry seems to
be frequently abused, essentially in order to reduce R-free. Generally,
when data CAN be merged in a higher symmetry, the only proper procedure in
going to a lower-symmetry structure is by expanding these higher-symmetry
data to a lower symmetry, and not by rescaling and merging the data in a
lower symmetry.

Zbyszek Otwinowski

> Dear all,
> We have solved the problem. Data processing in P1 looks better (six
> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
> in
> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
> of refinement (without put waters, ligands, etc.).
>
> Indeed, there were one more molecule in ASU, but the over-merged data in
> an orthorhombic lattice hid the correct solution.
>
> Thank you very much for all your suggestions, they were very important to
> solve this problem.
>
> Cheers,
>
> Andrey
>
> 2013/3/15 Andrey Nascimento 
>
>> *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 

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

[Jacob Keller]

Isn't lowering the symmetry equivalent to using multiple
models/conformations for one map? I remember seeing this done with the
infamous MSBA structure from a few years ago, so caveat emptor I guess. And
further, wouldn't using strict NCS make things equivalent to the
higher-symmetry space group? And then violating the NCS in places would
then just be equivalent to modelling multiple conformations, no?

JPK

On Tue, Mar 19, 2013 at 11:34 AM, Phoebe A. Rice  wrote:

> Hi Zbyszek,
>   If the issue is perfect twinning, I agree - good point!
>   But you don't want to confuse people who simply have
> nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit
> pedagogical here, but a lot of newbies read the BB).  We had a case of P31
> that was so close to P61 we actually solved the molecular replacement
> problem in P61, then expanded it back and re-rigid-bodied it.  We've played
> similar games with translational pseudo-symmetry (ignoring the weak spots
> at first).  In cases like that it is important to properly reprocess the
> data in the lower symmetry space group (or smaller unit cell) because there
> is real information in those small differences.  However, the point about
> Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags
> should be expanded by the xtal symmetry operators, not re-picked in the
> lower symmetry space group.
>Phoebe
>
> ++
>
> Phoebe A. Rice
> Dept. of Biochemistry & Molecular Biology
> The University of Chicago
> 773 834 1723; pr...@uchicago.edu
> http://bmb.bsd.uchicago.edu/Faculty_and_Research/
> http://www.rsc.org/shop/books/2008/9780854042722.asp
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek
> Otwinowski [zbys...@work.swmed.edu]
> Sent: Tuesday, March 19, 2013 9:37 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree
>
> It is a clear-cut case of crystal packing disorder. The tell-tale sign is
> that data can be merged in the higher-symmetry lattice, while the number
> of molecules in the asymmetric unit (3 in P21) is not divisible by the
> higher symmetry factor (2, by going from P21 to P21212).
> From my experience, this is more likely a case of order-disorder than
> merohedral twinning. The difference between these two is that structure
> factors are added for the alternative conformations in the case of
> order-disorder, while intensities (structure factors squared) are added in
> the case of merohedral twinning.
>
> Now an important comment on how to proceed in the cases where data can be
> merged in a higher symmetry, but the structure needs to be solved in a
> lower symmetry due to a disorder.
>
> !Such data needs to be merged in the higher symmetry,assigned R-free flag,
> and THEN expanded to the lower symmetry. Reprocessing the data in a lower
> symmetry is an absolutely wrong procedure and it will artificially reduce
> R-free, as the new R-free flags will not follow data symmetry!
>
> Moreover, while this one is likely to be a case of order-disorder, and
> these are infrequent, reprocessing the data in a lower symmetry seems to
> be frequently abused, essentially in order to reduce R-free. Generally,
> when data CAN be merged in a higher symmetry, the only proper procedure in
> going to a lower-symmetry structure is by expanding these higher-symmetry
> data to a lower symmetry, and not by rescaling and merging the data in a
> lower symmetry.
>
> Zbyszek Otwinowski
>
> > Dear all,
> > We have solved the problem. Data processing in P1 looks better (six
> > molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
> > in
> > ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
> > of refinement (without put waters, ligands, etc.).
> >
> > Indeed, there were one more molecule in ASU, but the over-merged data in
> > an orthorhombic lattice hid the correct solution.
> >
> > Thank you very much for all your suggestions, they were very important to
> > solve this problem.
> >
> > Cheers,
> >
> > Andrey
> >
> > 2013/3/15 Andrey Nascimento 
> >
> >> *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

