Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Richard Bayliss]

I third it - we used 3.2M + 5% glycerol ammonium sulfate to  
cryoprotect crystals grown in 1.2M ammonium suphate (Bayliss et al.  
JBC 2002). This really helped diffraction too. My tip when handling  
drops at very high salt is to make a little circle of wet tissue paper  
around your coverslip to make a humidity chamber. You'll have much  
longer to fish crystals out of the drop before salt crystals start to  
form.


Good luck
Richard


Dr Richard Bayliss
Royal Society University Research Fellow
& Career Development Team Leader
Section of Structural Biology
Institute of Cancer Research
237 Fulham Road
London SW3 6JB
UK

Tel: +44 (0)20 71535557
Fax: +44 (0)20 71535457




On 19 Aug 2009, at 7:20 PM, James Holton wrote:

I second that one.  Ammonium sulfate is one of my favorite cryos.  I  
recommend making up a saturated solution of ammonium sulfate, as it  
is actually a very good cryo all by itself, and then "dilute" it by  
adding the rest of the stuff in your condition (buffers, etc. and a  
little bit of water).  You want to be a little below saturation so  
that the salt does not grow crystals of its own while you are  
soaking.  This is especially important if your protein crystals are  
hexagonal rods!


-James Holton
MAD Scientist

Savvas Savvides wrote:

Hi Brenda
Try > 3M ammonium sulfate itself! We have tried that with great  
succes by
cryo-cooling xtals grown at 3.2M AS straight out of their  
crystallization
drops. You can consult the "M&M" section in Kyndt J. et al  
Biochemistry 2007

Jan 9;46(1):95-105 for a more detailed description of what we did.

Best of luck
Savvas

 Savvas Savvides L-ProBE, Unit for Structural Biology Ghent  
University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32- 
(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be 
 http://www.lprobe.ugent.be/xray.html




-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Schulman, Brenda
Sent: Wednesday, August 19, 2009 6:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hello!

I would be grateful for suggestions on cryoprotectants for crystals  
growing

in > 3M ammonium sulfate.

Thanks!

Brenda


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Re: [ccp4bb] Unknown density

[Mark J. van Raaij]

How about a 3-fold disordered metal ion?

Mark



On 19 Aug 2009, at 17:07, Sampath Natarajan wrote:


Dear All,

Currently I'm modeling one structure with 2.6A data. I could find an  
unknown density which I located in the interface between the three  
similar subunits. This density shows very strong peak, which appears  
until 10 sigma level in the difference map. It seems to be a metal  
ion. But, already I could find two Zn ions in the active site and  
refined well. The aspartic acid of each subunit has very close  
interaction with this density. You can see the pictures, which I  
have attached with this mail. The crystallization condition is 0.05M  
CaCl2 0.1M Bis-Tris pH 6.5, 30% (v/v) PEG monomethyl ether 550.  
Suggestion about this unknown density will be more helpful to find  
the molecule.


thanks a lot,

Regards,

Sampath

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

[Joerg Standfuss]

Hi Brenda,

Maybe you want to have a look at the cryoprotectant database for  
protein crystals.


http://idb.exst.jaxa.jp/db_data/protein/search-e.php

There you can search for your precipitant and get a list of  
compatible freezing conditions that worked before including the  
reference. It is a nice starting point and a good inspiration.


Joerg


On 19 Aug 2009, at 17:18, Schulman, Brenda wrote:


Hello!

I would be grateful for suggestions on cryoprotectants for crystals  
growing

in > 3M ammonium sulfate.

Thanks!

Brenda


Email Disclaimer:  www.stjude.org/emaildisclaimer


***
Jörg Standfuss
Medical Research Council
Laboratory of Molecular Biology
Hills Road
Cambridge CB2 0QH

[ccp4bb] Vacancy: Scientific Database Curator- PDBe

[Jawahar Swaminathan]

Dear All,
I wish to draw your attention to a recent post opening at the Protein 
Databank in Europe (http://www.ebi.ac.uk/pdbe/) for a Scientific 
Database Curator. Details of the job and application procedure below:

Thanks - Jawahar Swaminathan
PDBe, European Bioinformatics Institute,
Cambridge CB10 1SD UK
---

SCIENTIFIC DATABASE CURATOR***

Grade: 4, 5 or 6, depending on qualifications and experience
Duty Station: EMBL-EBI Hinxton, near Cambridge, UK
Commencing date: As soon as possible
Job description:
The high quality curation of PDB entries is essential in establishing 
the PDBe database as a world-leading source of protein-structure 
information. To maintain and extend this position, the PDBe team is 
looking for an expert biologist for a demanding role in database 
curation. The work will involve annotating preliminary PDB submissions 
and extracting biological information relevant to a given entry. In 
addition, curators will draw on their own areas of expertise in 
contributing to the development of methods and procedures. 

