[ccp4bb] New version SHELXL 2016/6 and PDB2INS

[George Sheldrick]

SHELXL may be used to refine both small and macromolecular structures 
against X-ray or neutron diffraction data, including non-merohedral 
twins. A new version 2016/6 of SHELXL may now be downloaded from the 
SHELX server. It has been well tested by about 20 volunteers to whom I 
am very grateful. A new version of Anna Luebben's program PDB2INS is 
also available there (for 64 bit systems only). PDB2INS makes the 
preparation of the /.ins/ and /.hkl /files to run macromolecular SHELXL 
refinements much easier. For most structures deposited in the PDB since 
2008 these two files can be generated automatically and no changes are 
needed to run SHELXL.


George

--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582

Re: [ccp4bb] cif-file macro?

[Ian Clifton]

Edwin Pozharski  writes:

> I expect this not to exist, but is there a way to define a variable in
> a cif-file (e.g. a global esd target for, say, angles)?

No, I don’t think either CIF or CIF2 have such variables/macros, I’m
afraid.
-- 
Ian Clifton ⚗ ℡: +44 1865 275677
Chemistry Research Laboratory ℻: +44 1865 285002
Oxford University : ian.clif...@chem.ox.ac.uk
Mansfield Road   Oxford OX1 3TA   UK

[ccp4bb] cif-file macro?

[Edwin Pozharski]

I expect this not to exist, but is there a way to define a variable in a
cif-file (e.g. a global esd target for, say, angles)?

I am certainly capable of putting together a bash script to emulate this,
so please don't bother with suggesting a workaround unless you really enjoy
that kind of thing :)

Cheers,

Ed

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Roger Rowlett]

I'll also recommend Buccaneer. You might try using a combination of 
PARROT for density modification and NCS averaging followed by 
autobuilding with BUCCANEER using initial phases from your MR solution. 
You only have two copies of the protein in the ASU, so you only get a 
modest boost in electron density averaging, but maybe every little bit 
helps. This approach was very successful for me with 8-fold NCS and a 
29% identity search model that gave poor but usable initial MR phases 
for 2.4A data.


___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 10/25/2016 9:19 AM, Dmytro Guzenko wrote:


Hi Vikram,

Try Buccaneer, it works much better at such resolution than Arp/Warp.

Launch it several times with different parameters (e.g. use original 
structure as seed/initial/nothing), then open the results together and 
merge the pieces that look best and have lowest b-factors. Use Buster 
for refinement of the merged model and iterate with Buccaneer again. 
That's what worked for me once to bootstrap a poor MR solution (~20% 
of the structure originally) to a near-complete model at 2.7A.


You are in a sense replicating what phenix.autobuild attempts to do, 
but you'll be able to do it better (and probably faster) manually.


Kind regards,

Dmytro.
On 25/10/16 14:16, Vikram Dalal wrote:

Hi everyone,

We are trying to solve a protein structure of 2.6 A. We have 
processed it with HKL2000. We have even tried processing with mosflm 
and xia2. It is in C2221 space group (checked by pointless) and data 
is not twinned. It has 31% identical with a search model and has 57% 
sequence coverage. There are 2 subunits in asu.


I did not get proper phases with MOLREP and phaser. I have tried 
Balbes (R free 50) and Mr BUMP (R free 54).But, density from balbes 
look some reasonable.
So i have refined it in ccp4i and phenix differently and then model 
build it by coot. My protein has 377 Amino acid. But, I stucked at R 
free 41 and now i have amino acid 50 to 369 in both chains, still 49 
amino acid at N terminal and 8 amino acid at C terminal are missing 
and some loops are missing in between too.


*I have tried the ARP/wARP, but it does not work for it. *
*
*
*I have even tried phase and build of phenix but condition remain 
same. i got stuck at R free at 42 and same around 49 amino acids at N 
terminal and 8 amino acids at C terminal and some internal loops 
still absent.*

*
*
*Thank you in advance.*



*
*

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Dmytro Guzenko]

Hi Vikram,

Try Buccaneer, it works much better at such resolution than Arp/Warp.