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

[Phoebe A. Rice]

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is
that data can be merged in the higher-symmetry lattice, while the number
of molecules in the asymmetric unit (3 in P21) is not divisible by the
higher symmetry factor (2, by going from P21 to P21212).
>From my experience, this is more likely a case of order-disorder than
merohedral twinning. The difference between these two is that structure
factors are added for the alternative conformations in the case of
order-disorder, while intensities (structure factors squared) are added in
the case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be
merged in a higher symmetry, but the structure needs to be solved in a
lower symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag,
and THEN expanded to the lower symmetry. Reprocessing the data in a lower
symmetry is an absolutely wrong procedure and it will artificially reduce
R-free, as the new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and
these are infrequent, reprocessing the data in a lower symmetry seems to
be frequently abused, essentially in order to reduce R-free. Generally,
when data CAN be merged in a higher symmetry, the only proper procedure in
going to a lower-symmetry structure is by expanding these higher-symmetry
data to a lower symmetry, and not by rescaling and merging the data in a
lower symmetry.

Zbyszek Otwinowski

> Dear all,
> We have solved the problem. Data processing in P1 looks better (six
> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
> in
> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
> of refinement (without put waters, ligands, etc.).
>
> Indeed, there were one more molecule in ASU, but the over-merged data in
> an orthorhombic lattice hid the correct solution.
>
> Thank you very much for all your suggestions, they were very important to
> solve this problem.
>
> Cheers,
>
> Andrey
>
> 2013/3/15 Andrey Nascimento 
>
>> *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

Re: [ccp4bb] Philosophical question

[Jacob Keller]

I never said QWERTY "just happened--" I said it was an "accident of
history," based on the belief that some people nowadays have stopped using
manual typewriters, and they nevertheless still use the QWERTY keyboard.
I.e., because of the way history unfolded, we are now locked into using a
non-ideal keyboard configuration. I am dubious whether this model, however,
would apply to the codon conventions.

Jacob

On Tue, Mar 19, 2013 at 10:44 AM, David Schuller  wrote:

>  On 03/19/13 10:34, Jacob Keller wrote:
>
> Never one to shrink from philosophizing, I wonder generally why the codon
> conventions are the way they are? Is it like the QWERTY keyboard--basically
> an historical accident-
>
>
> QWERTY didn't "just happen." It was designed. Don't kids today know how to
> use Wikipedia or Google?
>
> http://en.wikipedia.org/wiki/QWERTY
>
> "Still used to this day, the QWERTY layout was devised and created in the
> early 1870s by Christopher Latham 
> Sholes,
> a newspaper  editor and printer
> who lived in Milwaukee ...
> The solution was to place commonly used letter-pairs (like "th" or "st")
> so that their typebars were not neighboring, avoiding jams. Contrary to
> popular belief, the QWERTY layout was not designed to slow the typist down,
> [5] , but rather to
> speed up typing by preventing 
> jams.
> "
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>


-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***

Re: [ccp4bb] Philosophical question

[David Schuller]

On 03/19/13 10:34, Jacob Keller wrote:
Never one to shrink from philosophizing, I wonder generally why the 
codon conventions are the way they are? Is it like the QWERTY 
keyboard--basically an historical accident-


QWERTY didn't "just happen." It was designed. Don't kids today know how 
to use Wikipedia or Google?


http://en.wikipedia.org/wiki/QWERTY

"Still used to this day, the QWERTY layout was devised and created in 
the early 1870s by Christopher Latham Sholes 
, a newspaper 
 editor and printer who lived in 
Milwaukee ...
The solution was to place commonly used letter-pairs (like "th" or "st") 
so that their typebars were not neighboring, avoiding jams. Contrary to 
popular belief, the QWERTY layout was not designed to slow the typist 
down,^[5]  , but rather 
to speed up typing by preventing 
jams.^ "


--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] Philosophical question

[Bart Hazes]

Just search for genetic code evolution in pubmed and you will find tons of
literature on it. The main driving force appears to have been to minimize
physico-chemical changes in amino acid properties for frequent mutations.
In other words, if you take mutation rates at the single-nucleotide level
and use it to predict, via a codon table, the rates of amino acid mutations
you will find that it correlates strongly with the observed amino acid
rates.

