Re: [ccp4bb] Problem with installation in Fedora 25

[Murpholino Peligro]

I guess it was a problem with the directories name. (Changed the locale
when downloading... Did it all over again and it works).


2017-05-09 16:21 GMT-05:00 Murpholino Peligro :

> The installation process remains here
> ...(attached image)
>
> Is it doing something?
> Is there a way to solve this?
>
> Ps `uname -r` results in  `4.10.13-200.fc25.x86_64`
> Ps #2 This is a brand new Fedora installation. (tcsh installed)
>
>
>  x  x
>  ___
>
> Thanks
>

[ccp4bb] postdoctoral position available

[Tom Peat]

Dear Board Readers,

For anyone who might be interested, or know of interested persons, there is a 
postdoctoral position available at CSIRO:

https://career10.successfactors.com/sfcareer/jobreqcareer?jobId=39270=CSIRO

Best regards,  tom

[ccp4bb] Postdoc Position at Stockholm University, Sweden

[David Drew]

Postdoctoral position in membrane protein structural biology 

The laboratory of David Drew at Stockholm University is seeking an enthusiastic 
postdoc with an interest in elucidating molecular mechanisms of brain transport 
proteins using biochemical and biophysical approaches. We use diverse 
techniques to address brain transporter function, and use both X-ray 
crystallography and cryoEM for structural studies. Our department is equipped 
with a Talos Artica and Titan-Krios/K2 EM microscopes and has excellent support 
staff. The candidate should either hold or expect to hold a Ph.D. in the area 
of biochemistry or neurochemistry, with expertise in either cryoEM or X-ray 
crystallography. Expertise in mammalian cell culturing, and expression and 
purification of protein complexes would be advantageous. The Postdoc position 
is initially for 2 years with a possibility of extension. Please visit the lab 
website (http://www.su.se/english/profiles/drew-1.181696 
) before you apply and send 
your CV with a brief research statement as well as 2-3 contacts for references 
to dd...@dbb.su.se  before the 10th June.

Thank you.

David Drew

Wallenberg Academy Fellow
Department of Biochemistry and Biophysics
Stockholm University
Sweden

Ph: +46-8-162295
http://www.su.se/profiles/drew-1.181696 

[ccp4bb] Postdoc Position at Fox Chase Cancer Center, Philadelphia

[Wu, Jinhua]

A postdoctoral position is available at Fox Chase Cancer Center (Philadelphia, 
PA) for a highly motivated individual in the field of protein biochemistry, 
cell signaling, or structural biology.

The lab aims to understand the molecular basis for the cell signaling events 
that affect cell motility. The candidate should have strong experiences in 
protein biochemistry, molecular biology, and structural biology. Prior 
experiences in mammalian cell culture and confocal imaging are desired but not 
required. The candidate should obtain a Ph.D. degree in a related field in the 
last three years.

Fox Chase Cancer Center is a long-standing NCI-designated Comprehensive Cancer 
Center with multidisciplinary research interests from basic biological sciences 
to drug development and clinical studies. The research institute at FCCC is one 
of the top-ranked work places for postdoctoral researchers. It offers an 
outstanding research environment and strong support for career development.

Please send a cover letter describing previous research experience and current 
interests, an updated CV, and a list of three references to: 
Jinhua.wu(at)fccc.edu.


CONFIDENTIALITY NOTICE: This email communication may contain private, 
confidential, or legally privileged information intended for the sole use of 
the designated and/or duly authorized recipient(s). If you are not the intended 
recipient or have received this email in error, please notify the sender 
immediately by email and permanently delete all copies of this email including 
all attachments without reading them. If you are the intended recipient, secure 
the contents in a manner that conforms to all applicable state and/or federal 
requirements related to privacy and confidentiality of such information.

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

[ccp4bb] Staff Scientist Position in Vienna

[Kristina Djinovic Carugo]

**

*

*

*Staff scientist position*is available in the research group of Kristina 
Djinovic-Carugo in the Department for Structural and Computational 
Biology, Max. F. Perutz Laboratories at the University of Vienna, 
Austria. The main area of the group research is structural biology of 
F-actin based cytoskeleton, with main focus on striated muscle. We use 
integrative structural biology approach, combining various structural, 
molecular biology, biochemical and biophysical approaches.