Qualifications and experience: 
The postholder should hold a PhD in some area of structural biology or 
chemistry as well as have a broad knowledge in molecular biology. An 
indepth knowledge of biochemistry and/or protein structure analysis 
would be highly beneficial. The successful candidate will be computer 
literate and have proven experience of Linux/Unix, Emacs and molecular 
graphics software and scripting experience would be advantageous. 
Excellent communication and interpersonal skills are essential as is the 
ability to work as part of a team and pay particular attention to detail.  

Contract: An initial contract of 3 years will be offered to the 
successful candidate. This can be renewed, depending on circumstances at 
the time of the review.


Closing date:  13 September 2009
EMBL is an inclusive, equal opportunity employer offering attractive 
conditions and benefits appropriate to an international research 
organisation.


Please note that EMBL does not return CVs or attached documents to 
applicants.


**To apply, please send a CV (including names and addresses of referees) 
and covering letter, by email, quoting ref. no. W/09/071/EBI in the 
subject line, to: applicati...@ebi.ac.uk
General enquiries may be sent to applicati...@ebi.ac.uk or to the 
following address:

EMBL-EBI, Personnel, Wellcome Trust Genome Campus, Hinxton, CB10 1SD.
**--

Re: [ccp4bb] cyseteine modification

[Debajyoti Dutta]

Dear Sir,



Thank you all who have replied. I jast want to enquire that if there is any 
option in coot to introduce the OH bond with Cys residue as well as to 
introduce the secondary peptide like bond between carboxylic carbon with amino 
group of lys.



Sincerely

Debajyoti Dutta



On Wed, 19 Aug 2009 20:58:44 +0530  wrote

>







Dear Debajyoti



There is also the sulfenic acid species (-S-OH) which is actually

the first oxidized form of sulfhydryls on the way to sulfonic acid. However

sulfenic acids are very susceptible to further oxidation to sulfinic and 
sulphonic

acids, and therefore need a protective chemical environment to remain stable. 



See for example some previous work of ours on sulfenic and

sulfinic forms of active-site cysteines in glutathione reductase (Nature

Structure Biology vol 5, 267-271, 1998) and the corrresponding pdb entries 1dnc

and 1gsn.



Best regards



Savvas







 

Savvas Savvides 

L-ProBE, Unit for Structural Biology 

Ghent University 

K.L. Ledeganckstraat 35 

9000 Ghent, BELGIUM 

office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 

Email: savvas.savvi...@ugent.be 

http://www.lprobe.ugent.be/xray.html



















From: CCP4 bulletin board

[mailto:ccp...@jiscmail.ac.uk] On Behalf Of Debajyoti Dutta

Sent: Wednesday, August 19, 2009 4:42 PM

To: CCP4BB@JISCMAIL.AC.UK

Subject: [ccp4bb] cyseteine modification











Dear Sir,



Is there any other oxidation states of cysteine other than cysteine sulphinic

acid and cysteine sulphonic acid. In my protein, the cysteine molecule is

definitely overoxidized but the electron density is not corresponding to the

sulphinic or the sulphonic acid. The positive density looks as if it can

accomodate only one oxygen atom and not more.



Thank you for reply in advance. 



Sincerely

Debajyoti Dutta





 

  

  

  

 















>

>E-mail message checked by Spyware Doctor (6.1.0.447)Database version: 
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[ccp4bb] Postdoctoral position

[Lan Guan]

NIH-funded postdoctoral position is available immediately in a  
membrane protein transport and structural biology laboratory in the  
Department of Cell Physiology and Molecular Biophysics at Texas Tech  
University Health Sciences Center, Lubbock.  The laboratory is well  
equipped with the state-of–the-art equipment for robotic high  
throughput membrane protein crystallization.  Our current focus is on  
structure and mechanism of bacterial and human sugar transporters.   
Highly motivated candidates with a strong background in protein  
biochemistry or biophysics are encouraged to apply.  Experiences in  
molecular biology and/or protein X-ray crystallography are desirable.   
Competitive applicants should have demonstrated scientific  
productivity and innovation.  More information is available at http://www.ttuhsc.edu/SOM/physiology/faculty/Guan/guan.aspx 
 or http://www.webpages.ttu.edu/myousef/Guan.htm.  Salary is  
competitive based on experience. Please submit your CV through  
TTUHSC’s website (http://jobs.texastech.edu), requisition #79710, and  
the contact information for three references to Dr. Lan Guan (lan.g...@ttuhsc.edu 
).


