Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Actually this is taken care of in the BIOMOLECULE definition.
If the artist had used the principle biomolecule, it excludes the Fv fragments.

On 05/09/2017 01:08 PM, Edward A. Berry wrote:

In line with this, there are a number of pictures in the literature of the 
mitochondrial
electron transport chain, with the complexes lined up  in a row embedded in a 
membrane,
and with yeast complex III still having the Fv fragments it was crystallized 
with, attached.
Only obvious if you are familiar with the shape of the native structure, I 
guess.
Could have been avoided if the crystallographer had removed the Fv fragments 
before depositing,
but that would have been bad for the R-factors.

On 05/09/2017 12:49 PM, Ian Tickle wrote:


Hi Tristan

I'm not so sure.  The co-ordinates are the result of the experiment.  How other 
people choose to interpret those results is their affair.  Taking it to its 
logical conclusion suppose that we 'damage' the protein by mutating/deleting 
some residues or adding tags purely for the purpose of getting it to 
crystallise, do we report the structure of the protein as it is in the crystal, 
or do we report what it would have been if we hadn't messed with it ?  The 
choice is clear in that situation.

Cheers

-- Ian

On 9 May 2017 at 16:45, Tristan Croll > wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
model the structure as what it looks like after radiation damage has had its 
way with it, or what it must have looked like *before* the damage? I can see 
arguments both ways (and can sympathise with the former if you want to make 
radiation damage a subject of your manuscript), but this is going to lead to 
headaches for people who want to make use of the resulting coordinates to study 
the actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan


On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule 
(yes exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid  : "The hydration 
> equilibrium constant 
> 
at 25 °C is called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
> and ≈ 1.2×10^−3 in seawater 
>.^[6] 
> Hence, the majority of the carbon dioxide 
is not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

In line with this, there are a number of pictures in the literature of the 
mitochondrial
electron transport chain, with the complexes lined up  in a row embedded in a 
membrane,
and with yeast complex III still having the Fv fragments it was crystallized 
with, attached.
Only obvious if you are familiar with the shape of the native structure, I 
guess.
Could have been avoided if the crystallographer had removed the Fv fragments 
before depositing,
but that would have been bad for the R-factors.

On 05/09/2017 12:49 PM, Ian Tickle wrote:


Hi Tristan

I'm not so sure.  The co-ordinates are the result of the experiment.  How other 
people choose to interpret those results is their affair.  Taking it to its 
logical conclusion suppose that we 'damage' the protein by mutating/deleting 
some residues or adding tags purely for the purpose of getting it to 
crystallise, do we report the structure of the protein as it is in the crystal, 
or do we report what it would have been if we hadn't messed with it ?  The 
choice is clear in that situation.

Cheers

-- Ian

On 9 May 2017 at 16:45, Tristan Croll > wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
model the structure as what it looks like after radiation damage has had its 
way with it, or what it must have looked like *before* the damage? I can see 
arguments both ways (and can sympathise with the former if you want to make 
radiation damage a subject of your manuscript), but this is going to lead to 
headaches for people who want to make use of the resulting coordinates to study 
the actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan


On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule 
(yes exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid  : "The hydration 
> equilibrium constant 
> 
at 25 °C is called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
> and ≈ 1.2×10^−3 in seawater 
>.^[6] 
> Hence, the majority of the carbon dioxide 
is not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Tristan Croll]

A slightly different wrinkle on the perennial "do we model unresolved 
sidechains" debate, I guess. I would argue that in the case of mutations, tags 
etc. those are things you know were there before you started firing x-rays at 
your sample. In the case of the sidechain decarboxylation we know it's an 
artifact of the data collection method, and we correct for known artifacts in 
other contexts all the time.

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 9 May 2017, at 17:49, Ian Tickle  wrote:
> 
> 
> Hi Tristan
> 
> I'm not so sure.  The co-ordinates are the result of the experiment.  How 
> other people choose to interpret those results is their affair.  Taking it to 
> its logical conclusion suppose that we 'damage' the protein by 
> mutating/deleting some residues or adding tags purely for the purpose of 
> getting it to crystallise, do we report the structure of the protein as it is 
> in the crystal, or do we report what it would have been if we hadn't messed 
> with it ?  The choice is clear in that situation.
> 
> Cheers
> 
> -- Ian
> 
>> On 9 May 2017 at 16:45, Tristan Croll  wrote:
>> Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
>> model the structure as what it looks like after radiation damage has had its 
>> way with it, or what it must have looked like *before* the damage? I can see 
>> arguments both ways (and can sympathise with the former if you want to make 
>> radiation damage a subject of your manuscript), but this is going to lead to 
>> headaches for people who want to make use of the resulting coordinates to 
>> study the actual biology of your protein. Personally, I'd strongly prefer 
>> the latter approach.
>> 
>> Tristan
>> 
>> 
>>> On 2017-05-09 16:06, Edward A. Berry wrote:
 On 05/09/2017 06:18 AM, Ian Tickle wrote:
 We have seen almost identical density to Ed's for GLU side-chains, with 
 what looks like a linear molecule (yes exactly the size of CO2!) where the 
 carboxylate group would be and absolutely no density for the CG-CD bond.  
 So it's indeed very tempting to say that the CO2 is still there, and 
 presumably making the same H bonds that the carboxylate was making to hold 
 it there.  It would not be hydrated to carbonic acid, according to 
 https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration 
  equilibrium constant 
  at 25 °C is called 
 K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 
 1.7×10^−3 in pure water^[5] 
  and ≈ 
 1.2×10^−3 in seawater .^[6] 
  Hence, the 
 majority of the carbon dioxide is not converted into carbo
>>> n
>>> ic
 acid, remaining as CO_2 molecules.".
>>> 
>>> It looks like this ignores subsequent ionization of H2CO3 which would
>>> be quite spontaneous at neutral pH.  However the Wikipedia article
>>> also indicates the equilibrium is quite slow (which makes sense-
>>> otherwise why would carbonic anhydrase exist?) and it would be a great
>>> deal slower in vitreous ice at 100 K. Anyway, I had reached the same
>>> conclusion and have modeled a number of the troublesome glutamates as
>>> decarboxylated with CO2 hovering above. There is a problem that the
>>> remaining CG tends to push the CO2 a little out of the density in some
>>> cases, but not a severe clash and it may work itself out with further
>>> refinement or manual assistance.
>>> eab
> 