The Department for Structural and Computational Biology is equipped with 
state of the art instrumentation/facilities to carry out all steps from 
cloning, protein expression and purification, to X-ray diffraction, 
biomolecular NMR, as well as for a series of biophysical and optical 
spectroscopy techniques. The group has access to major European 
synchrotron high brilliance X-ray sources where it employs MX and SAXS 
beamlines. The Department is located on Vienna Campus Biocenter, which 
is the largest life sciences hub in Austria. Vienna is frequently ranked 
the world’s best city to live in. It is a United Nations city with a 
large English-speaking community.


*Job Responsibilities*

The successful candidate will participate in and support structural 
biology research in Djinovic-Carugo group, will assist in the training 
and supervision of undergraduate, graduate students and postdocs and 
will have some responsibilities in lab management (in particular 
operation and management of X-ray crystallography equipment) with help 
of a technical assistant and a senior staff scientist. The staff 
scientist will be involved in (but not limited to) conducting laboratory 
experiments related to all aspects and steps from sample preparation and 
characterisation to crystal growth, X-ray diffraction and solution 
scattering, structure solution, analysis and interpretation of 
scientific data, collaboration with other scientists and dissemination 
at external events. S/he will be able to develop her/his own projects 
primarily in the field of structural biology of cytoskeleton.


*Qualifications and experience*

We are looking for a highly motivated candidate who is enthusiastic to 
work in an ambitious and multidisciplinary team and fits the following 
profile:


**

a Ph.D. in biochemistry, molecular biology, biophysics, chemistry or a 
related field is required. Minimum 3-5 years post-doctoral experience in 
molecular biology, protein expression in prokaryotic and eukaryotic 
expression systems, purification, crystallisation with robotics and 
macromolecular crystallography is a must. Proven experience in protein 
structure determination using X-ray crystallography and small angle 
X-ray scattering (SAXS) are needed. Hands-on experience with biophysical 
techniques and complementary structural biology and biophysical methods 
(EM, ITC, SLS, themofluor, XL-MS, FCS, FRET or BiFC) will be considered 
an advantage. Good communication skills, organizational ability, project 
management experience and capacity to interact with other researchers 
are essential. Demonstrated potential in acquiring supplementary funding 
and teaching experience will be taken as merits.


*Contract*:A contract for maximum of 6 years will be offered to the 
successful candidate.


*Starting date:***October -**November 2017

*Closing date:***21^st May 2017

**

*Interviews planned for*: Starting beginning of June 2017.

*Further information*from Kristina Djinovic-Carugo 
; tel.: + 43 1 4277 52203


*Application details*: if you want to join an exiting project area and 
become a member of our interdisciplinary team please compile an 
application package containing (one pdf file):


-Cover letter

-CV

-List of publications

-Detailed summary of research experience and interests

-Contact information of three professional references

and submit your application to *Kristina Djinovic Carugo *(to /e-mail: 
*admin.v...@univie.ac.at*/)


--
Department of Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Vienna Biocenter (VBC), Campus Vienna Biocenter 5
A-1030 Vienna
Austria


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


--
Department of Structural and Computational Biology
Max F. Perutz Laboratories
University of Vienna
Vienna Biocenter (VBC), Campus Vienna Biocenter 5
A-1030 Vienna
Austria


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

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 
>>  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]

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] BSA as additive

[Avinash Punekar]

Dear H.Sin,

These references may help you: 

Meireles, M., Aimar, P., and Sanchez, V. (2004) Albumin denaturation during
ultrafiltration: effects of operating conditions and consequences on membrane
fouling, Biotech. Bioeng. 38, 528-534.

Schratter, P., (2004) Purification and concentration by ultrafiltration, Meth. 
Mol.Biol.: Protein Purification Protocols 244, 101-116

Sweryda-Krawiec, B., Devaraj, H., Jacob, G., and Hickman, J. J. (2004) A new
interpretation of serum albumin surface passivation, Langmuir 20, 2054-2056.

Adding 1% BSA to your buffer and pre-treating the centricon diafiltration 
membrane does prevent aggregation and improves protein yield by several fold.

Additionally, adding 50 mM L-Arginine (pH’ed to buffer) prevented aggregation 
and produced nice protein crystals.

Best wishes,
Avinash

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 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.
>
> 

[ccp4bb] Job Advertisement

[Ross Robinson]

​

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Group. You will be part of a small research team that aims to improve our 
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[ccp4bb] prepare stock solution of hampton heavy atom screen kits

[Liuqing Chen]

Hello everyone!
  I want to prepare heavy atom stock solution to soaking  my crystal,  what 
should i use to soluble the heavy atom Pt, Hg and other hampton heavy atom 
screen kits,  as i know someone not good soluble in water?
  can anyone give me some ideas!
sincerely
Liuqing chen

[ccp4bb] SUMMARY: looking for paper describing optimisation of crystals using a screen kit as additive

[Sebastiano Pasqualato]

Dear all,
as usual the bulletin board nails it.
The paper I was vaguely remembering was indeed the following:

Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):646-50
Crystallization of foot-and-mouth disease virus 3C protease: surface 
mutagenesis and a novel crystal-optimization strategy.
Birtley JR, Curry S.