Launch it several times with different parameters (e.g. use original 
structure as seed/initial/nothing), then open the results together and 
merge the pieces that look best and have lowest b-factors. Use Buster 
for refinement of the merged model and iterate with Buccaneer again. 
That's what worked for me once to bootstrap a poor MR solution (~20% of 
the structure originally) to a near-complete model at 2.7A.


You are in a sense replicating what phenix.autobuild attempts to do, but 
you'll be able to do it better (and probably faster) manually.


Kind regards,

Dmytro.
On 25/10/16 14:16, Vikram Dalal wrote:

Hi everyone,

We are trying to solve a protein structure of 2.6 A. We have processed 
it with HKL2000. We have even tried processing with mosflm and xia2. 
It is in C2221 space group (checked by pointless) and data is not 
twinned. It has 31% identical with a search model and has 57% sequence 
coverage. There are 2 subunits in asu.


I did not get proper phases with MOLREP and phaser. I have tried 
Balbes (R free 50) and Mr BUMP (R free 54).But, density from balbes 
look some reasonable.
So i have refined it in ccp4i and phenix differently and then model 
build it by coot. My protein has 377 Amino acid. But, I stucked at R 
free 41 and now i have amino acid 50 to 369 in both chains, still 49 
amino acid at N terminal and 8 amino acid at C terminal are missing 
and some loops are missing in between too.


*I have tried the ARP/wARP, but it does not work for it. *
*
*
*I have even tried phase and build of phenix but condition remain 
same. i got stuck at R free at 42 and same around 49 amino acids at N 
terminal and 8 amino acids at C terminal and some internal loops still 
absent.*

*
*
*Thank you in advance.*



*
*

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Eleanor Dodson]

Well - it sounds hopeful but still challenging..

First suggestion - are you SURE the SG is C2221 and not C222?
If your two molecules are related by a NC translation of x_tran, y_tran,
1/2  then you will get the apparent absences along the 0 0 l axis which
suggest SG C 2221 but the true SG could be C 222.

If you have run pointless/aimless/ctruncate, the log file will tell you of
any possible non-cryst translation.

Next - I usually see if there are any anomalous peaks in a difference
fourier. That is a very long shot but sometimes helps pin point S atoms and
encourages you that the solution is partially correct

Then I would try to do auto building with Buccaneer and see how far it
gets  - buccaneer tries to use NCS to improve the model .

Try those ideas first then maybe invoke parrot to do NCS averaging..
Eleanor





On 25 October 2016 at 13:16, Vikram Dalal  wrote:

> Hi everyone,
>
> We are trying to solve a protein structure of 2.6 A. We have processed it
> with HKL2000. We have even tried processing with mosflm and xia2. It is in
> C2221 space group (checked by pointless) and data is not twinned. It has
> 31% identical with a search model and has 57% sequence coverage. There are
> 2 subunits in asu.
>
> I did not get proper phases with MOLREP and phaser. I have tried Balbes (R
> free 50) and Mr BUMP (R free 54).But, density from balbes look some
> reasonable.
> So i have refined it in ccp4i and phenix differently and then model build
> it by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and
> now i have amino acid 50 to 369 in both chains, still 49 amino acid at N
> terminal and 8 amino acid at C terminal are missing and some loops are
> missing in between too.
>
> *I have tried the ARP/wARP, but it does not work for it. *
>
> *I have even tried phase and build of phenix but condition remain same. i
> got stuck at R free at 42 and same around 49 amino acids at N terminal and
> 8 amino acids at C terminal and some internal loops still absent.*
>
> *Thank you in advance.*
>
>
>
>
>
>
>>
>>
>>
>>
>>
>

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Mark J van Raaij]

Hi Vikram,

lots of questions for you:
- what do the images look like? Were there “contaminating” lattices? Did the 
spot shape look ok? Do they get worse later in the data collection, i.e. do you 
have radiation damage? Is the image quality regular or worse at certain angles?
- how good is the data near 2.6Å? Is the data anisotropic?
If the data is not so great, perhaps Rfree 41% is as good as you can get. 
Nevertheless:
- are you sure the molecular replacement solution is correct? Did you see 
density differences for residues you had to “mutate” in COOT that support the 
correctness of the molecular replacement solution?
- what does model validation say? Any regions that still need improving? Any 
major differences between the two monomers that are not explained by the 
density?
- did you try refining with local NCS in REFMAC? If not, this should get the 
Rfree down a bit more.
- residues 1-49 and 370-377 may be genuinely disordered and not modellable, how 
hard did you try?

