[ccp4bb] Probable bugs in acedrg

[Dale Tronrud]

Hi,

   I have been exploring a PDB model that contains an RNA molecule with
a ligand stuck on the 3' phosphate.  To perform real-space refinement in
Coot I needed to create a standard geometry CIF for the ligand and one
for the linkage between the ligand and an Adenine.  The CIF for the
ligand in CCP4 and the PDBe has (in my opinion) serious defects, but
with help from the masters at Global Phasing I was able to get a fine
set of restraints from the Grade server.

   To generate the linkage CIF I followed the very clear instructions at

https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/acedrg/acedrg.html

to use acedrg.  Long story short, one creates a one line description of
the link and feeds that into acedrg along with the two relevant CIFs and
Bob's your uncle.

   acedrg didn't work for me and I think I've uncovered two bugs that I
had to work-around to get my file.  I don't know who to report these
bugs to -- hence this letter.

   I typed the command to cause acedrg to produce my link, but it failed
saying (among other things)

The component A.cif contains a deloc or aromatic bond. It needs to be
kekulized.
You could use acedrg to do kekulication of the bonds.
Try getting A.cif from PDB/CCD and then use it as an input file
to run acedrg to generate a cif file for the ligand description.
e.g. You get the file A.cif from PDB/CCD and then,
acedrg -c A.cif -o A_fromAcedrg
...

The suggested second run of acedrg fails with the message (again truncated)

The system contains atoms of the following elements
P   O   C   N   H   "H5'
The input ligands/molecules contains metal or other heavier atoms
Acedrg currently deals with ligands/molecules with following elements only
C, N, O, S, P, B, F, Cl, Br, I, H

   There is no atom in A.cif from the CCP4 library with element "H5'.
It does contain an atom with the odd name "H5' ".  Knowing that many
programs have problems with embedded spaces in names, and
seeing no need for this embedded space, I changed the atom name and
tried again.  acedrg now completes the operation and writes out a new cif.

   This is bug #1.
   One might also consider the space in the name of this hydrogen atom
to be a bug, but A.cif is a widely used file and it works for it
principal purposes.

   Unfortunately, using the modified cif in the command for creating the
link still fails.  It appears that the kekulizer failed to kekulate.  To
investigate this kekulonic fault I examined the kekuleated cif and found
that the "delocalized" bonds of the phosphate were not affected by the
kekuliator.  I attempted to manually kekulinate it by changing these
bonds to a mixture of single and double bonds, with some trial and error
required to get an acceptable mix, and created a cif that was now
acceptably kekulinzed.  This file was used by acedrg and I got the link
I wanted.

   Bug #2 is the failure of acedrg's kekulinifier to remove all of the
delocalized bonds.

   I hope this note makes it to acedrg's developers and they find it
useful.  It may also be helpful to others attempting this task (at least
until the bugs are fixed).

Dale Tronrud



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Protein fold and the moonlighting function

[Peter Stogios]

I think you’re asking if two proteins with the same fold can have different 
activities.

As was previously mentioned, this is quite common.  The best characterized 
example is the TIM barrel fold - such proteins can be isomerases, transferases, 
hydrolases, lyases, or oxidoreductases, to name a few...



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Protein fold and the moonlighting function

[Jonathan Cooper]

 Hello, 
Re: "I think you’re asking about 2 proteins with a similar fold having similar 
but not exactly the same functions - yes, that does occur."
Indeed, I suspect it is very common, if not the norm. OK, so I now 
gather/remember moonlighting proteins are ones with more than one function - 
thank you for the link to the very interesting database. Is perhaps the 
questioner asking if homologues of a moonlighting protein also have similar 
moonlighting functions? If so, I'm out of my depth on that one.
Best wishes, Jon Cooper.On Sunday, 2 February 2020, 18:22:10 GMT, Jeffery, 
Constance J  wrote:  
 
 Hi Rajnandani,
I’m happy to help you with questions about moonlighting proteins.  My lab 
created the MoonProt Database (moonlightingproteins.org).  
I think you’re asking about 2 proteins with a similar fold having similar but 
not exactly the same functions - yes, that does occur.
Best regards,Connie Jeffery


On Feb 2, 2020, at 4:28 AM, Rajnandani Kashyap  
wrote:
Dear All
I am curious to know about the promiscuous activity of a protein based on their 
fold. Can a protein having same fold also have same function (say not 100% but 
some activity) as the homologous structure. 
Please also let me know few relevant PDB structures where such kind of activity 
is shown promising. 