But of course, the bulletin board goes even further, providing other 
interesting hints and food for thoughts:

Structure. 2003 Sep;11(9):1061-70.
Enhancing protein crystallization through precipitant synergy.
Majeed S, Ofek G, Belachew A, Huang CC, Zhou T, Kwong PD.

Acta Crystallogr F Struct Biol Commun. 2014 Sep;70(Pt 9):1117-26
Microseed matrix screening for optimization in protein crystallization: what 
have we learned?
D'Arcy A, Bergfors T, Cowan-Jacob SW, Marsh M

Structure. 2017 Jan 3;25(1):107-120
Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy 
for Isoform-Specific Mechanical Protection.
Pernigo S, Fukuzawa A, Beedle AE, Holt M, Round A, Pandini A, Garcia-Manyes S, 
Gautel M, Steiner RA.

http://www2.mrc-lmb.cam.ac.uk/groups/JYL/WWWrobots/PDF/PresentationsOther/additive-screening.pdf
 


Thanks everyone for the feedback and help!
May the crystals grow happily and diffract nicely,
ciao,
Sebastiano


> On 8 May 2017, at 15:18, Sebastiano Pasqualato 
>  wrote:
> 
> 
> Dear all,
> I recall a paper (or was is a talk at a conference?) describing the 
> optimisation of initial hits of crystallisation by using a standard screen 
> kit as additive.
> Something like setting the tray using the initial crystallisation hit 
> condition in the reservoir and mixing 75% of the hit condition with 25% of a 
> commercial sparse matrix screen kit with the protein in the drop.
> I can’t find the reference, can anybody help me?
> Thanks a lot,
> ciao,
> Sebastiano
> 
> 
> -- 
> Sebastiano Pasqualato, PhD
> Crystallography Unit
> Department of Experimental Oncology
> European Institute of Oncology
> IFOM-IEO Campus
> via Adamello, 16
> 20139 - Milano
> Italy
> 
> tel +39 02 9437 5167
> fax +39 02 9437 5990
> web http://is.gd/IEOXtalUnit 

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] convert hkl data to mtz

[Phil Evans]

You can do this in the data reduction task in ccp4i2, which runs Pointless, 
Aimless and Ctruncate
Phil

> On 7 May 2017, at 23:10, Careina Edgooms 
> <02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi
> 
> I have not been in the game of solving structures for years. In the past I 
> used the program combat in ccp4 to convert my hkl output file from SAINT into 
> mtz file. Now I don't see combat anymore as an option in ccp4 programs and 
> none of the programs recognise my SAINT output file. Please help
> 
> Careina

Re: [ccp4bb] how to run shelx C/D/E using CCP4 GUI on windows

[George Sheldrick]

Just to confirm what Eleanor said: all SHELX programs prefer to be given 
intensities but can accept F if the intensities have been lost. 
Converting intensities to F (e.g. with ctruncate) and back again 
degrades the statistics for the weak reflections and is discouraged. The 
same applies to PHASER and other modern programs.


George


On 05/08/2017 01:19 PM, Eleanor Dodson wrote:
I cant say anything about Windowssystems - jobs are MEANT to run on 
either.


But re Intensities - you will have intensities in an mtz file 
somewhere - all data processing outputs both amplitudes and intensities..


As a rule the conversion for I to F and back does not change the 
stronger intensities much and again as a rule, the SHELX chain will 
work. However the intermediate conversions are best avoided if you 
still have intensities available

Eleanor

On 8 May 2017 at 10:57, chen c > wrote:


Hi everyone,

Can anybody tell how to run shelx C/D/E within CCP4 GUI on windows
system? Moreover, since shelx C/D/E within CCP4 using mtz file
(structure factor) instead of sca file (intensity), would this
matters in tough conditions?

Thank you!

Best regards
Chen




-- 
Cheng Chen, Ph.D. Candidate

Laboratory of Structural Biology
Life Science Building,Tsinghua University
Beijing 100084
China
Tel:+86-10-62772291 
Fax:+86-10-62773145 
E-mail:che...@xtal.tsinghua.edu.cn


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--
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Dept. Structural Chemistry,
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Fax. +49-551-39-22582