Re: [ccp4bb] peroxy-glutamate?

[Ian Tickle]

Hi Tristan

I'm not so sure.  The co-ordinates are the result of the experiment.  How
other people choose to interpret those results is their affair.  Taking it
to its logical conclusion suppose that we 'damage' the protein by
mutating/deleting some residues or adding tags purely for the purpose of
getting it to crystallise, do we report the structure of the protein as it
is in the crystal, or do we report what it would have been if we hadn't
messed with it ?  The choice is clear in that situation.

Cheers

-- Ian

On 9 May 2017 at 16:45, Tristan Croll  wrote:

> Hmm... this is a bit of a philosophical pickle in my mind. Do we want to
> model the structure as what it looks like after radiation damage has had
> its way with it, or what it must have looked like *before* the damage? I
> can see arguments both ways (and can sympathise with the former if you want
> to make radiation damage a subject of your manuscript), but this is going
> to lead to headaches for people who want to make use of the resulting
> coordinates to study the actual biology of your protein. Personally, I'd
> strongly prefer the latter approach.
>
> Tristan
>
>
> On 2017-05-09 16:06, Edward A. Berry wrote:
>
>> On 05/09/2017 06:18 AM, Ian Tickle wrote:
>>
>>> We have seen almost identical density to Ed's for GLU side-chains, with
>>> what looks like a linear molecule (yes exactly the size of CO2!) where the
>>> carboxylate group would be and absolutely no density for the CG-CD bond.
>>> So it's indeed very tempting to say that the CO2 is still there, and
>>> presumably making the same H bonds that the carboxylate was making to hold
>>> it there.  It would not be hydrated to carbonic acid, according to
>>> https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration <
>>> https://en.wikipedia.org/wiki/Hydrate> equilibrium constant <
>>> https://en.wikipedia.org/wiki/Equilibrium_constant> at 25 °C is called
>>> K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3
>>> in pure water^[5] >> /Carbonic_acid#cite_note-HS-5> and ≈ 1.2×10^−3 in seawater <
>>> https://en.wikipedia.org/wiki/Seawater>.^[6] <
>>> https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6> Hence, the
>>> majority of the carbon dioxide is not converted into carbo
>>>
>> n
>> ic
>>
>>> acid, remaining as CO_2 molecules.".
>>>
>>
>> It looks like this ignores subsequent ionization of H2CO3 which would
>> be quite spontaneous at neutral pH.  However the Wikipedia article
>> also indicates the equilibrium is quite slow (which makes sense-
>> otherwise why would carbonic anhydrase exist?) and it would be a great
>> deal slower in vitreous ice at 100 K. Anyway, I had reached the same
>> conclusion and have modeled a number of the troublesome glutamates as
>> decarboxylated with CO2 hovering above. There is a problem that the
>> remaining CG tends to push the CO2 a little out of the density in some
>> cases, but not a severe clash and it may work itself out with further
>> refinement or manual assistance.
>> eab
>>
>

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Hmm... this is a bit of a philosophical pickle in my mind.


I agree.
Right now I want as accurate a model as possible to improve the phases for 
interpretation of a few remaining bits. I haven't decided what to deposit- 
maybe three separate structures:
1. Conservatively modeled: Everything that I can't model is left unmodelled.
2. Speculative: every little green blob is filled with partial-occupancy water, 
methanol, ethanol, acetate/bicarbonate, isopropanol, glycerol, Tris, or PEG 
fragments. If peroxy-glutamate fits better, put it.
3. repaired model- rebuild the damaged glutamates, cycteines and methionines. Average the two 
heterotetramers in the asymmetric unit to make one BioMolecule, and do a few ps of molecular 
dynamics to eliminate crystallization artifacts. (Since this would now be a "solution 
structure" I wouldn't be expected to report R-factor or deposit diffraction data for this 
one). More likely it would be rejected as a "Model".

eab

On 05/09/2017 11:45 AM, Tristan Croll wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to model 
the structure as what it looks like after radiation damage has had its way with 
it, or what it must have looked like *before* the damage? I can see arguments 
both ways (and can sympathise with the former if you want to make radiation 
damage a subject of your manuscript), but this is going to lead to headaches 
for people who want to make use of the resulting coordinates to study the 
actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan

On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule (yes 
exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration  
equilibrium constant  at 25 °C is called K_h , which in 
the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
 and ≈ 1.2×10^−3 in seawater 
.^[6] 
 Hence, the majority of the carbon dioxide is 
not converted into car

b
o

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

I would think the first goal is to model the observed data correctly, and then 
afterwards an accurate "before" model could be inferred.

