Basic Tutorial for VMD

The VMD interface consists of 3 windows

VMD Main – Main area with options to load molecules, change display options, and various analysis options

VMD OpenGL Display – The display window, you should see VMD spinning in red, green and blue.

A Terminal Window – displays the logfile, accepts commands

Loading a Molecule

On the VMD Main window, select “File” -> “New Molecule”

This brings up a new window called the Molecule File Browser, click on “Browse”, and browse to your TOPLOGY file, it will normally end in .prmtop. The filetype will be automatically recognized as AMBER7 Parm.  Click on “Load”.

The terminal window should give some output like this:

Info) Analyzing structure ...

Info)    Atoms: 510

Info)    Bonds: 532

Info)    Angles: 0  Dihedrals: 0  Impropers: 0  Cross-terms: 0

Info)    Bondtypes: 0  Angletypes: 0  Dihedraltypes: 0  Impropertypes: 0

Info)    Residues: 26

Info)    Waters: 0

Info)    Segments: 1

Info)    Fragments: 2   Protein: 0   Nucleic: 0

Go back to the Molecule File Browser and click “Browse”, then select your trajectory file, this should end in .mdcrd. VMD will automatically select “AMBER coordinates” because of the MDCRD file extension. This will be fine if you used the “nobox” command in ptraj. But if you have a periodic box, then you should change this option to “AMBER coordinates with periodic box”

Only use the periodic box if your trajectory file defines this

Postdoctoral position at the University of Chicago

From: Robert Keenan
Date: 26 August 2011 17:17
Subject: [ccp4bb] Postdoctoral position at the University of Chicago


Postdoctoral position at the University of Chicago

The Keenan lab in the Department of Biochemistry & Molecular Biology at the University of Chicago seeks to recruit an outstanding postdoctoral scientist with a strong interest in membrane protein biogenesis. The main goal of the lab is to understand, in molecular mechanistic detail, how membrane proteins are targeted to, and inserted into biological membranes. We do this using a combination of structural, biochemical and genetic approaches.

This position offers an excellent opportunity for an experienced protein biochemist to complement his or her expertise using modern tools for structural analysis. The focus of the project is on a series of soluble and integral membrane proteins (and their complexes), which coordinate targeting and insertion of eukaryotic membrane proteins. The postdoc will benefit from the highly collaborative University of Chicago community, and our regular access to the nearby Advanced Photon Source (APS) at Argonne National Laboratory.

Candidates should have (or expect) a Ph.D. in biochemistry or a related field, and have strong experience in molecular biology, protein expression, purification, crystallization and functional analysis. A significant publication record is an advantage.

To apply, please email a PDF of your CV to bkeenan@uchicago.edu.

For more information, please visit the lab website:  http://keenanlab.bsd.uchicago.edu

Spherulites and PEG3350

From: Jan van Agthoven
Date: 24 August 2011 19:05


Dear all,

I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration?

Thanks, 

----------
From: Savvas Savvides
Date: 24 August 2011 19:40

Dear Jan

I would recommend running the following protocol on your spherulites. Just pretend that they are crystals :)

This was posted some time ago on the ccp4bb.

best regards

Savvas

On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete <  wrote:  > Hi Ivan, > > there are several tests (e.g. Izit dye, crush test) you can do discern > protein from salt crystals but what was always very informative to me (and > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the > crystals using the following protocol: > > - select a drop which contains some substantial crystalline material. The > crystals can be many and small (crystal shower) or few and large. > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother > liquor containing a 10% higher concentration of precipitant) > - transfer all the crystalline material from the drop into the PCR-tube > using a pipet (use stabilizing buffer from the PCR tube to collect all > crystals) > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the > crystals under the microscope. They should be at the bottom of the PCR-tube. > - Remove as much as supernatant as you can (make sure not to remove your > crystals), add stabilizing buffer to wash the crystals, and centrifuge again > - repeat this washing protocol a few times > - after the final washing step, add Laemli-buffer to the crystals and use > this sample to load the SDS-PAGE gel > - include a positive (eg. solubilize another drop directly in > Laemli-buffer) and a negative (final washing buffer) control > - use silver staining to visualize the protein > > This always works for me. If you don't see a band at this point I would be > worried that it is salt. You could then choose to do a Western blot instead > of silver staining to increase the sensitivity. Make sure to include control > samples then. > > Kind regards, > > Kenneth Verstraete > L-PROBE > Ghent University > Belgium

----------
From: Gregory Verdon
Date: 24 August 2011 19:42



You should check these "spherulites" on SDS-PAGE gel to make sure that these contain your protein. Then you can start thinking about optimization.

