Re: [ccp4bb] Coot keybinding in a Mac

[Paul Emsley]

On 19/03/2018 21:18, Nikolas wrote:


I'm solving my first structure ever (yay!) 


Yay!

on a MAC OS 


Boo!


and they keybindings doesn't work.


Boo.

I thought that 
they would come with the CCP4 suite but I've found also Paul's and Bernhard's files on COOT wiki and tried 
to create the .coot file but it didn't work. Unfortunately I'm a Windows power user (you can throw eggs at 
me later) and I'm not super familiar with UNIX/MAC yet.


Hmm.

Space bar for navigating through residues works fine as well as "O" for changing chain. The "W" for adding 
waters and the "G" for centering on the blob doesn't.


Extensions -> Settings -> Install Template Key-bindings

Yay?

P.

Re: [ccp4bb] A question related to Fe-S proteins

[PULSARSTRIAN]

Dear Professor Ethan,

I apologize, for the partial
information provided by me in the second email. For the *in vivo* assembled
(as-purified) protein, we have confirmed the [4Fe-4S] cluster by CD and
EPR. From the 415/280 ratio and Ferrozine assay, we have confirmed there is
one [4Fe-4S] cluster / per three monomers.

 So, we anticipate the [4Fe-4S] cluster between 4
cysteines out of 6 cysteines form the helices-1 of three monomers. There
are also few examples in the PDB, for homo-trimeric proteins having only
one [4Fe-4S] cluster. Yet so far, I have not found any example with
different oligomeric states for the *in vivo* assembled (as-purified)
protein VS the *in vitro* reconstituted one.


Regards,

Bhanu



On Mon, Mar 19, 2018 at 11:56 PM, Ethan A Merritt 
wrote:

> On Monday, March 19, 2018 2:20:50 PM PDT PULSARSTRIAN wrote:
> > Dear Professor Diana,
> >
> > Thank you very much for your
> > comment, and we will certainly look at, if ‘domain swapping’ is any
> reason
> > for the trimeric nature of the protein.
> >
> >   There are two cysteines in helix-1 and two
> cysteines
> > in helix 3. From our mutational studies (C to S), we confirmed in the *in
> > vivo *assembled protein, that only helix-1 (the first two cysteines) form
> > the Fe-S cluster. So, its altogether a completely new structure compared
> to
> > reconstituted protein.
>
> Your description of the in vivo characterization seems to be inconsistent.
>
> 1) 4Fe-4S cluster formed in vivo from 2 copies of helix-1, each
> contributing 2 Cys.
> 2) resulting in a trimer
>
> How would that work?
>
> Or is it that you are proposing that there is a single 4Fe/4S center per
> trimer,
> formed stochastically from 4 of the 6 available Cys residues?
>
> You don't mention having EPR for the in vivo assembly.
> Have you determined the Fe/S stoichiometry in the trimer?
>
> Ethan
>
> >
> > Regards,
> >
> > Bhanu
> >
> >
> >
> > On Mon, Mar 19, 2018 at 10:13 PM, Diana Tomchick <
> > diana.tomch...@utsouthwestern.edu> wrote:
> >
> > > Is it possible that you have a case of domain swapping that causes the
> > > trimeric assembly?
> > >
> > > Diana
> > >
> > > **
> > > Diana R. Tomchick
> > > Professor
> > > Departments of Biophysics and Biochemistry
> > > University of Texas 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 Mar 19, 2018, at 3:07 PM, PULSARSTRIAN 
> > > wrote:
> > >
> > > Dear all,
> > >
> > > Sorry for the slightly off-topic question.
> > >
> > >I am working on a non-native, *de novo* [4Fe-4S]
> > > protein, designed as a four-helix bundle. The * in vitro* reconstituted
> > > protein assembles with [4Fe-4S] (confirmed by EPR) and exists in
> > > monomer-dimer configuration (confirmed by SEC). These results have been
> > > already published.
> > >
> > >Recently we could get the [4Fe-4S] assembly directly
> from
> > > the *E. coli* (*in vivo* assembly). Everything is as expected (compared
> > > to reconstituted protein), except the oligomerization state. The
> protein
> > > assembles as trimer, in contrast to monomer-dimer configuration of the
> > > reconstituted protein. The trimeric nature of the *in vivo* assembled
> > > protein has been confirmed by SEC, SEC-SLS and SAXS.
> > >
> > >
> > >  *So, my question is, has anyone encountered such
> situation,
> > > where the As-purified Fe-S protein having a completely different
> oligomeric
> > > state compared to the in vitro reconstitution protein? *
> > >
> > >
> > > Looking forward to hearing for some examples and/or references.
> > >
> > >
> > > Regards,
> > >
> > > Bhanu
> > >
> > >
> > > --
> > >
> > > UT Southwestern
> > >
> > > Medical Center
> > >
> > > The future of medicine, today.
> > >
> >
> >
> >
> >
>
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> MS 357742,   University of Washington, Seattle 98195-7742
>
>