 

[ccp4bb] Vacancy for crystallographer at Boston Univ School of Medicine

[Hwai-Chen Guo]

Dear All,

 I wish to bring your attention to a crystallographer postdoctoral opening 
at Boston University School of Medicine. Please see the job description below. 
Thanks,

HC Guo

Postdoctoral Fellow Position Open
Boston University School of Medicine
Department: Physiology and Biophysics
Location: Boston, Mass
URL: biophysics.bumc.bu.edu/faculty/guo/index.html
Start Date: immediately
Duration: 1-3 years
Description: A postdoctoral position is open for highly motivated individuals 
to work on biochemical and crystallographic studies of enzymes involved in 
post-translational processings and immunity. Some crystals are already 
available. The work may involve any aspect of protein purification, 
mutagenesis, crystal growth, data collection, model building and refinement. 
Suitable candidates will also have the chance to participate in the biochemical 
characterization of the proteins. A background in protein 
expression/biochemistry and/or x-ray structure determination is prerequisite. 
Experienced macromolecular crystallographers are preferred, but highly 
motivated recent graduates with a strong background in protein biochemistry and 
interested in learning crystallography are also encouraged to apply. The 
laboratory is fully equipped for biochemical experiments, and with 
state-of-the-art crystallographic facilities. In addition, the National 
Synchrotron Light Source at the Brookhaven National Laboratory is only 5-hour 
driving/ferry away. Available immediately, applications will be accepted till 
positions filled.
Other details: Equal Opportunity Employer 
Please submit: Cover letter with curriculum vita and the names and addresses 
(including e-mail addresses) of three referees
Person to contact: Dr. Hwai-Chen Guo
Surface mail address: 700 Albany Street, Boston, MA 02118
Email address: hc...@bu.edu
Phone number: 617-638-4023

Re: [ccp4bb] xplot 84 driver and "Bad plot84 file format" error

[Brad Bennett]

Hi all-
Thanks to Charles Ballard, who sent me an xplot driver file with
optimization turned off. Once I copied this into my
/usr/local/ccp4-6.1.2/bin folder, sourced and reloaded ccp4i, the plots
opened up without a problem. Please note that, at least in my hands, just
updating to CCP4 version 6.1.2 did not fix the problem.

Thanks again-
Brad

On Wed, Aug 19, 2009 at 9:09 PM, William G. Scott <
wgsc...@chemistry.ucsc.edu> wrote:

> Did you make the plt file on the same platform?  If not, try regenerating
> it (I never managed to get byte-swapping to work).
> Seems to work ok in 6.1.2 fink install (as does xloggraph
> and xccpjiffy2idraw).
>
>
> On Wed, Aug 12, 2009 at 10:49 AM, Brad Bennett wrote:
>
>> Hi all-
>> CCP4 distribution: 6.1.1
>> OS: Mac OS X, version 10.5.7 (Intel)
>>
>> I wanted to look at a plot output (.plt file) from a POLARRFN run invoked
>> within ccp4i. When attempting to open the plot file, I get a "Bad plot84
>> file format" but the archaic xplot84 driver window does open just with no
>> plot visible. I notice from the CCP4 BB archives that this was a problem for
>> Macs back in CCP4 version 6.0.2 and the fix was to get updated binaries or
>> download a tar ball from the ftp site. But those files are specific for
>> 6.0.2, right? It seems the xplot84 problem has persisted to version 6.1.1?
>> Or is it a Mac Intel compiler issue? I looked in /usr/local/ccp4-6.1.1/bin
>> and there is an xplot84driver but no xloggraph and xccpjiffy2idraw.  Any
>> patch available for 6.1.1 or am I missing something?
>>
>> Thanks!
>> Brad
>>
>
>
>
> --
> -
>
>
> William G. Scott
>
> contact info:  
> http://chemistry.ucsc.edu/~wgscott
>
> Please reply to:  wgsc...@chemistry.ucsc.edu
>
>
>
>

[ccp4bb] Postdoctoral position at UCLA on membrane protein structural biology

[vincent Chaptal]

Posted on behalf of Dr. Jeff Abramson:


   A postdoctoral position is available in the laboratory of
Dr. Jeff Abramson at the Department of Physiology, University of
California Los Angeles (UCLA) for a highly motivated individual with a
strong interest in structure and function of membrane transport
proteins. This is an NIH-funded project which aims to use crystal
structures as templates to design and implement state-of-the-art
biophysical techniques for tracking conformational changes in the sodium
galactose transporter (SGLT) during its transport process.