It seems that it would be extremely helpful to this end to add another column 
to the .pdb format: a "time constant" for radiation damage for each atom. When 
set to 0, there would be no decay (default, toggled off in refinement?), and 
negative and positive values could denote exponential decay (carboxyl) or 
appearance (CO2). But then, of course, one would also have to set up refinement 
to use unmerged data. But shouldn't this be done at some point anyway, now that 
we have the cyber-power to do it?

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tristan 
Croll
Sent: Tuesday, May 09, 2017 11:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to model 
the structure as what it looks like after radiation damage has had its way with 
it, or what it must have looked like *before* the damage? I can see arguments 
both ways (and can sympathise with the former if you want to make radiation 
damage a subject of your manuscript), but this is going to lead to headaches 
for people who want to make use of the resulting coordinates to study the 
actual biology of your protein. 
Personally, I'd strongly prefer the latter approach.

Tristan

On 2017-05-09 16:06, Edward A. Berry wrote:
> On 05/09/2017 06:18 AM, Ian Tickle wrote:
>> We have seen almost identical density to Ed's for GLU side-chains, 
>> with what looks like a linear molecule (yes exactly the size of CO2!) 
>> where the carboxylate group would be and absolutely no density for 
>> the CG-CD bond.  So it's indeed very tempting to say that the CO2 is 
>> still there, and presumably making the same H bonds that the 
>> carboxylate was making to hold it there.  It would not be hydrated to 
>> carbonic acid, according to 
>> https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration 
>> <https://en.wikipedia.org/wiki/Hydrate> equilibrium constant 
>> <https://en.wikipedia.org/wiki/Equilibrium_constant> at 25 °C is 
>> called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 
>> ] ≈ 1.7×10^−3 in pure water^[5] 
>> <https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5> and ≈
>> 1.2×10^−3 in seawater <https://en.wikipedia.org/wiki/Seawater>.^[6]
>> <https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6> Hence, 
>> the majority of the carbon dioxide is not converted into carbo
> n
> ic
>> acid, remaining as CO_2 molecules.".
> 
> It looks like this ignores subsequent ionization of H2CO3 which would 
> be quite spontaneous at neutral pH.  However the Wikipedia article 
> also indicates the equilibrium is quite slow (which makes sense- 
> otherwise why would carbonic anhydrase exist?) and it would be a great 
> deal slower in vitreous ice at 100 K. Anyway, I had reached the same 
> conclusion and have modeled a number of the troublesome glutamates as 
> decarboxylated with CO2 hovering above. There is a problem that the 
> remaining CG tends to push the CO2 a little out of the density in some 
> cases, but not a severe clash and it may work itself out with further 
> refinement or manual assistance.
> eab

Re: [ccp4bb] peroxy-glutamate?

[Tristan Croll]

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
model the structure as what it looks like after radiation damage has had 
its way with it, or what it must have looked like *before* the damage? I 
can see arguments both ways (and can sympathise with the former if you 
want to make radiation damage a subject of your manuscript), but this is 
going to lead to headaches for people who want to make use of the 
resulting coordinates to study the actual biology of your protein. 
Personally, I'd strongly prefer the latter approach.


Tristan

On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:
We have seen almost identical density to Ed's for GLU side-chains, 
with what looks like a linear molecule (yes exactly the size of CO2!) 
where the carboxylate group would be and absolutely no density for the 
CG-CD bond.  So it's indeed very tempting to say that the CO2 is still 
there, and presumably making the same H bonds that the carboxylate was 
making to hold it there.  It would not be hydrated to carbonic acid, 
according to https://en.wikipedia.org/wiki/Carbonic_acid : "The 
hydration  equilibrium constant 
 at 25 °C is 
called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] 
≈ 1.7×10^−3 in pure water^[5] 
 and ≈ 
1.2×10^−3 in seawater .^[6] 
 Hence, 
the majority of the carbon dioxide is not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule (yes 
exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration  
equilibrium constant  at 25 °C is called K_h , which in 
the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
 and ≈ 1.2×10^−3 in seawater 
.^[6] 
 Hence, the majority of the carbon dioxide is 
not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would be quite 
spontaneous at neutral pH.  However the Wikipedia article also indicates the 
equilibrium is quite slow (which makes sense- otherwise why would carbonic 
anhydrase exist?) and it would be a great deal slower in vitreous ice at 100 K. 
Anyway, I had reached the same conclusion and have modeled a number of the 
troublesome glutamates as decarboxylated with CO2 hovering above. There is a 
problem that the remaining CG tends to push the CO2 a little out of the density 
in some cases, but not a severe clash and it may work itself out with further 
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Ian Tickle]

Hi Jacob

On 9 May 2017 at 14:03, Keller, Jacob  wrote:

> Wouldn’t the not-bonded CO2 have a new steric clash with the CG, though?
>

I think the CG can easily swing out of the way, since it's now only
attached to the CB.


> And what happened to the radical that was presumably generated?
>

Good question: if the GLU was (-) charged as seems likely then the
bond-breaking X-ray photon would generate a fleeting -CH2(-) carbanion at
the CG, not a radical.  This would need to rapidly pick up an H+ from
somewhere - but there must be plenty of those around!


> Also, I would think solvent-exposed side chains would be more prone to
> diffusion than buried ones.
>

Agree but what matters is the absolute rate of diffusion, not the rate in
protein v. vitrified solvent.  If the CO2 is happy where it is why should
it diffuse away?  After all presumably the carboxylate was happy in that
position.