Regards
Rajnandani Kashyap
PhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
  



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] Post-doctoral position in cryo-EM at IOCB Prague

[Sebastian Zoll]

Job description

A fellowship-funded postdoc position is open in Dr Sebastian Zoll’s group at 
the Institute of Organic Chemistry and Biochemsitry (IOCB) in Prague. Dr Zoll’s 
group is using a combination of biochemical, biophysical, immunological and 
structural biology techniques to understand the mechanisms of host-pathogen 
interactions. The succesful candidate will use Cryo-EM to determine the 
structure of a viral capsid protein in complex with its cellular receptor. As 
part of this process the applicant will generate monoclonal antibody and 
nanobody libraries as tools as well as for therapeutic applications.

 

Requirements

-PhD in structural biology using Cryo-EM as the main tool. Expertise in 
grid preparation, microscope operation and data processing in RELION is 
essential. Knowledge in X-ray crystallography and SAXS is a bonus but not 
necessary.

-Experience in expression construct design, protein purification and 
biophysical techniques to analyse protein-protein interactions (ITC or SPR). 

The project is supported by a multidisciplinary network of international 
collaborators and provides excellent opportunities to receive additional 
training in foreign host institutions.

The fellowship is granted for one year with possible extension by one more 
year. The earliest starting date is May 2020. For further information please 
see:

 

https://www.uochb.cz/en/iocb-fellowships 

 

The institute and the city

IOCB Prague is the leading scientific research institution in the Czech 
Republic providing a

dynamic, international environment that brings together researchers from 
various fields such as structural biology, molecular biology, medicinal and 
computational chemistry.

Due to its long-standing history in applied science and strong ties with the 
pharmaceutical industry, IOCB enjoys an excellent funding situation and offers 
access to facilities for all modern structural and biophysical methods.

The IOCB Cryo-EM facility housing a 300 keV high-resolution and 200 keV 
screening cryo-electron microscope will become operational next year. In the 
meanwhile IOCB researchers enjoy privileged access to the EM facilities 
(screening microscopes and Titan Krios G3) at CEITEC.

IOCB is situated near the city centre in a vibrating, historical quarter of 
Prague boasting beautiful architecture and a rich array of public amenities 
such as cafés, pubs, parks and grocery stores. Transport links by metro and 
tram are excellent with short distances to both airport as well as old town and 
Letna park with its stunning views over the Vltava river.

 

How to apply?

Please send a short motivation statement (max. 500 words) and a copy of your CV 
to sebastian.z...@uochb.cas.cz

Feel free to contact Sebastian for informal enquiries.

 

Dr Sebastian Zoll

Junior Group Leader

Institute of Organic Chemistry and Biochemistry

Czech Academy of Sciences

Flemingovo namesti 542/2, 166 10 Prague 6

Czech Republic

Phone: +420 220 183 591

Email: sebastian.z...@uochb.cas.cz

Facebook: https://www.facebook.com/IOCBPrague


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Mixed oligomeric states in crystallo

[Artem Evdokimov]

5FRT

We got scooped on this one a long time ago - but back in 2015 it was very
gratifying to see that the academic group who published this structure have
also obtained an ASU with two dimers and one monomer - same exact story as
what we saw. The difference can be attributed to the movement of a short
helix.

Nature is cruel, but interesting.

Artem

On Fri, Jan 31, 2020, 10:33 AM Kluenemann, Thomas <
thomas.kluenem...@helmholtz-hzi.de> wrote:

> Dear all,
>
>
>
> We recently solved a the structure of a small c-type cytochrome. We
> observed, that of the eleven chains in the asymmetric unit ten form 3D
> domain swapped dimers by exchanging an α-helix. The eleventh  chain is
> present as a monomer. Based on the anomalous iron signal and the chain
> tracing we are sure that no chain was missed.
>
> I tried to find other examples in the PDB, were one crystal is made of
> different homo- or heterooligomers.  I only found proteins with partial
> occupied peptide binding sites, which is not what I am looking for. Does
> anyone know of a case were the presence of different homo- or
> heterooligomers is required to form the crystal?
>
>
>
> Best regards,
>
> Thomas Klünemann
>
>
>
>
>
> --
>
> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124
> Braunschweig | www.helmholtz-hzi.de
>
> Vorsitzende des Aufsichtsrates: Frau MinDir'in Prof. Dr. Veronika von
> Messling
> Stellvertreter: MinDirig Rüdiger Eichel, Niedersächsisches Ministerium für
> Wissenschaft und Kultur
> Geschäftsführung: Prof. Dr. Dirk Heinz; Silke Tannapfel
> Gesellschaft mit beschränkter Haftung (GmbH)
> Sitz der Gesellschaft: Braunschweig
> Handelsregister: Amtsgericht Braunschweig, HRB 477
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Mixed oligomeric states in crystallo

[Jose Duarte]

Here's a few examples:

https://www.rcsb.org/structure/1A2K
The crystal has an A3B2 stoichiometry. The paper describes how they find
A2B2 in solution, concluding the extra A molecule comes from "fortuitous
packing" in the crystal

https://www.rcsb.org/structure/2RBL
A design protein, but with very similar crystal packing as described in the
original post: a domain-swapped dimer and a non-swapped monomer.

https://www.rcsb.org/structure/1A99
Both dimers and monomers in the crystal



On Fri, 31 Jan 2020 at 09:46, Diana Tomchick <
diana.tomch...@utsouthwestern.edu> wrote:

> Here’s two examples of heterooligomers that crystallized in a lattice with
> an extra monomer of one of the proteins. In both cases this was an
> unexpected result, but easily explained due to the low micro molar
> affinities for the complex.
>
> 4PKY
> 2CJS
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Jan 31, 2020, at 9:23 AM, Kluenemann, Thomas <
> thomas.kluenem...@helmholtz-hzi.de> wrote:
>
>
> EXTERNAL MAIL
>
> Dear all,
>
> We recently solved a the structure of a small c-type cytochrome. We
> observed, that of the eleven chains in the asymmetric unit ten form 3D
> domain swapped dimers by exchanging an α-helix. The eleventh  chain is
> present as a monomer. Based on the anomalous iron signal and the chain
> tracing we are sure that no chain was missed.
> I tried to find other examples in the PDB, were one crystal is made of
> different homo- or heterooligomers.  I only found proteins with partial
> occupied peptide binding sites, which is not what I am looking for. Does
> anyone know of a case were the presence of different homo- or
> heterooligomers is required to form the crystal?
>
> Best regards,
> Thomas Klünemann
>
>
>
> --
>
> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124
> Braunschweig | www.helmholtz-hzi.de
>
> Vorsitzende des Aufsichtsrates: Frau MinDir'in Prof. Dr. Veronika von
> Messling
> Stellvertreter: MinDirig Rüdiger Eichel, Niedersächsisches Ministerium für
> Wissenschaft und Kultur
> Geschäftsführung: Prof. Dr. Dirk Heinz; Silke Tannapfel
> Gesellschaft mit beschränkter Haftung (GmbH)
> Sitz der Gesellschaft: Braunschweig
> Handelsregister: Amtsgericht Braunschweig, HRB 477
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> CAUTION: This email originated from outside UTSW. Please be cautious of
> links or attachments, and validate the sender's email address before
> replying.
>
>
> --
>
> UT Southwestern
>
> Medical Center
>
> The future of medicine, today.
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Protein fold and the moonlighting function

[Jeffery, Constance J]

Hi Rajnandani,

I’m happy to help you with questions about moonlighting proteins.  My lab 
created the MoonProt Database 
(moonlightingproteins.org).

I think you’re asking about 2 proteins with a similar fold having similar but 
not exactly the same functions - yes, that does occur.

Best regards,
Connie Jeffery

On Feb 2, 2020, at 4:28 AM, Rajnandani Kashyap 
mailto:kashyap.rajnand...@gmail.com>> wrote:

Dear All

I am curious to know about the promiscuous activity of a protein based on their 
fold. Can a protein having same fold also have same function (say not 100% but 
some activity) as the homologous structure.

Please also let me know few relevant PDB structures where such kind of activity 
is shown promising.


Regards

Rajnandani Kashyap
PhD Student




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[Artem Evdokimov]

Thank you for the clarification!