Hope this helps,

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij







> On 25 Oct 2016, at 14:16, Vikram Dalal  wrote:
> 
> Hi everyone,
> 
> We are trying to solve a protein structure of 2.6 A. We have processed it 
> with HKL2000. We have even tried processing with mosflm and xia2. It is in 
> C2221 space group (checked by pointless) and data is not twinned. It has 31% 
> identical with a search model and has 57% sequence coverage. There are 2 
> subunits in asu.
> 
> I did not get proper phases with MOLREP and phaser. I have tried Balbes (R 
> free 50) and Mr BUMP (R free 54).But, density from balbes look some 
> reasonable. 
> So i have refined it in ccp4i and phenix differently and then model build it 
> by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and now i 
> have amino acid 50 to 369 in both chains, still 49 amino acid at N terminal 
> and 8 amino acid at C terminal are missing and some loops are missing in 
> between too. 
> 
> I have tried the ARP/wARP, but it does not work for it. 
> 
> I have even tried phase and build of phenix but condition remain same. i got 
> stuck at R free at 42 and same around 49 amino acids at N terminal and 8 
> amino acids at C terminal and some internal loops still absent.
> 
> Thank you in advance.
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Stefano Trapani]

 

Dear Pascal 

have a look at : 

https://doi.org/10.1107/S0907444910002763 

Acta Cryst. (2010). D66, 514-521
Macromolecular crystal data phased by negative-stained
electron-microscopy reconstructions
S. Trapani, G. Schoehn, J. Navaza and C. Abergel 

Best, 

---
Stefano Trapani

Maître de Conférences
http://www.cbs.cnrs.fr/index.php/fr/personnel?PERS=Stefano%20Trapani
-
Centre de Biochimie Structurale (CBS)
29 rue de Navacelles
34090 MONTPELLIER Cedex, France

Tel : +33 (0)4 67 41 77 29
Fax : +33 (0)4 67 41 79 13
-
Université de Montpellier
CNRS UMR 5048
INSERM UMR 1054
-

Le 2016-10-25 03:49, Pascal Egea a écrit : 

> Dear All, 
> 
> I would like to know if it is possible to use a low resolution EM 
> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
> help molecular replacement in a 4.5A resolution X-ray diffraction data set of 
> the same complex 
> I am aware of the possibility of using low resolution cryoEM maps for MR as 
> described in the review from Jackson et al in Nature Protocols but I was 
> wondering if there is an intrinsically impossibility for negative stain 
> reconstructions. 
> 
> Any thoughts or advice will be greatly appreciated.
> 
> Best, 
> -- 
> 
> Pascal F. Egea, PhD
> Assistant Professor
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> Boyer Hall room 356
> 
> 611 Charles E Young Drive East 
> Los Angeles CA 90095
> office (310)-983-3515
> lab (310)-983-3516
> email pegea at mednet.ucla.edu [1] 
> -- 
> This message has been scanned for viruses and 
> dangerous content by MAILSCANNER [2], and is 
> believed to be clean.
 

Links:
--
[1] http://mednet.ucla.edu
[2] http://www.mailscanner.info/

-- 
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.

Re: [ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Vikram Dalal]

Hi everyone,

We are trying to solve a protein structure of 2.6 A. We have processed it
with HKL2000. We have even tried processing with mosflm and xia2. It is in
C2221 space group (checked by pointless) and data is not twinned. It has
31% identical with a search model and has 57% sequence coverage. There are
2 subunits in asu.