----------
From: Regina Kettering
Date: 24 August 2011 19:46

Something to consider is the quality of the PEG 3350.  We have found that different qualities of PEG 3350 can give different results, depending on the type and amount of contaminants.  What used to be the Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax).  We use high quality PEG 3350 for normal screening, but switch to the highest quality grade we can get for optimizing.

Regina

---

From: Jan van Agthoven
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350

----------
From: Jan van Agthoven
Date: 24 August 2011 20:08



Thanks for the protocole and advice! I'll put my spherulites on gel. It will make things clear.

Jan

----------
From: Prince, D Bryan
Date: 24 August 2011 20:09

Something else to try would be the Protic Ionic Liquid kit from Hampton. I recently had crystals of a protein that would only grow as laminated stacks of plates. Optimizing the conditions and using an additive screen didn't improve crystal morphology. I tried the PIL kit from Hampton and was able to get single, thick plates in several conditions. At the ACA meeting in Hawaii a few years back, there was a poster about ageing your PEG solutions by microwaving them and letting them cool on the bench. This was the only way that the poster's author could get reproducible crystals of her target protein.

Good Luck!

Bryan

From: CCP4 bulletin board On Behalf Of Regina Kettering
Sent: Wednesday, August 24, 2011 2:47 PM

Subject: Re: [ccp4bb] spherulites and PEG3350

----------
From: Tom Peat
Date: 24 August 2011 22:48

You might to consider that PEG 3350 has phosphate contamination, so playing around with small amounts of phosphate (or removing it) might be worthwhile.
Cheers, tom

From: Regina Kettering
Sent: Thursday, August 25, 2011 04:46 AM

Postdoc Position at EMBL Grenoble

From: Matthias Haffke
Date: 26 August 2011 10:24
Subject: [ccp4bb] Postdoc Position at EMBL Grenoble

The Berger laboratory at the European Molecular Biology Laboratory (EMBL) Grenoble, France seeks to recruit an outstanding postdoctoral scientist in structural biology with a research focus directed towards the structure of macromolecular assemblies. The major theme within the group is the structural molecular biology of multiprotein complexes in human gene expression. For this purpose, we are also very active in the development and implementation of advanced recombinant expression technologies (Fitzgerald et al. Nat. Methods 2006; Bieniossek et al. Nat. Methods 2009; Kriz et al. Nat. Communications 2010). Notably our MultiBac technology is our unique asset for producing large multisubunit transcription factors in the quality and quantity required for structural analyses (Berger et al. Nat. Biotechnol. 2004, Imasaki et al. Nature 2011). A number of highly purified complexes are available in the laboratory. Information about our work can be obtained from http://www.embl.fr/research/unit/berger/index.html.

This position requires a Ph.D. in biochemistry or a related field, with a strong background in molecular biology and protein biochemistry. The applicant has experience in structure determination projects from start to finish, experience in expressing and purifying protein complexes and /or Electron Microcopy or X-ray crystallography are advantageous. The candidate is a highly motivated individual who enjoys working as part of a collaborative and multidisciplinary, dynamic and interactive team.  

The laboratory is excellently situated in the structural biology environment at the Polygone Scientifique in Grenoble. Access to our Eukaryotic Expression Facility (EEF), synchrotron X-ray radiation (ESRF), modern biophysical instrumentation (surface plasmon resonance, dynamic light scattering, isothermal calorimetry, analytical ultracentrifugation), and to electron microscopy is provided. Complementary collaborations with leading laboratories in the field are in place.

The position is available immediately and funded for 2 years. It can be extended dependent on performance.

To apply please send your CV, a statement of research interests, and names (including email address) of at least two referees by email to Dr. Imre Berger (iberger@embl.fr). Applications will be accepted until the position is filled.