Bart

On Tue, Mar 19, 2013 at 8:34 AM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:

> Never one to shrink from philosophizing, I wonder generally why the codon
> conventions are the way they are? Is it like the QWERTY keyboard--basically
> an historical accident--or is there some more beautiful reason? One might
> argue that since basically all organisms share the convention (are there
> exceptions, even?), that it must be the "best of all possible" conventions.
> I have often wondered whether maybe this particular convention allows for
> the most effective pathways between proteins of significant function, e.g.,
> through the fewest mutations perhaps? One certainly cannot maintain that
> every possible protein sequence has been made at some time or another in
> the history of the biological world (go quantitate!) so there must be a way
> to ensure that mostly the "best" ones got made. On the other hand, since
> many organisms share DNA, maybe they had to "agree" on a system (I think
> this is the dogma?). Was there a "United Organisms" convention at some
> point, reminiscent of "Les Immortels" of the French language or POSIX or
> something, to ensure compliance? What was the penalty for non-compliance?
>
> Anyway, I like the question about the methionines,
>
> Jacob
>
> On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry wrote:
>
>> Opher Gileadi wrote:
>>
>>> Hi Theresa,
>>>
>>> To add to Anat's comments: Although the AUG codon for the first
>>> methionine and all other methionines in a protein coding sequence look the
>>> same, they are read in a very different way by the ribosomal machinery. The
>>> first AUG is recognized by the initiation complex, which includes the
>>> separate small ribosomal subunit (40s), a special tRNA-methionine, and
>>> initiation factors (proteins) including eIF2. This leads to assembly of a
>>> complete ribosome and initiation of protein synthesis. Subsequently, in the
>>> process of elongation, AUG codons are read by a different tRNA, which is
>>> brought to the 80s ribosome bound to a protein called elongation factor 1a.
>>> This is an oversimplification, of course, but the point is that the
>>> initiation codon (=the first amino acid to be incorporated to the protein)
>>> is read by a special tRNA, hence the universal use of methionine.
>>>
>>> Opher
>>>
>>>  Yes, but why methionine? Half the time it has to be removed by
>> N-terminal peptidase to give a small first residue, or by leader sequence
>> processing. Why use a big expensive amino acid instead of choosing one of
>> the glycine codons? Is there an obvious reason, or just "it had to be
>> something, and Met happened to get selected"?
>>
>> And why sometimes alternate start codons can be used? and why doesn't
>> initiation occur also at methionines in the middle of proteins? I'm
>> guessing it has to do with 5' untranslated region and ribosome binding
>> sites. So could the start codon actually be anything you want, provided
>> there is a strong ribosome binding site there?
>>
>> Just being philosophical, and not afraid to display my ignorance,
>> eab
>>
>
>
>
> --
> ***
>
> Jacob Pearson Keller, PhD
>
> Looger Lab/HHMI Janelia Farms Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> email: kell...@janelia.hhmi.org
>
> ***
>



-- 

Bart Hazes
Associate Professor
Dept. of Medical Microbiology & Immunology
University of Alberta

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

[Zbyszek Otwinowski]

It is a clear-cut case of crystal packing disorder. The tell-tale sign is
that data can be merged in the higher-symmetry lattice, while the number
of molecules in the asymmetric unit (3 in P21) is not divisible by the
higher symmetry factor (2, by going from P21 to P21212).
>From my experience, this is more likely a case of order-disorder than
merohedral twinning. The difference between these two is that structure
factors are added for the alternative conformations in the case of
order-disorder, while intensities (structure factors squared) are added in
the case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be
merged in a higher symmetry, but the structure needs to be solved in a
lower symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag,
and THEN expanded to the lower symmetry. Reprocessing the data in a lower
symmetry is an absolutely wrong procedure and it will artificially reduce
R-free, as the new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and
these are infrequent, reprocessing the data in a lower symmetry seems to
be frequently abused, essentially in order to reduce R-free. Generally,
when data CAN be merged in a higher symmetry, the only proper procedure in
going to a lower-symmetry structure is by expanding these higher-symmetry
data to a lower symmetry, and not by rescaling and merging the data in a
lower symmetry.