I admit this is all pure speculation, but our map is remarkably similar to
Ed's.

Cheers

-- Ian

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

Wouldn’t the not-bonded CO2 have a new steric clash with the CG, though? And 
what happened to the radical that was presumably generated?

Also, I would think solvent-exposed side chains would be more prone to 
diffusion than buried ones.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle
Sent: Tuesday, May 09, 2017 6:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?


Hi Andrew
We have seen almost identical density to Ed's for GLU side-chains, with what 
looks like a linear molecule (yes exactly the size of CO2!) where the 
carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably 
making the same H bonds that the carboxylate was making to hold it there.  It 
would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The 
hydration<https://en.wikipedia.org/wiki/Hydrate> equilibrium 
constant<https://en.wikipedia.org/wiki/Equilibrium_constant> at 25 °C is called 
Kh, which in the case of carbonic acid is [H2CO3]/[CO2] ≈ 1.7×10−3 in pure 
water[5]<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5> and ≈ 
1.2×10−3 in 
seawater<https://en.wikipedia.org/wiki/Seawater>.[6]<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6>
 Hence, the majority of the carbon dioxide is not converted into carbonic acid, 
remaining as CO2 molecules.".
Also, diffusion of hydrated HCl in crystalline (hexagonal) ice is apparently 
negligible at 110 K according to this paper: "Depth-Profiling and Diffusion 
Measurements in Ice Films Using Infrared Laser Resonant Desorption", F.E. 
Livingston, J.A. Smith & S.M. George, Anal. Chem., 2000, 72 (22), 5590–9,DOI: 
10.1021/ac000724t.  Quoting their observations: "at T = 110 K show that the HCl 
hydrate interlayer is initially well localized at t = 0.  The temperature of 
the H2O/ HCl/ H2O sandwich structure was then raised to T = 190 K for t = 120 s 
with a constant H2O backing pressure and subsequently cooled rapidly to ∼110 K 
to terminate further HCl diffusion.”.  Now of course measurements of hydrated 
HCl in crystalline ice may have absolutely no relevance to CO2 in a protein and 
vitreous ice.  It's known that ions diffuse more rapidly in vitreous than 
crystalline ice because the diffusion mechanism requires 'hopping' between H2O 
vacancies and there are far fewer of these in crystalline ice.
Cheers
-- Ian



On 4 May 2017 at 11:25, Andrew Leslie 
<and...@mrc-lmb.cam.ac.uk<mailto:and...@mrc-lmb.cam.ac.uk>> wrote:
Dear Ed,

  I find your electron density quite interesting, because generally 
(I think, I would be happy to be corrected on this) when de-carboxylation of 
Asp/Glu occurs due to radiation damage, there is no evidence of what happens to 
the resulting CO2 group. One interpretation of this is that it diffuses away 
from the side chain and is effectively totally disordered, so no electron 
density is seen, but I was surprised that this would always be the case, 
especially as I would have thought that diffusion would be quite limited at 
100K (maybe I’m wrong about that too, but that is supposed to be one reason why 
radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew


> On 3 May 2017, at 22:19, Edward A. Berry 
> <ber...@upstate.edu<mailto:ber...@upstate.edu>> wrote:
>
>
>
> On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
>> Dear Ed,
>>
>>  Have you considered the possibility that it could be a water
>> stepping in to fill the void created by partial decarboxylation of the
>> glutamate? That could be easily modelled, refined, and tested for its
>> ability to flatten the difference map.
>>
>>  Gerard.
>>
> Actually some of them do appear decarboxylated. Is that something that can 
> happen? In the crystal, or as radiation damage?
> However when there is density for the carboxylate (figure), it appears 
> continuous and linear, doesn't break up into spheres at H-bonding distance - 
> almost like the CO2 is still sitting there- but I guess it would get hydrated 
> to bicarbonate. I could use azide. Or maybe waters with some disorder.
> Thanks,
> eab
>
> Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> comparison, not part of the model.
>
> 

Re: [ccp4bb] peroxy-glutamate?

[Ian Tickle]

Hi Andrew

We have seen almost identical density to Ed's for GLU side-chains, with
what looks like a linear molecule (yes exactly the size of CO2!) where the
carboxylate group would be and absolutely no density for the CG-CD bond.
So it's indeed very tempting to say that the CO2 is still there, and
presumably making the same H bonds that the carboxylate was making to hold
it there.  It would not be hydrated to carbonic acid, according to
https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration
 equilibrium constant
 at 25 °C is called Kh,
which in the case of carbonic acid is [H2CO3]/[CO2] ≈ 1.7×10−3 in pure water
[5]  and ≈
1.2×10−3 in seawater .[6]
 Hence, the
majority of the carbon dioxide is not converted into carbonic acid,
remaining as CO2 molecules.".

Also, diffusion of hydrated HCl in crystalline (hexagonal) ice is
apparently negligible at 110 K according to this paper: "Depth-Profiling
and Diffusion Measurements in Ice Films Using Infrared Laser Resonant
Desorption", F.E. Livingston, J.A. Smith & S.M. George, Anal. Chem., 2000,
72 (22), 5590–9,*DOI: *10.1021/ac000724t.  Quoting their observations: "*at
T* = 110 K show that the HCl hydrate interlayer is initially well localized
at *t* = 0.  The temperature of the H2O/ HCl/ H2O sandwich structure was
then raised to *T* = 190 K for *t* = 120 s with a constant H2O backing
pressure and subsequently cooled rapidly to ∼110 K to terminate further HCl
diffusion.”.  Now of course measurements of hydrated HCl in crystalline ice
may have absolutely no relevance to CO2 in a protein and vitreous ice.
It's known that ions diffuse more rapidly in vitreous than crystalline ice
because the diffusion mechanism requires 'hopping' between H2O vacancies
and there are far fewer of these in crystalline ice.