-- 
B4U

Re: [ccp4bb] A question related to Fe-S proteins

[Ethan A Merritt]

On Monday, March 19, 2018 2:20:50 PM PDT PULSARSTRIAN wrote:
> Dear Professor Diana,
> 
> Thank you very much for your
> comment, and we will certainly look at, if ‘domain swapping’ is any reason
> for the trimeric nature of the protein.
> 
>   There are two cysteines in helix-1 and two cysteines
> in helix 3. From our mutational studies (C to S), we confirmed in the *in
> vivo *assembled protein, that only helix-1 (the first two cysteines) form
> the Fe-S cluster. So, its altogether a completely new structure compared to
> reconstituted protein.

Your description of the in vivo characterization seems to be inconsistent.

1) 4Fe-4S cluster formed in vivo from 2 copies of helix-1, each contributing 2 
Cys. 
2) resulting in a trimer

How would that work?

Or is it that you are proposing that there is a single 4Fe/4S center per trimer,
formed stochastically from 4 of the 6 available Cys residues?

You don't mention having EPR for the in vivo assembly.
Have you determined the Fe/S stoichiometry in the trimer?

Ethan

> 
> Regards,
> 
> Bhanu
> 
> 
> 
> On Mon, Mar 19, 2018 at 10:13 PM, Diana Tomchick <
> diana.tomch...@utsouthwestern.edu> wrote:
> 
> > Is it possible that you have a case of domain swapping that causes the
> > trimeric assembly?
> >
> > Diana
> >
> > **
> > Diana R. Tomchick
> > Professor
> > Departments of Biophysics and Biochemistry
> > University of Texas 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 Mar 19, 2018, at 3:07 PM, PULSARSTRIAN 
> > wrote:
> >
> > Dear all,
> >
> > Sorry for the slightly off-topic question.
> >
> >I am working on a non-native, *de novo* [4Fe-4S]
> > protein, designed as a four-helix bundle. The * in vitro* reconstituted
> > protein assembles with [4Fe-4S] (confirmed by EPR) and exists in
> > monomer-dimer configuration (confirmed by SEC). These results have been
> > already published.
> >
> >Recently we could get the [4Fe-4S] assembly directly from
> > the *E. coli* (*in vivo* assembly). Everything is as expected (compared
> > to reconstituted protein), except the oligomerization state. The protein
> > assembles as trimer, in contrast to monomer-dimer configuration of the
> > reconstituted protein. The trimeric nature of the *in vivo* assembled
> > protein has been confirmed by SEC, SEC-SLS and SAXS.
> >
> >
> >  *So, my question is, has anyone encountered such situation,
> > where the As-purified Fe-S protein having a completely different oligomeric
> > state compared to the in vitro reconstitution protein? *
> >
> >
> > Looking forward to hearing for some examples and/or references.
> >
> >
> > Regards,
> >
> > Bhanu
> >
> >
> > --
> >
> > UT Southwestern
> >
> > Medical Center
> >
> > The future of medicine, today.
> >
> 
> 
> 
> 