Zbyszek Otwinowski

> Dear all,
> We have solved the problem. Data processing in P1 looks better (six
> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
> in
> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
> of refinement (without put waters, ligands, etc.).
>
> Indeed, there were one more molecule in ASU, but the over-merged data in
> an orthorhombic lattice hid the correct solution.
>
> Thank you very much for all your suggestions, they were very important to
> solve this problem.
>
> Cheers,
>
> Andrey
>
> 2013/3/15 Andrey Nascimento 
>
>> *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*
>>
>


Zbyszek Otwinowski
UT Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390-8816
Tel. 214-645-6385
Fax. 214-645-6353

Re: [ccp4bb] Philosophical question

[Jacob Keller]

Never one to shrink from philosophizing, I wonder generally why the codon
conventions are the way they are? Is it like the QWERTY keyboard--basically
an historical accident--or is there some more beautiful reason? One might
argue that since basically all organisms share the convention (are there
exceptions, even?), that it must be the "best of all possible" conventions.
I have often wondered whether maybe this particular convention allows for
the most effective pathways between proteins of significant function, e.g.,
through the fewest mutations perhaps? One certainly cannot maintain that
every possible protein sequence has been made at some time or another in
the history of the biological world (go quantitate!) so there must be a way
to ensure that mostly the "best" ones got made. On the other hand, since
many organisms share DNA, maybe they had to "agree" on a system (I think
this is the dogma?). Was there a "United Organisms" convention at some
point, reminiscent of "Les Immortels" of the French language or POSIX or
something, to ensure compliance? What was the penalty for non-compliance?

Anyway, I like the question about the methionines,

Jacob

On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry  wrote:

> Opher Gileadi wrote:
>
>> Hi Theresa,
>>
>> To add to Anat's comments: Although the AUG codon for the first
>> methionine and all other methionines in a protein coding sequence look the
>> same, they are read in a very different way by the ribosomal machinery. The
>> first AUG is recognized by the initiation complex, which includes the
>> separate small ribosomal subunit (40s), a special tRNA-methionine, and
>> initiation factors (proteins) including eIF2. This leads to assembly of a
>> complete ribosome and initiation of protein synthesis. Subsequently, in the
>> process of elongation, AUG codons are read by a different tRNA, which is
>> brought to the 80s ribosome bound to a protein called elongation factor 1a.
>> This is an oversimplification, of course, but the point is that the
>> initiation codon (=the first amino acid to be incorporated to the protein)
>> is read by a special tRNA, hence the universal use of methionine.
>>
>> Opher
>>
>>  Yes, but why methionine? Half the time it has to be removed by
> N-terminal peptidase to give a small first residue, or by leader sequence
> processing. Why use a big expensive amino acid instead of choosing one of
> the glycine codons? Is there an obvious reason, or just "it had to be
> something, and Met happened to get selected"?
>
> And why sometimes alternate start codons can be used? and why doesn't
> initiation occur also at methionines in the middle of proteins? I'm
> guessing it has to do with 5' untranslated region and ribosome binding
> sites. So could the start codon actually be anything you want, provided
> there is a strong ribosome binding site there?
>
> Just being philosophical, and not afraid to display my ignorance,
> eab
>



-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***

Re: [ccp4bb] Philosophical question

[Edward A. Berry]

Opher Gileadi wrote:

Hi Theresa,

To add to Anat's comments: Although the AUG codon for the first methionine and 
all other methionines in a protein coding sequence look the same, they are read 
in a very different way by the ribosomal machinery. The first AUG is recognized 
by the initiation complex, which includes the separate small ribosomal subunit 
(40s), a special tRNA-methionine, and initiation factors (proteins) including 
eIF2. This leads to assembly of a complete ribosome and initiation of protein 
synthesis. Subsequently, in the process of elongation, AUG codons are read by a 
different tRNA, which is brought to the 80s ribosome bound to a protein called 
elongation factor 1a. This is an oversimplification, of course, but the point 
is that the initiation codon (=the first amino acid to be incorporated to the 
protein) is read by a special tRNA, hence the universal use of methionine.