Cheers

-- Ian



On 4 May 2017 at 11:25, Andrew Leslie  wrote:

> Dear Ed,
>
>   I find your electron density quite interesting, because
> generally (I think, I would be happy to be corrected on this) when
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no
> evidence of what happens to the resulting CO2 group. One interpretation of
> this is that it diffuses away from the side chain and is effectively
> totally disordered, so no electron density is seen, but I was surprised
> that this would always be the case, especially as I would have thought that
> diffusion would be quite limited at 100K (maybe I’m wrong about that too,
> but that is supposed to be one reason why radiation damage is less at 100K).
>
> If the residual density is due to partial de-carboxylation, then I would
> have expected density for the CG-CD bond, which is not present (at your
> chosen contour level).
>
> Do many of your Glu side chains have the residual density?
>
> Best wishes,
>
> Andrew
>
>
> > On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> >
> >
> >
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >>
> >>  Have you considered the possibility that it could be a water
> >> stepping in to fill the void created by partial decarboxylation of the
> >> glutamate? That could be easily modelled, refined, and tested for its
> >> ability to flatten the difference map.
> >>
> >>  Gerard.
> >>
> > Actually some of them do appear decarboxylated. Is that something that
> can happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears
> continuous and linear, doesn't break up into spheres at H-bonding distance
> - almost like the CO2 is still sitting there- but I guess it would get
> hydrated to bicarbonate. I could use azide. Or maybe waters with some
> disorder.
> > Thanks,
> > eab
> >
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown
> for comparison, not part of the model.
> >
> > 
>

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

As you suggest, it depends on the contour level. Looking through a list of 17 
dicarboxylates that I found problematic, there were 4 (actually two and their 
ncs-mates) that showed disconnected density for the carboxylate at 1.4 sigma as 
in the figure I sent. Going up to 2 sigma, four more became disconnected and 
linear (backbone density is continuous to 3.5 sigma). Going down to 0.6 sigma, 
disconnected residual density showed up for several more. So I guess there are 
varying degrees of decarboxylation, and varying extent of retention of the 
fragment.  I have the impression these are mostly on the protein/solvent 
boundary, which could explain their disorder, but perhaps would also expose 
them to greater concentration of radicals generated in the solvent channels(?).

Ed

On 05/04/2017 06:25 AM, Andrew Leslie wrote:

Dear Ed,

   I find your electron density quite interesting, because 
generally (I think, I would be happy to be corrected on this) when 
de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
evidence of what happens to the resulting CO2 group. One interpretation of this 
is that it diffuses away from the side chain and is effectively totally 
disordered, so no electron density is seen, but I was surprised that this would 
always be the case, especially as I would have thought that diffusion would be 
quite limited at 100K (maybe I’m wrong about that too, but that is supposed to 
be one reason why radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew



On 3 May 2017, at 22:19, Edward A. Berry  wrote:



On 05/03/2017 02:46 PM, Gerard Bricogne wrote:

Dear Ed,

  Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

  Gerard.


Actually some of them do appear decarboxylated. Is that something that can 
happen? In the crystal, or as radiation damage?
However when there is density for the carboxylate (figure), it appears 
continuous and linear, doesn't break up into spheres at H-bonding distance - 
almost like the CO2 is still sitting there- but I guess it would get hydrated 
to bicarbonate. I could use azide. Or maybe waters with some disorder.
Thanks,
eab

Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
comparison, not part of the model.

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

I have seen anomalous "flecks" bespangling an protein's internal cavity which 
had a couple of cysteines in it, and I assumed that these were liberated 
sulphurs (they were not Fourier-truncation-like.) I would agree with Andrew 
that diffusion should not be large, but alighting on the nearest perch should 
be possible, no? Attractive and repulsive forces remain just as strong at 100K, 
so although motions are not driven by thermal sampling or random-walking, they 
would still be driven by local forces.

Further, I would think that the likeliest local perches would appear as new 
sites, for the CO2 as well as for sulphurs.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of DUMAS 
Philippe (VIE)
Sent: Thursday, May 04, 2017 6:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?

 
Le Jeudi 4 Mai 2017 12:25 CEST, Andrew Leslie <and...@mrc-lmb.cam.ac.uk> a 
écrit: 

Dear Andrew,
We looked in details to this problem of diffusion at ca. 100 K with bromine in 
"Ennifar et al., Acta D58(2002)1262" and we concluded

"It was attempted to derive a value for the diffusion coefficient of the free 
bromine species (most likely Br-) in amorphous ice at 100±110 K. This failed 
because the diffusion was much too rapid compared with both the radiolysis and 
datacollection timescales to permit such a determination."