I did not get proper phases with MOLREP and phaser. I have tried Balbes (R
free 50) and Mr BUMP (R free 54).But, density from balbes look some
reasonable.
So i have refined it in ccp4i and phenix differently and then model build
it by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and
now i have amino acid 50 to 369 in both chains, still 49 amino acid at N
terminal and 8 amino acid at C terminal are missing and some loops are
missing in between too.

*I have tried the ARP/wARP, but it does not work for it. *

*I have even tried phase and build of phenix but condition remain same. i
got stuck at R free at 42 and same around 49 amino acids at N terminal and
8 amino acids at C terminal and some internal loops still absent.*

*Thank you in advance.*






>
>
>
>
>

[ccp4bb] suggestion for structure solution of a protein with low sequence identity

[Vikram Dalal]

Hi everyone,

We are trying to solve a protein structure of 2.6 A. We have processed it
with HKL2000. We have even tried processing with mosflm and xia2. It is in
C2221 space group (checked by pointless) and data is not twinned. It has
31% identical with a search model and has 57% sequence coverage. There are
2 subunits in asu.

I did not proper phases with MOLREP and phaser. I have tried Balbes (R free
50) and Mr BUMP (R free 54).But, density from balbes look some reasonable.
So i have refined it in ccp4i and phenix differently and then model build
it by coot. My protein has 377 Amino acid. But, I stucked at R free 41 and
now i have amino acid 50 to 369 in both chains, still 49 amino acid at N
terminal and 8 amino acid at C terminal are missing.

*I have tried the ARP/wARP, but it does not work for it. *



Thanks & Regards,


VIKRAM DALAL
Research Scholar
Macromolecular Crystallographic Unit
Department of Biotechnology
Indian Institute of Technology
Roorkee (INDIA)

Re: [ccp4bb] metal electron density detection

[Kabasakal, Burak V]

If figured out by yourself, this web page would help;


http://tanna.bch.ed.ac.uk/

METAL COORDINATION SITES IN PROTEINS
tanna.bch.ed.ac.uk
METAL COORDINATION SITES IN PROTEINS (last updated August 2011) This website 
assembles information about the geometry and constitution of metal coordination 
groups in ...

Best regards,


Burak



From: CCP4 bulletin board  on behalf of Robbie Joosten 

Sent: 25 October 2016 11:02:27
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] metal electron density detection

Hi Ansuman,

Do you really want to do this in an automated way? Wouldn't you rather figure 
it out for yourself?
There is some automation in Phenix for ion building, but you still need to 
verify yourself. The CheckMyMetal server is quite good for that, but beware 
that to some extent you find what you model because you severely bias the metal 
coordination. Mespeus has a lot of info about common coordination for specific 
metals from the PDB, but the dataset is not filtered. Which metals are you 
talking about, the ones from your crystallization condition, or the 'native' 
ions. If you don't add the metal you can try identifying it from adsorption 
spectrum. You can also try metal substitution. Anomalous data helps a lot too, 
especially if you can compare different elements. You can also look close 
homologs, but I've seen cases where this did not give the right answer.

Cheers,
Robbie

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ansuman 
biswas
Sent: Tuesday, October 25, 2016 11:05
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] metal electron density detection

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

Re: [ccp4bb] metal electron density detection

[Robbie Joosten]

Hi Ansuman,

 

Do you really want to do this in an automated way? Wouldn’t you rather figure 
it out for yourself?

There is some automation in Phenix for ion building, but you still need to 
verify yourself. The CheckMyMetal server is quite good for that, but beware 
that to some extent you find what you model because you severely bias the metal 
coordination. Mespeus has a lot of info about common coordination for specific 
metals from the PDB, but the dataset is not filtered. Which metals are you 
talking about, the ones from your crystallization condition, or the ‘native’ 
ions. If you don’t add the metal you can try identifying it from adsorption 
spectrum. You can also try metal substitution. Anomalous data helps a lot too, 
especially if you can compare different elements. You can also look close 
homologs, but I’ve seen cases where this did not give the right answer. 