Postdoctoral positions in Geneva

From: stéphane Thore
Date: 25 August 2011 07:57

We offer 4 Postdoctoral Positions in Structural Biology in Geneva (Switzerland) and Grenoble (France).

These positions are available immediately in the laboratories of Profs. Thanos Halazonetis, Robbie Loewith and Stéphane Thore at the Molecular Biology department and the group of Dr. Christiane Schaffitzel at the EMBL in Grenoble. We seek highly motivated and outstanding biochemists or structural biologists to perform structure / function studies of large kinases and their associated factors using X-ray crystallography and electron microscopy techniques. These complexes are at the core of multiple cellular responses like DNA damage, growth control or non-sense-mediated decay. Defaults in these pathways are linked to diseases including cancer, cystic fibrosis and others. Many  targeted proteins are highly relevant for drug discovery.

The positions are initially funded for three years. Strong candidates will be encouraged and supported to apply for competitive fellowships. Candidate should be within three years from obtaining their PhD degree. A strong background in crystallography or in electron microscopy documented by an excellent publication record is a plus for the candidates.

Applications with CV, contact information for three references and a description of the candidate's past research experience should be sent to stephane.thore@unige.ch

With best regards,

Stéphane Thore, Ph. D

Assistant Professor

Department of Molecular Biology

University of Geneva, Science III

30, Quai Ernest-Ansermet

1211 Geneva 4

Tel: + 41 (0) 22 379 61 27

Finding obsolete pdb entries

From: Simon Kolstoe
Date: 24 August 2011 11:14

Dear ccp4bb,

About 6 years ago I noted a couple of structures I was interested in were removed from the pdb. I saw in a recent email discussion that it is possible to access obsolete entries, however unfortunately I do not have the pdb code of the structure I am interested in - and neither does the original publication list the pdb code. Is there a way of searching withdrawn/obsolete entries for author name, macromolecule etc. or are structures that were withdrawn over 5 years ago lost for good?

Thanks,

Simon

----------
From: Gerard DVD Kleywegt

!Hola! Actually, you can. Tom Oldfield has written a web interface to query the PDBe search database directly using SQL - it lives here:

    http://www.ebi.ac.uk/pdbe-as/pdbedatabase/

If you want to look for obsoleted entries by molecule name, paste the following into the SQL box:

Select * from pdb_status WHERE status_code in ('OBS')  and
upper(TITLE) like '%ISOMERASE%';

Hit the "Submit SQL" button and you will get a list of hits with the word "ISOMERASE" in their title. Hit the "Get" (Text) button to save the complete list of hits.

If you know one of the authors, try the following query instead:

Select * from pdb_status WHERE status_code in ('OBS')  and
upper(AUTHOR_LIST) like '%JONES%';

!No hay de que!

--Gerard

Modeling ligands in binding pockets when the density is weak.

From: Francis E Reyes
Date: 23 August 2011 19:36

Seems to be a quiet day on the BB, so I propose this question:


Suppose you have a ligand in the binding pocket and some mediocre data (3 A or so), the 'core' of the ligand is well defined in 2Fo-Fc map  using the model phases of your protein, however there are 'chains/tails' of the ligand which are not.  Composite omit or simulated annealing omit maps do not produce density for these 'chains'

The question here is how the chains/tails should be modeled (if at all).


[1] Model in the core, but remove the atoms for the chains  (and conclude the diffraction data do not support interactions with the protein and subsequent experiments are needed (higher resolution data, biochemical data, etc)).

or

[2] Model in the chains/tails noting that potential hydrogen bond donors/acceptors on the protein are within hydrogen bonding distance to the chains/tails. You do this and subsequent refinement still does not produce the expected density for the chains.


or

[3] Your solution here.



 If this situation has been discussed before, please let me know .

F

---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

----------
From: Ed Pozharski

So you need an earthquake :)

This is similar, imho, to the issue of disordered side chains:

https://docs.google.com/spreadsheet/gform?key=0Ahe0ET6Vsx-kdHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc&hl=en_US&gridId=0#chart

https://docs.google.com/spreadsheet/viewform?hl=en_US&formkey=dHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc6MQ#gid=0



--
"Hurry up before we all come back to our senses!"
                          Julian, King of Lemurs

----------
From: Bosch, Juergen

(3a) You could give GraphENT a shot first and see if you can do magic on visualizing the remaining bits of density.