Best regards
Philippe Dumas
 
> Dear Ed,
> 
>   I find your electron density quite interesting, because 
> generally (I think, I would be happy to be corrected on this) when 
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
> evidence of what happens to the resulting CO2 group. One interpretation of 
> this is that it diffuses away from the side chain and is effectively totally 
> disordered, so no electron density is seen, but I was surprised that this 
> would always be the case, especially as I would have thought that diffusion 
> would be quite limited at 100K (maybe I’m wrong about that too, but that is 
> supposed to be one reason why radiation damage is less at 100K).
> 
> If the residual density is due to partial de-carboxylation, then I would have 
> expected density for the CG-CD bond, which is not present (at your chosen 
> contour level).
> 
> Do many of your Glu side chains have the residual density?
> 
> Best wishes,
> 
> Andrew
> 
> 
> > On 3 May 2017, at 22:19, Edward A. Berry <ber...@upstate.edu> wrote:
> > 
> > 
> > 
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >> 
> >>  Have you considered the possibility that it could be a water

> >> stepping in to fill the void created by partial decarboxylation of 
> >> the glutamate? That could be easily modelled, refined, and tested 
> >> for its ability to flatten the difference map.
> >> 
> >>  Gerard.
> >> 
> > Actually some of them do appear decarboxylated. Is that something that can 
> > happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears 
> > continuous and linear, doesn't break up into spheres at H-bonding distance 
> > - almost like the CO2 is still sitting there- but I guess it would get 
> > hydrated to bicarbonate. I could use azide. Or maybe waters with some 
> > disorder.
> > Thanks,
> > eab
> > 
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> > comparison, not part of the model.
> > 
> > 
 
 
 
 

Re: [ccp4bb] peroxy-glutamate?

[VIE]

Le Jeudi 4 Mai 2017 12:25 CEST, Andrew Leslie  a 
écrit:

Dear Andrew,
We looked in details to this problem of diffusion at ca. 100 K with bromine in 
"Ennifar et al., Acta D58(2002)1262" and we concluded

"It was attempted to derive a value for the diffusion coefficient of the free 
bromine species (most likely Br-) in amorphous ice at 100±110 K. This failed 
because the diffusion was much too rapid compared with both the radiolysis and 
datacollection timescales to permit such a determination."

Best regards
Philippe Dumas

> Dear Ed,
>
>   I find your electron density quite interesting, because 
> generally (I think, I would be happy to be corrected on this) when 
> de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
> evidence of what happens to the resulting CO2 group. One interpretation of 
> this is that it diffuses away from the side chain and is effectively totally 
> disordered, so no electron density is seen, but I was surprised that this 
> would always be the case, especially as I would have thought that diffusion 
> would be quite limited at 100K (maybe I’m wrong about that too, but that is 
> supposed to be one reason why radiation damage is less at 100K).
>
> If the residual density is due to partial de-carboxylation, then I would have 
> expected density for the CG-CD bond, which is not present (at your chosen 
> contour level).
>
> Do many of your Glu side chains have the residual density?
>
> Best wishes,
>
> Andrew
>
>
> > On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> >
> >
> >
> > On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >> Dear Ed,
> >>
> >>  Have you considered the possibility that it could be a water

> >> stepping in to fill the void created by partial decarboxylation of the
> >> glutamate? That could be easily modelled, refined, and tested for its
> >> ability to flatten the difference map.
> >>
> >>  Gerard.
> >>
> > Actually some of them do appear decarboxylated. Is that something that can 
> > happen? In the crystal, or as radiation damage?
> > However when there is density for the carboxylate (figure), it appears 
> > continuous and linear, doesn't break up into spheres at H-bonding distance 
> > - almost like the CO2 is still sitting there- but I guess it would get 
> > hydrated to bicarbonate. I could use azide. Or maybe waters with some 
> > disorder.
> > Thanks,
> > eab
> >
> > Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> > comparison, not part of the model.
> >
> > 

Re: [ccp4bb] peroxy-glutamate?

[Andrew Leslie]

Dear Ed,

  I find your electron density quite interesting, because generally 
(I think, I would be happy to be corrected on this) when de-carboxylation of 
Asp/Glu occurs due to radiation damage, there is no evidence of what happens to 
the resulting CO2 group. One interpretation of this is that it diffuses away 
from the side chain and is effectively totally disordered, so no electron 
density is seen, but I was surprised that this would always be the case, 
especially as I would have thought that diffusion would be quite limited at 
100K (maybe I’m wrong about that too, but that is supposed to be one reason why 
radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew


> On 3 May 2017, at 22:19, Edward A. Berry  wrote:
> 
> 
> 
> On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
>> Dear Ed,
>> 
>>  Have you considered the possibility that it could be a water
>> stepping in to fill the void created by partial decarboxylation of the
>> glutamate? That could be easily modelled, refined, and tested for its
>> ability to flatten the difference map.
>> 
>>  Gerard.
>> 
> Actually some of them do appear decarboxylated. Is that something that can 
> happen? In the crystal, or as radiation damage?
> However when there is density for the carboxylate (figure), it appears 
> continuous and linear, doesn't break up into spheres at H-bonding distance - 
> almost like the CO2 is still sitting there- but I guess it would get hydrated 
> to bicarbonate. I could use azide. Or maybe waters with some disorder.
> Thanks,
> eab
> 
> Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> comparison, not part of the model.
> 
> 

Re: [ccp4bb] peroxy-glutamate?

[Keller, Jacob]

Damage-Selective (DamSel) map?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bellini, 
Dom
Sent: Wednesday, May 03, 2017 6:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] peroxy-glutamate?


or RDM (raddam detection map), better known as raddamap? :)



BW,



D


From: CCP4 bulletin board <CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>> 
on behalf of Pavel Afonine <pafon...@gmail.com<mailto:pafon...@gmail.com>>
Sent: 03 May 2017 22:51:48
To: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
Subject: Re: [ccp4bb] peroxy-glutamate?