First of all, many proteins will exhibit this sort of behavior when stored
in liquid form at moderately low temperature.

One solution is to store in glycerol (35 percent and up) at -20C. Or in
deep freeze at -80C or in LN2.

The one remaining question that would be useful to address is the degree of
homogeneity in your sample. Is 100% of your protein folded and functional?
We have seen cases where a small population of poorly folded protein would
give rise to aggregates that eventually seed the entire sample into
aggregation. Removing these seeds early in the game was key to keeping the
whole sample happy. In a few very curious cases we were able to achieve
this by controlled heating (e.g. 55C for 5 minutes) of the sample followed
by ultracentrifugation or some other separative technique. The idea is to
accelerate aggregation of the busted protein and then to crash the
aggregate out. This is of course somewhat extreme and cannot be recommended
as a general solution to every issue of this sort.

Addition of the right detergent can also help. Separation via another
orthogonal matrix is often helpful as well.

Artem


On Sun, Feb 2, 2020, 4:22 AM Jon Hughes 
wrote:

> Thanks for you interest. Ok, here are some more details.
>
> The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa)
> holophytochrome, produced with a C-terminal His6 tag together with its
> co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red
> photochromic (that is, photoactive) such that, as a 2 component sensory
> histidine autokinase / phosphotransferase, its kinase activity can be
> switched on and off by appropriate light pulses. Thus it is unambiguously
> functional. It is also highly soluble (10 mg/ml is no problem) – but
> subsequently (over days and weeks) it aggregates (irrespective of the
> photostate) to form a fluffy precipitate.
>
> Incidentally, I believe that most SHPK's and indeed most phytochromes have
> aggregation problems like this.
>
> Beyond urea being a less potent chaotrope than guanidinium/HCl, the
> different chemical actions of the two might give a hint as to what causes
> the aggregation.
>
> Cheers
>
> jon
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *Artem
> Evdokimov
> *Gesendet:* Sonntag, 2. Februar 2020 00:46
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] Urea vs. Guanidinium/HCl
>
>
>
> More details would be helpful. Do you know whether your protein is folded
> and active to begin with? Many partially folded proteins behave in a way
> that resembles your experience... Urea is a less potent denaturant mole for
> mole than GuHCl so it is not super surprising that it behaves differently.
>
>
>
> Artem
>
>
>
> On Sat, Feb 1, 2020, 6:22 PM Jon Hughes <
> jon.hug...@bot3.bio.uni-giessen.de> wrote:
>
> Hello everyone,
> We work on a protein that tends to aggregate. The process is slowed but not
> stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
> dissolves the aggregate readily, urea just turns it into an amorphous
> chewing-gum-like mass. Does that info provide anyone with a clue as to why
> the aggregation occurs and maybe suggest how to stop it in a way that would
> not thwart crystal formation?
> Best,
> jon
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Protein fold and the moonlighting function

[00000c2488af9525-dmarc-request]

Can you possibly expand a bit on exactly what you are asking?Jon CooperOn 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

On 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

On 2 Feb 2020 09:28, Rajnandani Kashyap  wrote:Dear AllI am curious to know about the promiscuous activity of a protein based on their fold. Can a protein having same fold also have same function (say not 100% but some activity) as the homologous structure. Please also let me know few relevant PDB structures where such kind of activity is shown promising. RegardsRajnandani KashyapPhD Student


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[00000c2488af9525-dmarc-request]