Opher

Yes, but why methionine? Half the time it has to be removed by N-terminal peptidase to give a small first residue, or by 
leader sequence processing. Why use a big expensive amino acid instead of choosing one of the glycine codons? Is there 
an obvious reason, or just "it had to be something, and Met happened to get selected"?


And why sometimes alternate start codons can be used? and why doesn't initiation occur also at methionines in the middle 
of proteins? I'm guessing it has to do with 5' untranslated region and ribosome binding sites. So could the start codon 
actually be anything you want, provided there is a strong ribosome binding site there?


Just being philosophical, and not afraid to display my ignorance,
eab

Re: [ccp4bb] DNA Restraints or Cif file for Phenix refinement

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Yurong,

the three letter code ABN stands for BENZYLAMINE. Do you know how this
made it into your PDB-file? It's rather unlikely coot put it there by
"the simple mutate of nucleic acids", and more likely that coot's
"simple mutate" actually IS compatible with Phenix.

Best wishes,
Tim


On 03/19/2013 02:15 PM, Yurong Wen wrote:
> Dear All, Recently I generated a dataset of DNA protein complex. I
> built the DNA model with the simple mutate of nucleic acids,
> however when I tried to refine my structure, I came to the fatal
> error below with unknown bonded energy atom. (Similar to the phenix
> Tutorial 10: Generating ligand coordinates and restraints for
> structure refinement): Number of atoms with unknown nonbonded
> energy type symbols: 16 " N   ABN A 246 " " C   ABN A 246 " " C1
> ABN A 246 " " C2  ABN A 246 " " C3  ABN A 246 " " C4  ABN A 246 " "
> C5  ABN A 246 " " C6  ABN A 246 " " N   ABN A 247 " " C   ABN A 247
> " Further, Phenix.ready and Phenix.eLBOW were used to generated the
> restraints information, however nothing extra restraints
> information was got phenix.eLBOW. I assumed that the phenix.eLBOW
> recognized the nucleic acids from the coot “simple mutate” as
> compatible with Phenix but still I can’t use phenix.refine to
> refine this model.
> 
> So how can I force the phenix.eLBOW to generate the restraints? or
> is there any other software could be used to generate the cif
> file?
> 
> Many thanks, Yurong

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

GPG Key ID = A46BEE1A
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[ccp4bb] DNA Restraints or Cif file for Phenix refinement

[Yurong Wen]

Dear All,
Recently I generated a dataset of DNA protein complex.
I built the DNA model with the simple mutate of nucleic acids, however when I 
tried to refine my structure, I came to the fatal error below with unknown 
bonded energy atom.
(Similar to the phenix Tutorial 10: Generating ligand coordinates and 
restraints for structure refinement):
Number of atoms with unknown nonbonded energy type symbols: 16
" N   ABN A 246 "
" C   ABN A 246 "
" C1  ABN A 246 "
" C2  ABN A 246 "
" C3  ABN A 246 "
" C4  ABN A 246 "
" C5  ABN A 246 "
" C6  ABN A 246 "
" N   ABN A 247 "
" C   ABN A 247 "
Further, Phenix.ready and Phenix.eLBOW were used to generated the restraints 
information, however nothing extra restraints information was got phenix.eLBOW.
I assumed that the phenix.eLBOW recognized the nucleic acids from the coot 
“simple mutate” as compatible with Phenix but still I can’t use phenix.refine 
to refine this model.

So how can I force the phenix.eLBOW to generate the restraints? or is there any 
other software could be used to generate the cif file?

Many thanks,
Yurong

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

[Andrey Nascimento]

Dear all,
We have solved the problem. Data processing in P1 looks better (six
molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules in
ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
of refinement (without put waters, ligands, etc.).