The successful applicant will hold a PhD and have ample knowledge in
expression, cloning and purification of membrane proteins as well as
versed in modern macromolecular crystallographic methods. The candidate
must possess excellent written and oral communication skills, and will
be expected to work in a diverse and highly collaborative environment.

The Lab is well equipped for membrane protein biochemistry and
structural biology including state-of-the-art crystallization robotics.
(http://www.physiology.ucla.edu/Labs/Abramson/index.html ) We also have
full access to the Molecular Biology Institute’s (MBI) X-ray core
facility featuring a Rigaku FRD generator using the RAXIS IV++ and
Quantum 4 CCD detectors. There is a large computational cluster and
monthly access to the ALS synchrotron in Berkley, California. UCLA has a
strong and highly collaborative macromolecular crystallography program
in place with 6 faculties and 50 students, staff and postdocs.

Los Angeles is a cosmopolitan city with 330 days a year of perfect
weather. Sunny beaches and hiking trails are just a few minutes away and
skiing within hours. There are also number of museums and playhouses
within walking distance from the university.

Funding support is available immediately for a minimum of 2 years
through the NIH grant mechanism. If interested, please send a CV, three
references, and a cover letter explaining your research interests to
Jeff Abramson: _jabram...@mednet.ucla.edu
_


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Unauthorized redisclosure or failure to maintain confidentiality may subject 
you to federal and state penalties. If you are not the intended recipient, 
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Re: [ccp4bb] electron density map

[Artem Evdokimov]

Hi,

 

Hard to say w/o moving the thing around, but it could be: pep flips, or even
(less likely) wrong chain direction. Incidentally, some of thr the side
chains 'just don't look right to me' - interpret that one as you wish but
e.g. the leucine in the middle seems particularly wrong (try auto-find best
conformer in Coot, I bet the opposite conformer is picked as the best).The
very bottom-most residue's carbonyl doesn't seem to be in the density
either.

 

Suggestions:

 

1.  Delete the top and bottom-most residues and execute real space
refinement a few times - drag the carbonyls around manually and see what
happens
2.  Replace all residues with glycine, do the real space refinement, and
then a few rounds of Refmac - see what comes up and rebuild sidechains.

 

Artem

 

"Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone" 

 Jorge Luis Borges

 

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mike
England
Sent: Thursday, August 20, 2009 7:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] electron density map

 

Hi all,

 

I will appreciate the comments on the 2Fo-Fc (1.5 sigma) and Fo-Fc (3.0
sigma) (at coot) maps as shown in attached picture .

 

I am working at 3.0 A resolution (MR phasing with more than 80% homology)
and current Rfree  is 0.27 and  FOM of 0.8.

 

How to interpret the positive density (green) in Fo-Fc map which overlaps
with the C-alpha tube? The side-chains of the polypeptide around this
regions seems to be properly fitting and registry of the sequence seems to
be OK.

 

Is this due to some kind of model bias? 

 

Thanks in advance.

 

Mike

Re: [ccp4bb] electron density map

[James Stroud]

I agree with Artem. I also don't think you have a register problem.  
All of the residues show typical 2fofc density for their types,  
especially considering the 3A data. The disappearing act of the asp  
carboxyl is typical of solvent exposed aspartates. Assuming the  
register is correct, you have a carbonyl flip somewhere throwing off  
the whole segment. The phe is telltale. The 2 dimensionality and  
limited region of the picture makes a full diagnosis difficult.


Also, the 2fofc is heavily biased. Don't trust it when it tells you  
where things are. I find 2fofcs essentially useless. Your milage may  
vary.


James


On Aug 20, 2009, at 5:24 PM, Mike England wrote:


Hi all,

I will appreciate the comments on the 2Fo-Fc (1.5 sigma) and Fo-Fc  
(3.0 sigma) (at coot) maps as shown in attached picture .


I am working at 3.0 A resolution (MR phasing with more than 80%  
homology) and current Rfree  is 0.27 and  FOM of 0.8.


How to interpret the positive density (green) in Fo-Fc map which  
overlaps with the C-alpha tube? The side-chains of the polypeptide  
around this regions seems to be properly fitting and registry of the  
sequence seems to be OK.


Is this due to some kind of model bias?

Thanks in advance.

Mike