Hello, have you tried eluting from nickel-NTA straight into glycerol buffer so the protein has absolutely minimal time on it's own? Also, 10 mg/ml is quite a high concentration in my book. What is in your purification buffer? Just interested. Hope your competitors aren't reading all this! Best wishes.Jon CooperOn 2 Feb 2020 12:13, John Newitt  wrote:Have you tried maintaining the protein in a solution with a low concentration of excess cofactor? Light vs dark? Excess reducing agent or different reducing agent or oxygen-depleted and under argon?JohnOn Feb 2, 2020, at 4:22 AM, Jon Hughes  wrote:Thanks for you interest. Ok, here are some more details. The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) holophytochrome, produced with a C-terminal His6 tag together with its co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red photochromic (that is, photoactive) such that, as a 2 component sensory histidine autokinase / phosphotransferase, its kinase activity can be switched on and off by appropriate light pulses. Thus it is unambiguously functional. It is also highly soluble (10 mg/ml is no problem) – but subsequently (over days and weeks) it aggregates (irrespective of the photostate) to form a fluffy precipitate. Incidentally, I believe that most SHPK's and indeed most phytochromes have aggregation problems like this. Beyond urea being a less potent chaotrope than guanidinium/HCl, the different chemical actions of the two might give a hint as to what causes the aggregation.Cheersjon Von: CCP4 bulletin board  Im Auftrag von Artem EvdokimovGesendet: Sonntag, 2. Februar 2020 00:46An: CCP4BB@JISCMAIL.AC.UKBetreff: Re: [ccp4bb] Urea vs. Guanidinium/HCl More details would be helpful. Do you know whether your protein is folded and active to begin with? Many partially folded proteins behave in a way that resembles your experience... Urea is a less potent denaturant mole for mole than GuHCl so it is not super surprising that it behaves differently. Artem On Sat, Feb 1, 2020, 6:22 PM Jon Hughes  wrote:Hello everyone,We work on a protein that tends to aggregate. The process is slowed but notstopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCldissolves the aggregate readily, urea just turns it into an amorphouschewing-gum-like mass. Does that info provide anyone with a clue as to whythe aggregation occurs and maybe suggest how to stop it in a way that wouldnot thwart crystal formation?Best,jon To unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link:https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] Post-doctoral position in cryo-EM at IOCB Prague

[Sebastian Zoll]

Job description 

A fellowship-funded postdoc position is open in Dr Sebastian Zoll’s group at 
the Institute of Organic Chemistry and Biochemsitry (IOCB) in Prague. Dr Zoll’s 
group is using a combination of biochemical, biophysical, immunological and 
structural biology techniques to understand the mechanisms of host-pathogen 
interactions. The succesful candidate will use Cryo-EM to determine the 
structure of a viral capsid protein in complex with its cellular receptor. As 
part of this process the applicant will generate monoclonal antibody and 
nanobody libraries as tools as well as for therapeutic applications.

 
Requirements

-PhD in structural biology using Cryo-EM as the main tool. Expertise in 
grid preparation, microscope operation and data processing in RELION is 
essential. Knowledge in X-ray crystallography and SAXS is a bonus but not 
necessary. 

-Experience in expression construct design, protein purification and 
biophysical techniques to analyse protein-protein interactions (ITC or SPR). 

The project is supported by a multidisciplinary network of international 
collaborators and provides excellent opportunities to receive additional 
training in foreign host institutions.



The fellowship is granted for one year with possible extension by one more 
year. The earliest starting date is May 2020. For further information please 
see:

https://www.uochb.cz/en/iocb-fellowships 

 
The institute and the city 

IOCB Prague is the leading scientific research institution in the Czech 
Republic providing a dynamic, international environment that brings together 
researchers from various fields such as structural biology, molecular biology, 
medicinal and computational chemistry.

Due to its long-standing history in applied science and strong ties with the 
pharmaceutical industry, IOCB enjoys an excellent funding situation and offers 
access to facilities for all modern structural and biophysical methods.

The IOCB Cryo-EM facility housing a 300 keV high-resolution and 200 keV 
screening cryo-electron microscope will become operational next year. In the 
meanwhile IOCB researchers enjoy privileged access to the EM facilities 
(screening microscopes and Titan Krios G3) at CEITEC.

IOCB is situated near the city centre in a vibrating, historical quarter of 
Prague boasting beautiful architecture and a rich array of public amenities 
such as cafés, pubs, parks and grocery stores. Transport links by metro and 
tram are excellent with short distances to both airport as well as old town and 
Letna park with its stunning views over the Vltava river.

 
How to apply?

Please send a short motivation statement (max. 500 words) and a copy of your CV 
to sebastian.z...@uochb.cas.cz

Feel free to contact Sebastian for informal enquiries.

 
Dr Sebastian Zoll

Junior Group Leader

Institute of Organic Chemistry and Biochemistry

Czech Academy of Sciences

Flemingovo namesti 542/2, 166 10 Prague 6

Czech Republic

Phone: +420 220 183 591

Email: sebastian.z...@uochb.cas.cz

Facebook: https://www.facebook.com/IOCBPrague


To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[John Newitt]

Have you tried maintaining the protein in a solution with a low concentration 
of excess cofactor? Light vs dark? Excess reducing agent or different reducing 
agent or oxygen-depleted and under argon?