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

[ccp4bb] Coot keybinding in a Mac

[Nikolas]

Hello everyone,

I'm solving my first structure ever (yay!) on a MAC OS and they keybindings
doesn't work. I thought that they would come with the CCP4 suite but I've
found also Paul's and Bernhard's files on COOT wiki and tried to create the
.coot file but it didn't work. Unfortunately I'm a Windows power user (you
can throw eggs at me later) and I'm not super familiar with UNIX/MAC yet.

Space bar for navigating through residues works fine as well as "O" for
changing chain. The "W" for adding waters and the "G" for centtering on the
blob doesn't.

Any tips or hints on how can I fix the bindings?

Many thanks,
Nikk

Re: [ccp4bb] A question related to Fe-S proteins

[PULSARSTRIAN]

Dear Professor Diana,

Thank you very much for your
comment, and we will certainly look at, if ‘domain swapping’ is any reason
for the trimeric nature of the protein.

  There are two cysteines in helix-1 and two cysteines
in helix 3. From our mutational studies (C to S), we confirmed in the *in
vivo *assembled protein, that only helix-1 (the first two cysteines) form
the Fe-S cluster. So, its altogether a completely new structure compared to
reconstituted protein.


Regards,

Bhanu



On Mon, Mar 19, 2018 at 10:13 PM, Diana Tomchick <
diana.tomch...@utsouthwestern.edu> wrote:

> Is it possible that you have a case of domain swapping that causes the
> trimeric assembly?
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas 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 Mar 19, 2018, at 3:07 PM, PULSARSTRIAN 
> wrote:
>
> Dear all,
>
> Sorry for the slightly off-topic question.
>
>I am working on a non-native, *de novo* [4Fe-4S]
> protein, designed as a four-helix bundle. The * in vitro* reconstituted
> protein assembles with [4Fe-4S] (confirmed by EPR) and exists in
> monomer-dimer configuration (confirmed by SEC). These results have been
> already published.
>
>Recently we could get the [4Fe-4S] assembly directly from
> the *E. coli* (*in vivo* assembly). Everything is as expected (compared
> to reconstituted protein), except the oligomerization state. The protein
> assembles as trimer, in contrast to monomer-dimer configuration of the
> reconstituted protein. The trimeric nature of the *in vivo* assembled
> protein has been confirmed by SEC, SEC-SLS and SAXS.
>
>
>  *So, my question is, has anyone encountered such situation,
> where the As-purified Fe-S protein having a completely different oligomeric
> state compared to the in vitro reconstitution protein? *
>
>
> Looking forward to hearing for some examples and/or references.
>
>
> Regards,
>
> Bhanu
>
>
> --
>
> UT Southwestern
>
> Medical Center
>
> The future of medicine, today.
>



-- 
B4U

Re: [ccp4bb] A question related to Fe-S proteins

[Diana Tomchick]

Is it possible that you have a case of domain swapping that causes the trimeric 
assembly?

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas 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 Mar 19, 2018, at 3:07 PM, PULSARSTRIAN 
> wrote:

Dear all,
Sorry for the slightly off-topic question.
   I am working on a non-native, de novo [4Fe-4S] protein, 
designed as a four-helix bundle. The in vitro reconstituted protein assembles 
with [4Fe-4S] (confirmed by EPR) and exists in monomer-dimer configuration 
(confirmed by SEC). These results have been already published.
   Recently we could get the [4Fe-4S] assembly directly from the E. 
coli (in vivo assembly). Everything is as expected (compared to reconstituted 
protein), except the oligomerization state. The protein assembles as trimer, in 
contrast to monomer-dimer configuration of the reconstituted protein. The 
trimeric nature of the in vivo assembled protein has been confirmed by SEC, 
SEC-SLS and SAXS.