Dear Gerard,

I am sure others
are certain to propose a cooler name for that very same type of map
some day ;-) .

a free tip: how about DDM (Decarboxylation Detector Map)?

All the best,
Pavel

Re: [ccp4bb] peroxy-glutamate?

[Pavel Afonine]

Dear Gerard,

I am sure others
> are certain to propose a cooler name for that very same type of map
> some day ;-) .
>

a free tip: how about DDM (Decarboxylation Detector Map)?

All the best,
Pavel

Re: [ccp4bb] peroxy-glutamate?

[Gerard Bricogne]

Dear Ed,

 Thank you for the picture. The decarboxylation of GLU and ASP
side-chains is perhaps the most ubiquitous manifestation of radiation
damage. We have introduced a feature in our autoPROC processing
package whereby, if redundancy in the unmerged data is sufficient to
allow reasonably complete and non-overlapping "early" and "late"
data(sub)sets to be defined, we output "early-minus-late" difference
coefficients that can later be picked up by autoBUSTER to compute the
corresponding difference map. This is a great way of pin-pointing
acidic side-chains, and also sulphur-containing ones. I am sure others
are certain to propose a cooler name for that very same type of map
some day ;-) .

 Here, you seem to have a clear case of partial decarboxylation.
You could try reprocessing your images with autoPROC and computing an
early-minus-late map, just to see what it looks like. If your dataset
complies with the modern low-transmission, high-redundancy paradigm,
it should be very straightforward. You are welcome to get in touch
off-list about this.


 With best wishes,
 
  Gerard.

--
On Wed, May 03, 2017 at 05:19:25PM -0400, Edward A. Berry wrote:
> 
> 
> On 05/03/2017 02:46 PM, Gerard Bricogne wrote:
> >Dear Ed,
> >
> >  Have you considered the possibility that it could be a water
> >stepping in to fill the void created by partial decarboxylation of the
> >glutamate? That could be easily modelled, refined, and tested for its
> >ability to flatten the difference map.
> >
> >  Gerard.
> >
> Actually some of them do appear decarboxylated. Is that something that can 
> happen? In the crystal, or as radiation damage?
> However when there is density for the carboxylate (figure), it appears 
> continuous and linear, doesn't break up into spheres at H-bonding distance - 
> almost like the CO2 is still sitting there- but I guess it would get hydrated 
> to bicarbonate. I could use azide. Or maybe waters with some disorder.
> Thanks,
> eab
> 
> Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
> comparison, not part of the model.
> 

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

On 05/03/2017 02:46 PM, Gerard Bricogne wrote:

Dear Ed,

  Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

  Gerard.


Actually some of them do appear decarboxylated. Is that something that can 
happen? In the crystal, or as radiation damage?
However when there is density for the carboxylate (figure), it appears 
continuous and linear, doesn't break up into spheres at H-bonding distance - 
almost like the CO2 is still sitting there- but I guess it would get hydrated 
to bicarbonate. I could use azide. Or maybe waters with some disorder.
Thanks,
eab

Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
comparison, not part of the model.

Re: [ccp4bb] peroxy-glutamate?

[Gerard Bricogne]

Dear Ed,

 Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

 Gerard.

--
On Wed, May 03, 2017 at 02:29:11PM -0400, Edward A. Berry wrote:
> Thanks to all who replied. Yes, in some of the other cases density between Cd 
> and Cg is conspicuously weak. From preliminary tests it looks like dual 
> conformations, in some cases together with correlated waters, will account 
> for the density adequately. No Zebras here!
> eab
> 
> On 05/03/2017 04:26 AM, Matthew Merski wrote:
> >Have you tried just a double conformation of the Glu?  Its hard to tell in 
> >your pictures but it looks like there might be less than perfect density for 
> >the CG-CD bond?  If you just try adding a second conf of the Glu that might 
> >work too (and perhaps be a horse rather than a zebra making your hoofprints?)
> >
> >Matthew Merski
> >Crystallochemistry Laboratory
> >Univ. of Warsaw
> >
> >On Wed, May 3, 2017 at 6:29 AM, Edward A. Berry  >> wrote:
> >
> >I'm finishing up refinement of a 1.8A structure (R's 0.17, 0.20) , and 
> > among the largest peaks in the difference map are small spherical blobs 
> > that seem to be attached (1.46 A here) to carboxylate O's (Figures). Are 
> > these likely artifacts? If not, how can I interpret/model them? One idea is 
> > that the acid has reacted with peroxide from the PEG to make the 
> > (hydro)peroxy-acid. I don't know how stable that would be, and I don't see 
> > any peroxyglutamate in Ligand Depot or HIC-Up. Another guess would be acid 
> > hydroxamate but I don't know how that would be generated. Methyl ester 
> > seems to be ruled out by the proximity of the two water molecules (2.45 and 
> > 2.48 A here) suggesting the mystery atom is an H-bond acceptor or donor. 
> > However since the occupancy seems to be < 1, the waters may be there only 
> > when the atom is not.
> >I guess another possibility is there is a lot of motion in the plane of 
> > the carboxylate (up and down here) which cannot be modeled by my isotropic 
> > B-factors. In some cases the green blobs appear on both sides of the 
> > carboxylate (but that could also be alternate conformations of 
> > peroxyglutamate).
> >
> >The difference map (mFo-DFc, green) is contoured at 3 sigma (.06 
> > e-/A^3). The difference peak is 5.4 sigma (0.1 e/A3).
> >The 2mFo-DFc map is contoured at 1.5 sigma (0.1 e/A3). 2mFo-DFc density 
> > extends to the difference peak if I contour down at 0.64 sigma (0.04 e/A3, 
> > third figure).
> >
> >If I put an O atom there, link it with plenty of slack, and refine 
> > occupancy, it goes to 1.54 A from the carboxylate O and refines to 
> > occupancy 0.35, B-factor 15 (carboxylate O is 30). Now it is reached by 
> > 2mFo-DFc density at 1.5 sigma (0.1 e/A3).
> >Any suggestions would be welcome.
> >eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Thanks to all who replied. Yes, in some of the other cases density between Cd 
and Cg is conspicuously weak. From preliminary tests it looks like dual 
conformations, in some cases together with correlated waters, will account for 
the density adequately. No Zebras here!
eab