 

Cheers,

Robbie

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ansuman 
biswas
Sent: Tuesday, October 25, 2016 11:05
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] metal electron density detection

 

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

Re: [ccp4bb] metal electron density detection

[Nicolas FOOS]

Dear Ansuman,

this could help you, it explain how it's working on Phenix.refine

Echols, N., Morshed, N., Afonine, P.V., McCoy, A.J., Miller, M.D., Read, 
R.J., Richardson, J.S., Terwilliger, T.C., and Adams, P.D. (2014). 
Automated identification of elemental ions in macromolecular crystal 
structures. Acta Crystallographica D /70/, 1104–1114.


Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 25/10/2016 11:04, ansuman biswas wrote:

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

Re: [ccp4bb] metal electron density detection

[David Briggs]

Hi Ansuman,

Phenix.refine* will place metal ions if you ask it to:


   - *Ion placement* extends solvent picking to build elemental ions such
   as calcium and zinc. To enable this option, simply enter a list of the
   elements to search for; this will also enable solvent picking as a first
   step. This option is not recommended if you have data worse than 3.0A
   resolution, and works best with anomalous data.

 https://www.phenix-online.org/documentation/reference/refine_gui.html

*other refinement programs are available.

HTH,

Dave


On Tue, 25 Oct 2016 at 10:08 ansuman biswas <
0d48627c03ca-dmarc-requ...@jiscmail.ac.uk> wrote:

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

-- 


[image: --]

David Briggs PhD
[image: https://]about.me/david_briggs

[ccp4bb] metal electron density detection

[ansuman biswas]

Hi all,

Is there any program (like ARP/wARP) available to locate the
correct metal atom automatically in the electron density ?

best,

Ansuman

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Savvas Savvides]

Dear Pascal,

the EM2DAM program from the ATSAS package for SAXS 
(https://www.embl-hamburg.de/biosaxs/manuals/em2dam.html 
) can convert any EM 
envelope to a dummy-atom model in pdb format.
Also, the recent SUPALM algorithm might provide additional options as well.
http://journals.iucr.org/j/issues/2016/03/00/ap5002/ap5002.pdf 



best wishes
Savvas


Savvas Savvides
Ghent University, L-ProBE
VIB Inflammation Research Center (IRC)
Technology Park 927, B-9052 Ghent (Zwijnaarde), Belgium
+32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype: 
savvas.savvides_skype
http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx 




> On 25 Oct 2016, at 10:00, Randy Read  wrote:
> 
> Dear Pascal,
> 
> I'm assuming that you're talking about using the negative stain image as an 
> MR model.  I don't recall hearing of this having ever worked (though I would 
> be very interested of course if anyone has managed to do this!), but my 
> intuition is that it's not going to work.  Negative stain just gives you an 
> external shape, whereas a cryo-EM reconstruction has internal features as 
> well.
> 
> Presumably you don't have atomic models of the individual components of the 
> complex?  If you did, using those directly for MR would be my first choice, 
> but you could also consider making a pseudo-atomic model by docking them into 
> the shape of the negative stain image.  Such a model would add substantial 
> higher-resolution information.
> 
> Best wishes and good luck,
> 
> Randy Read
> 
>> On 25 Oct 2016, at 02:49, Pascal Egea > > wrote:
>> 
>> Dear All,
>> 
>> I would like to know if it is possible to use a low resolution EM 
>> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
>> help molecular replacement in a 4.5A resolution X-ray diffraction data set 
>> of the same complex
>> I am aware of the possibility of using low resolution cryoEM maps for MR as 
>> described in the review from Jackson et al in Nature Protocols but I was 
>> wondering if there is an intrinsically impossibility for negative stain 
>> reconstructions.
>> 
>> Any thoughts or advice will be greatly appreciated.
>> 
>> Best,
>> 
>> -- 
>> Pascal F. Egea, PhD
>> Assistant Professor
>> UCLA, David Geffen School of Medicine
>> Department of Biological Chemistry
>> Boyer Hall room 356
>> 611 Charles E Young Drive East
>> Los Angeles CA 90095
>> office (310)-983-3515
>> lab  (310)-983-3516
>> email pegea at mednet.ucla.edu 
> 
> --
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research  Tel: + 44 1223 336500
> Wellcome Trust/MRC Building   Fax: + 44 1223 336827
> Hills RoadE-mail: rj...@cam.ac.uk 
> 
> Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk 
> 

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Alexandre OURJOUMTSEV]

Dear Pascal,

A while ago when I did  my tests on MR at a very low resolution, I did it both 
with “shaped” envelopes and with the “flat” ones. It did work for both (with 
simulated data, which is obviously not the same as the experimental), but MR 
with the “shaped” models was more robust.