 So, my question is, has anyone encountered such situation, where 
the As-purified Fe-S protein having a completely different oligomeric state 
compared to the in vitro reconstitution protein?

Looking forward to hearing for some examples and/or references.

Regards,
Bhanu




UT Southwestern


Medical Center



The future of medicine, today.

[ccp4bb] A question related to Fe-S proteins

[PULSARSTRIAN]

Dear all,

Sorry for the slightly off-topic question.

   I am working on a non-native, *de novo* [4Fe-4S]
protein, designed as a four-helix bundle. The *in vitro* reconstituted
protein assembles with [4Fe-4S] (confirmed by EPR) and exists in
monomer-dimer configuration (confirmed by SEC). These results have been
already published.

   Recently we could get the [4Fe-4S] assembly directly from
the *E. coli* (*in vivo* assembly). Everything is as expected (compared to
reconstituted protein), except the oligomerization state. The protein
assembles as trimer, in contrast to monomer-dimer configuration of the
reconstituted protein. The trimeric nature of the *in vivo* assembled
protein has been confirmed by SEC, SEC-SLS and SAXS.


 *So, my question is, has anyone encountered such situation,
where the As-purified Fe-S protein having a completely different oligomeric
state compared to the in vitro reconstitution protein? *


Looking forward to hearing for some examples and/or references.


Regards,

Bhanu

[ccp4bb] ReNaFoBiS. Oleron 2018. There are only few places left

[VIE]

ReNaFoBiS. Oleron 2018. There are only few places left.

The 5th RéNaFoBiS Oléron workshop, dedicated to Integrative Structural Biology, 
will take place on the Oléron island (France), from June the 1st until June the 
8th, 2018. The main objective of this workshop is to offer a theoretical and 
practical training in the different techniques used in integrative structural 
biology (X-ray diffraction, small angle X-ray scattering, NMR, cryo-electron 
microscopy, sample preparation for structural biology,  biophysical methods to 
study and characterize macromolecular interactions). The goals are to explain 
and illustrate, to an audience mainly composed of doctoral students and young 
researchers, the contributions and limitations of each method with a strong 
emphasis on their complementarity and future developments. Engineers and 
Technicians (public and private sector employers) are welcome.
The official language of the workshop is French but presentations may be given 
in English, depending on the overall profile of the students. For practicals, 
English and French speaking groups may be organised.
Registration is open on the web site:
https://ecolebios2018.sciencesconf.org/

There are only few places left. Deadline for registration: April 9, 2018 
(maximum number of participants: 25)
Applications will be evaluated upon deposition.
More information on the French Initiative ReNaFoBiS : http://www.renafobis.fr/
Best  regards

Jean Cavarelli

National ReNaFoBiS Coordinator


ReNaFoBiS. 5ème École de Biologie Structurale Intégrative
Oléron – du 1 au 8 juin 2018

Il reste quelques places. Inscriptions sur le site 
https://ecolebios2018.sciencesconf.org

Cette cinquième école propose une formation théorique et appliquée aux 
différentes approches utilisées en biologie structurale (diffraction et 
diffusion des rayons X, RMN, cryo-microscopie, préparations des échantillons en 
vue des études structurales, interactions macromoléculaires). Elle mettra 
l’accent sur l’intégration de plusieurs de ces méthodes pour répondre aux 
grandes questions de la biologie fonctionnelle à l’échelle atomique.
Pour un public de doctorants ou de jeunes chercheurs, cette formation montrera 
les apports et les limites de chaque méthode et leur complémentarité. Elle 
inclura des sessions théoriques le matin et des travaux pratiques en groupes 
l’après-midi.
Cette école est ouverte aux techniciens et ingénieurs (domaine académique et 
industriel) dans la cadre de la formation continue. Les conférences seront 
données principalement en français. Les supports des présentations seront en 
anglais, afin de permettre aux participants non-francophones de suivre plus 
facilement. Lors des sessions pratiques (TP), des groupes anglophones pourront 
être proposés si besoin.
Dépôt des dossiers avant le 9 avril 2018.
Le nombre de places étant limité (25 participants), les participants seront 
sélectionnés sur la base d'un CV et d'une lettre de motivation. Les dossiers 
seront examinés et validés au fur et à mesure de leur déposition.
Plus d'informations sur le site de l'école 
https://ecolebios2018.sciencesconf.org et sur le site  de ReNaFoBiS  
http://www.renafobis.fr/
Tres cordialement