Indeed, there were one more molecule in ASU, but the over-merged data in
an orthorhombic lattice hid the correct solution.

Thank you very much for all your suggestions, they were very important to
solve this problem.

Cheers,

Andrey

2013/3/15 Andrey Nascimento 

> *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*
>

[ccp4bb] Register for Conference on Myofibrillar Z-disk Structure and Dynamics

[Kristina Djinovic Carugo]

 Register now for the EMBL Conference on


 Myofibrillar Z-disk Structure and Dynamics


 Where? - EMBL Hamburg, Germany


 When? - Monday 14 - Thursday 17 October 2013

This conference aims to bring together world leading experts and young 
researchers from across the fields of sarcomeric proteins and muscle 
diseases interested in exchanging ideas, creating new collaborations and 
working together to advance research in these key areas.


This event covers five interconnecting themes:
1)  protein-protein interactions in the sarcomere - structure and 
function
2)  structure, conformational plasticity and mechanics of sarcomeric 
proteins

3)  signalling, dynamics, and development
4)  skeletal and cardiac muscle disease

For more information and to register, please go to:
http://www.embl-hamburg.de/training/events/2013/SSS13-01/index.html
We look forward to seeing you in Hamburg!

Kristina Djinovic Carugo, University of Vienna, Austria
Dieter Fürst, University of Bonn, Germany
Matthias Gautel, King's College London, United Kingdom
Matthias Wilmanns, EMBL-Hamburg, Germany



--
___

Kristina Djinovic-Carugo
Department of Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

e-mail:kristina.djino...@univie.ac.at
Phone: +43-1-4277-52203/52201
Mobile: +43-664-602 77-522 03
Fax: +43-1-4277-9522

Re: [ccp4bb] Philosophical question

[Opher Gileadi]

Hi Theresa,

To add to Anat's comments: Although the AUG codon for the first methionine and 
all other methionines in a protein coding sequence look the same, they are read 
in a very different way by the ribosomal machinery. The first AUG is recognized 
by the initiation complex, which includes the separate small ribosomal subunit 
(40s), a special tRNA-methionine, and initiation factors (proteins) including 
eIF2. This leads to assembly of a complete ribosome and initiation of protein 
synthesis. Subsequently, in the process of elongation, AUG codons are read by a 
different tRNA, which is brought to the 80s ribosome bound to a protein called 
elongation factor 1a. This is an oversimplification, of course, but the point 
is that the initiation codon (=the first amino acid to be incorporated to the 
protein) is read by a special tRNA, hence the universal use of methionine.

Opher

[ccp4bb] postdoc vacancy

[Nicola G A Abrescia]

Dear All,



We have a postdoc vacancy in our group [Abrescia Lab. at the CIC bioGUNE


in Bilbao-Spain  www.cicbiogune.es ;

http://www.ikerbasque.net/nicola.abrescia]
that we would like to cover with a candidate jointly applying for
extra-mural fellowship (national and international).

Our group uses both X-ray crystallography and cryo-EM to determine the
structures of viruses and large macromolecular complexes.

Potential candidates should possess a recent PhD/DPhil in a relevant
subject and
be highly interested or competent in X-ray crystallography and/or
electron microscopy with particular emphasis on hybrid methods.
Experience in molecular biology and biochemistry (cloning, protein
purification etc.) is a plus.

For informal enquiries please email: 
nabres...@cicbiogune.es



To apply: Please send as a PDF your current CV including a brief
description of research interests and accomplishments, and the names and
addresses of two referees by email directly to
 nabres...@cicbiogune.es





Best wishes,

Nicola Abrescia







Dr. Nicola G. A. Abrescia

Basque Foundation for Science (Ikerbasque)

Structural Biology Unit

CIC bioGUNE

Bizkaia Technology Park, Bld 800

48160 Derio - Spain



Tel. +34 946 572 523 (ext. 307)

http://www.cicbiogune.es/secciones/investigacion/miembros.php?idioma=en&;
miembro=163&unidad=5&subgrupo=51













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