Some traces of this are in the article in Acta Cryst D51 (1995) 888-895.

Best regards,

Sacha Urzhumtsev


De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Pascal 
Egea
Envoyé : mardi 25 octobre 2016 03:49
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] MR phasing using Negative Stain EM reconstruction

Dear All,

I would like to know if it is possible to use a low resolution EM 
reconstruction of a complex obtained in negative stain EM (not cryo EM) to help 
molecular replacement in a 4.5A resolution X-ray diffraction data set of the 
same complex
I am aware of the possibility of using low resolution cryoEM maps for MR as 
described in the review from Jackson et al in Nature Protocols but I was 
wondering if there is an intrinsically impossibility for negative stain 
reconstructions.

Any thoughts or advice will be greatly appreciated.

Best,

--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu

Re: [ccp4bb] MR phasing using Negative Stain EM reconstruction

[Randy Read]

Dear Pascal,

I'm assuming that you're talking about using the negative stain image as an MR 
model.  I don't recall hearing of this having ever worked (though I would be 
very interested of course if anyone has managed to do this!), but my intuition 
is that it's not going to work.  Negative stain just gives you an external 
shape, whereas a cryo-EM reconstruction has internal features as well.

Presumably you don't have atomic models of the individual components of the 
complex?  If you did, using those directly for MR would be my first choice, but 
you could also consider making a pseudo-atomic model by docking them into the 
shape of the negative stain image.  Such a model would add substantial 
higher-resolution information.

Best wishes and good luck,

Randy Read

> On 25 Oct 2016, at 02:49, Pascal Egea  wrote:
> 
> Dear All,
> 
> I would like to know if it is possible to use a low resolution EM 
> reconstruction of a complex obtained in negative stain EM (not cryo EM) to 
> help molecular replacement in a 4.5A resolution X-ray diffraction data set of 
> the same complex
> I am aware of the possibility of using low resolution cryoEM maps for MR as 
> described in the review from Jackson et al in Nature Protocols but I was 
> wondering if there is an intrinsically impossibility for negative stain 
> reconstructions.
> 
> Any thoughts or advice will be greatly appreciated.
> 
> Best,
> 
> -- 
> Pascal F. Egea, PhD
> Assistant Professor
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> Boyer Hall room 356
> 611 Charles E Young Drive East
> Los Angeles CA 90095
> office (310)-983-3515
> lab  (310)-983-3516
> email pegea at mednet.ucla.edu 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

[ccp4bb] Postdoctoral positions at the University of Limerick

[Tewfik Soulimane]

*Postdoctoral positions – Molecular Structural Biology at the University of
Limerick, Ireland*



Post-doctoral positions are available for highly motivated, hard-working
candidates to join a research group interested in investigating the
structure and function of selected membrane proteins. The funded projects
will involve biochemical and biophysical characterization of macromolecular
complexes, including structure determination by X-ray crystallography.

Minimum Requirements
Applicants should hold a PhD in Biochemistry or related disciplines and
have significant experience in molecular cloning, protein production and
X-ray crystallography, preferably on membrane proteins. Additional
background in enzymatic assay is considered is an asset. Interested
applicants should send curriculum vitae, a summary of past research
experience and accomplishments and contact information of three referees to
Tewfik Soulimane, molecular structural biology group, University of
Limerick, Ireland (Email: tewfik.soulim...@ul.ie)





*Prof Dr Tewfik Soulimane*

*Course Director Industrial Biochemistry*

*Department of Chemical and Environmental Sciences*

*Materials and Surface Science Institute*

*Univeristy of Limerick*

*Limerick*

*Ireland*

*Tel.: +353- 61-234133*

*Mobile: +353-86-1238746*