Jean Cavarelli
Coordinateur National ReNaFoBiS


--

Jean Cavarelli
Professor of Structural Biology
Tél : +33 (0)3 69 48 52 74
jean.cavare...@unistra.fr
Structural Biology of Epigenetic Targets
Integrated Structural Biology Department
IGBMC. UMR7104 CNRS-UDS, INSERM U964.
F - 67404 Illkirch

[ccp4bb] CCP-EM: Instruct Workshop for High Resolution Model Building and Refinement (4th Icknield)

[Tom Burnley]

Dear all,

Further to lasts week message...:

1) Mea culpa... to my shame I forget to include 'Instruct' in the title of
the workshop.  They are kindly supporting this workshop.

2) Places to register interest are filling up rapidly so if you would like
to come please register soon:

cvent.com/d/btq6g6

This course is aimed at structural biologists with high resolution EM maps
ready for / in the process of model building and refinement. This four day
course (1st-4th May) will host some of the leading software developers and
provide ample contact time to allow delegates to discuss their data in
detail alongside traditional lectures and tutorials. It will cover all
aspects of model building including: map optimisation, docking, automated
model building, medium resolution refinement, high resolution refinement,
interactive refinement, validation and deposition.

The course will be held at the RAL / Diamond Campus, Harwell, UK, site of
the eBIC national facility. Registration is free and we will provide food
and three nights accommodation at the Bear Hotel in nearby Wantage (nights
of 1st, 2nd and 3rd May). Please note that during the application process
you will be asked if you would like to make an accommodation request - if
you would like accommodation provided for you free of charge please select
yes and let us know which nights you would like. If you do not want
accommodation provided for you please select no. Delegates are asked to
meet their own travel costs. Please see the agenda for full course times.

The course is limited to 20 delegates who will be selected from all
applicants.  A maximum of 50 applicants can register interest.

Applications will close on ***Wednesday 28th March*** and the 20 successful
applicants will be informed the following day. Please note the short
registration time as we would like to strongly encourage applicants to work
on 'live' datasets.

* Registration site
cvent.com/d/btq6g6

* Confirmed tutors / topics:

Tristan Croll (Cambridge)
ISOLDE: Interactive model building/real-space refinement based on
interactive molecular dynamics

Kevin Cowtan (York)
Buccaneer: automated model building

Paul Emsley (MRC-LMB Cambridge)
Coot: macromolecular model building, model completion and validation

Arjen Jakobi (Delft)
LocScale: scaling tool using prior model information to improve map
contrast

Oleg Kovalevskiy (Murshudov group, MRC-LMB Cambridge)
Refmac: refinement of high-resolution EM structures

Ardan Patwardhan (EBI)
EMBD: deposition in EMBD

Joerg Schaarschmidt (Bonvin Group, Utrecht)
Powerfit: Fast and sensitive rigid-body fitting into cryo-EM density maps

Maya Topf (Birkbeck)
FlexEM: fitting and refinement of atomic structures guided by cryoEM density


Best wishes,

Tom



-- 
Dr Tom Burnley, PhD
CCP-EM | @ccp_em * | *www.ccpem.ac.uk

Science and Technology Facilities Council (STFC)
The Research Complex At Harwell
Rutherford Appleton Laboratory, R92
OX11 0FA
01235 56 7871