On 05/03/2017 04:26 AM, Matthew Merski wrote:

Have you tried just a double conformation of the Glu?  Its hard to tell in your 
pictures but it looks like there might be less than perfect density for the 
CG-CD bond?  If you just try adding a second conf of the Glu that might work 
too (and perhaps be a horse rather than a zebra making your hoofprints?)

Matthew Merski
Crystallochemistry Laboratory
Univ. of Warsaw

On Wed, May 3, 2017 at 6:29 AM, Edward A. Berry > wrote:

I'm finishing up refinement of a 1.8A structure (R's 0.17, 0.20) , and among 
the largest peaks in the difference map are small spherical blobs that seem to be 
attached (1.46 A here) to carboxylate O's (Figures). Are these likely artifacts? 
If not, how can I interpret/model them? One idea is that the acid has reacted with 
peroxide from the PEG to make the (hydro)peroxy-acid. I don't know how stable that 
would be, and I don't see any peroxyglutamate in Ligand Depot or HIC-Up. Another 
guess would be acid hydroxamate but I don't know how that would be generated. 
Methyl ester seems to be ruled out by the proximity of the two water molecules 
(2.45 and 2.48 A here) suggesting the mystery atom is an H-bond acceptor or donor. 
However since the occupancy seems to be < 1, the waters may be there only when 
the atom is not.
I guess another possibility is there is a lot of motion in the plane of the 
carboxylate (up and down here) which cannot be modeled by my isotropic 
B-factors. In some cases the green blobs appear on both sides of the 
carboxylate (but that could also be alternate conformations of peroxyglutamate).

The difference map (mFo-DFc, green) is contoured at 3 sigma (.06 e-/A^3). 
The difference peak is 5.4 sigma (0.1 e/A3).
The 2mFo-DFc map is contoured at 1.5 sigma (0.1 e/A3). 2mFo-DFc density 
extends to the difference peak if I contour down at 0.64 sigma (0.04 e/A3, 
third figure).

If I put an O atom there, link it with plenty of slack, and refine 
occupancy, it goes to 1.54 A from the carboxylate O and refines to occupancy 
0.35, B-factor 15 (carboxylate O is 30). Now it is reached by 2mFo-DFc density 
at 1.5 sigma (0.1 e/A3).
Any suggestions would be welcome.
eab

Re: [ccp4bb] peroxy-glutamate?

[Tristan Croll]

Peroxyacids are unlikely - they're very reactive/unstable under normal 
conditions. Is it possible your crystal is just at unusually low pH so these 
acids are protonated? That makes the carbon-oxygen bond lengths asymmetric, 
possibly by enough to explain your blobs.

Tristan

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 3 May 2017, at 05:29, Edward A. Berry  wrote:
> 
> I'm finishing up refinement of a 1.8A structure (R's 0.17, 0.20) , and among 
> the largest peaks in the difference map are small spherical blobs that seem 
> to be attached (1.46 A here) to carboxylate O's (Figures). Are these likely 
> artifacts? If not, how can I interpret/model them? One idea is that the acid 
> has reacted with peroxide from the PEG to make the (hydro)peroxy-acid. I 
> don't know how stable that would be, and I don't see any peroxyglutamate in 
> Ligand Depot or HIC-Up. Another guess would be acid hydroxamate but I don't 
> know how that would be generated. Methyl ester seems to be ruled out by the 
> proximity of the two water molecules (2.45 and 2.48 A here) suggesting the 
> mystery atom is an H-bond acceptor or donor. However since the occupancy 
> seems to be < 1, the waters may be there only when the atom is not.
> I guess another possibility is there is a lot of motion in the plane of the 
> carboxylate (up and down here) which cannot be modeled by my isotropic 
> B-factors. In some cases the green blobs appear on both sides of the 
> carboxylate (but that could also be alternate conformations of 
> peroxyglutamate).
> 
> The difference map (mFo-DFc, green) is contoured at 3 sigma (.06 e-/A^3). The 
> difference peak is 5.4 sigma (0.1 e/A3).
> The 2mFo-DFc map is contoured at 1.5 sigma (0.1 e/A3). 2mFo-DFc density 
> extends to the difference peak if I contour down at 0.64 sigma (0.04 e/A3, 
> third figure).
> 
> If I put an O atom there, link it with plenty of slack, and refine occupancy, 
> it goes to 1.54 A from the carboxylate O and refines to occupancy 0.35, 
> B-factor 15 (carboxylate O is 30). Now it is reached by 2mFo-DFc density at 
> 1.5 sigma (0.1 e/A3).
> Any suggestions would be welcome.
> eab
> 
> 
>