John

> On Feb 2, 2020, at 4:22 AM, Jon Hughes  
> wrote:
> 
> 
> Thanks for you interest. Ok, here are some more details.
> The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) 
> holophytochrome, produced with a C-terminal His6 tag together with its 
> co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red 
> photochromic (that is, photoactive) such that, as a 2 component sensory 
> histidine autokinase / phosphotransferase, its kinase activity can be 
> switched on and off by appropriate light pulses. Thus it is unambiguously 
> functional. It is also highly soluble (10 mg/ml is no problem) – but 
> subsequently (over days and weeks) it aggregates (irrespective of the 
> photostate) to form a fluffy precipitate.
> Incidentally, I believe that most SHPK's and indeed most phytochromes have 
> aggregation problems like this.
> Beyond urea being a less potent chaotrope than guanidinium/HCl, the different 
> chemical actions of the two might give a hint as to what causes the 
> aggregation.
> Cheers
> jon
>  
> Von: CCP4 bulletin board  Im Auftrag von Artem 
> Evdokimov
> Gesendet: Sonntag, 2. Februar 2020 00:46
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] Urea vs. Guanidinium/HCl
>  
> More details would be helpful. Do you know whether your protein is folded and 
> active to begin with? Many partially folded proteins behave in a way that 
> resembles your experience... Urea is a less potent denaturant mole for mole 
> than GuHCl so it is not super surprising that it behaves differently.
>  
> Artem
>  
> On Sat, Feb 1, 2020, 6:22 PM Jon Hughes  
> wrote:
> Hello everyone,
> We work on a protein that tends to aggregate. The process is slowed but not
> stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
> dissolves the aggregate readily, urea just turns it into an amorphous
> chewing-gum-like mass. Does that info provide anyone with a clue as to why
> the aggregation occurs and maybe suggest how to stop it in a way that would
> not thwart crystal formation?
> Best,
> jon 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] Protein fold and the moonlighting function

[Rajnandani Kashyap]

Dear All

I am curious to know about the promiscuous activity of a protein based on
their fold. Can a protein having same fold also have same function (say not
100% but some activity) as the homologous structure.

Please also let me know few relevant PDB structures where such kind of
activity is shown promising.


Regards

Rajnandani Kashyap
PhD Student



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[Jon Hughes]

Thanks for you interest. Ok, here are some more details. 

The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) 
holophytochrome, produced with a C-terminal His6 tag together with its 
co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red 
photochromic (that is, photoactive) such that, as a 2 component sensory 
histidine autokinase / phosphotransferase, its kinase activity can be switched 
on and off by appropriate light pulses. Thus it is unambiguously functional. It 
is also highly soluble (10 mg/ml is no problem) – but subsequently (over days 
and weeks) it aggregates (irrespective of the photostate) to form a fluffy 
precipitate. 

Incidentally, I believe that most SHPK's and indeed most phytochromes have 
aggregation problems like this. 

Beyond urea being a less potent chaotrope than guanidinium/HCl, the different 
chemical actions of the two might give a hint as to what causes the aggregation.

Cheers

jon

 

Von: CCP4 bulletin board  Im Auftrag von Artem Evdokimov
Gesendet: Sonntag, 2. Februar 2020 00:46
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Urea vs. Guanidinium/HCl

 

More details would be helpful. Do you know whether your protein is folded and 
active to begin with? Many partially folded proteins behave in a way that 
resembles your experience... Urea is a less potent denaturant mole for mole 
than GuHCl so it is not super surprising that it behaves differently.

 

Artem

 

On Sat, Feb 1, 2020, 6:22 PM Jon Hughes mailto:jon.hug...@bot3.bio.uni-giessen.de> > wrote:

Hello everyone,
We work on a protein that tends to aggregate. The process is slowed but not
stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
dissolves the aggregate readily, urea just turns it into an amorphous
chewing-gum-like mass. Does that info provide anyone with a clue as to why
the aggregation occurs and maybe suggest how to stop it in a way that would
not thwart crystal formation?
Best,
jon 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB 
 =1

 

  _  

To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB 
 =1 




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1