[ccp4bb] FW: [ccp4bb] Resolution mismatch aimless/refmac

[Orru, Dr. Roberto]

Dear All,

Thanks everyone for the suggestions and clarifications. As Eleanor and Garib 
wrote, I cannot find real differences in map quality or statistics after 
refinement depending if I set or not the resolution edges.
This is more a matter of how the remarks in the pdb are recorded for 
validation/deposition for which a big red exclamation mark is pointing to the 
mismatch values. This is happening only when the ccp4i2 interface is used for 
refmab but not with ccp4i. Maybe a script problem for the pdb remark data 
extrapolation?
Also if I manually set the resolution I found that the pdb remark is still not 
correct exactly (in ccp4i2). But when I ran with the same files a refmac 
refinement using the ccp4i interface, everything is running smoothly and 
perfectly with no "error" at all.

Just as an open question: As seems more a pdb file writing issue than a 
refinement problem, correcting the pdb file manually writing with a text editor 
in in the remark the exact low resolution, could be also a fix or can be 
considered as faking the file? ( I didn't test it yet, just mornings idea...)

All the best,
Roberto

---
Dr. Roberto Orru, Ph.D.

Insitute of molecular biology (IMB) and
JGU - Institut für Molekulare Physiologie (AG Prof. Wolf)
Mainz, Germany

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Garib 
Murshudov
Sent: Sunday, March 18, 2018 5:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Resolution mismatch aimless/refmac


On 18 Mar 2018, at 16:02, Eleanor Dodson 
> wrote:

Does it matter? The refinement will only use observed reflections.

It should not matter for refinement and map calculations (needs to be checked 
carefully). However it does matter for deposition when multiple entries are 
analysed and the PDB deposition software (rightly so) says that you have 
information mismatch. And it confuses users and most of all it confuses 
deposition. It would confuse me.

Best wishes,
Garib


As Andrew says your output from dataprocessing includes a complete list of all 
possible indices to the upper resolution limit with a Free R flag assigned to 
the indices. This makes it easier to keep the same FreeR assignments for all 
your sets of measurements, which is a good thing..

There is a library call which REFMAC could use which would return the actual 
limits of the observed data - that small modification to the program would give 
a more sensible log file for the refinement.

At present the REFMAC outputs a reflection list will include all indices,  with 
the Fcalc PHIcalc information listed for missing data . I find that quite 
useful in some cases, but if you do not wish to use the terms there are ways of 
excluding them from subsequent calculations..

Eleanor



On 18 March 2018 at 12:04, Garib Murshudov 
> wrote:
Dear All,

As far as I know it could happen when you are using i2. We are working to fix 
this problem. At the moment the best solution is to use advanced options in the 
refmac interface and define resolution limits explicitly. For example you can 
add in the advanced options:


resolution 37 2.5

where these resolution limits are from the aimless output. Low resolution limit 
should not affect refinement behaviour, however it may cause problem during 
deposition.

We will have better solution soon. I2 has many good features. Please report if 
you see some misbehaviours.

Best wishes,
Garib

P.S. you can also use advanced options to do things that are not available on 
the interface yet.
1) refinement against electron diffraction. Add

source EC MB   #  electron form factor will be used using Mott-Bethe formula

2) you can add occupancy refinement

etc


On 18 Mar 2018, at 10:46, "Orru, Dr. Roberto" 
> wrote:

Dear All,

I am noticing that the low resolution in the reflections files after scaling 
with aimless and after refinement with refmac does not coincide.
In a case I have 37A with aimless but for some reason refmac is 67A.

Any idea?
All the best,
R.

Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge
CB2 0QH UK
Web http://www.mrc-lmb.cam.ac.uk,
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/



Dr Garib N Murshudov
MRC-LMB
Francis Crick Avenue
Cambridge
CB2 0QH UK
Web http://www.mrc-lmb.cam.ac.uk,
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/