You could also have multiple conformations, try refining with a low occupancy first and see what you get back.

(3b) use AFitt

Jürgen

P.S. I would set the occupancy to zero for those parts which you don't see density but I would never remove them unless you know that your ligands has been degraded.

......................

Jürgen Bosch

Johns Hopkins Bloomberg School of Public Health

Department of Biochemistry & Molecular Biology

Johns Hopkins Malaria Research Institute

615 North Wolfe Street, W8708

Baltimore, MD 21205

----------
From: David Schuller

Had one already, thanks.

--
=======================================================================
All Things Serve the Beam
=======================================================================
                              David J. Schuller
                              modern man in a post-modern world
                              MacCHESS, Cornell University
                              

From: Edward A. Berry


Apparently sent from the vicinity of U. Maryland and JHSPH, thanks.

----------
From: <Herman.Schreuder


Dear Francis,

Although I am a member of the "never truncate a disordered side chain"
camp, I think for ligands it is quite a different story.

For me a random disordered lysine on the protein surface is completely
uninteresting, except if one wants to examine the electrostatic surface.
A non-expert end-user is either not aware of truncations and ends up
with wrong results, or has to laboriously put back the side chains the
crystallographer laboriously had removed before.

However, a bound ligand is very different. Determining its binding mode
is usually THE goal of the study and every hydrogen bond with the
protein is discussed in great detail. Chemists and modelers use these
structures to design more potent ligands and theoreticians use these
structures to improve their force fields. Also biochemists and
biologists may be tempted to draw all kind of conclusions about
important interactions where in fact there may be none.

Especially when the ligand has designed to make a certain interaction
and in absence of experimental data you model the same interaction, the
chemist and modeler will be very happy and immediately jump on it to
design more of the same. I have seen many cases where theoretically, the
ligand would be able to make a wonderful interaction with the protein,
but that flexible side chains on the protein or ligand just did not want
to give up their freedom (entropy) to become locked in such an
interaction. Not seeing flexible parts of a ligand is not resolution
dependent. At higher resolution you see even less of the flexible parts
since there is less model bias possible.

So my approach: If there is weak or even very weak but real density for
the flexible parts of the ligands (I usually scroll down to 0.6 sigma),
I build the part, or build 2 or 3 conformations in case of discrete
disorder. Here I think I take more liberties then most of my colleagues.


However, if no convincing (weak) density is present above the noise
level I remove the undefined parts and do not even consider to leave
them in with occupancy zero. They will appear on the display of the end
user and give the impression that an interaction is present where there
is none. If the ligand would make the interaction, it would be visible
in the electron density maps.

My choice is definitively [1], but before rushing to get more
experimental data I would first put some brain power in it: maybe it is
better for binding not to fix certain flexible side chains (less entropy
loss), flexibility may endow the protein with broader substrate/ligand
specificity, there may be crystal contacts which prevent the correct
binding mode, components of the crystallization buffer may interfere
with proper ligand binding etc.

Best,
Herman

----------
From: Ed Pozharski

The second link above happens to point at the form editing page or such,
so please disregard (my googlebox is getting flooded with requests to
share it).  If you want to cast a belated vote, use this link

https://spreadsheets.google.com/viewform?hl=en&formkey=dHVNa3VodUtfbVQtZ2pnUFcxQkx6RHc6MQ

To see the results, see this one.
Ed.

--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
                                               Julian, King of Lemurs

----------
From: Dale Tronrud


  I agree with Herman, and would like to add to his list of
explanations why density may not be observed.  It is possible
that the compound binding to your protein simply doesn't contain
those bits without density.  I have known cases where the
compound in the vial does not match the label on the vial.  In
addition I've had cases where bits were cleaved from the compound
at some point before binding.  Sometimes you can't see it because
it simply isn't there.

Dale Tronrud

----------
From: Shya Biswas

Hi Francis,

Once I had asked Pavel Afonine the same questions and these were his suggestions but most of these can be implemented in phenix...

I guess there is no general/unified procedure to do this, and in most of cases the tools and outcomes vary case by case.

Some general points:

- Removing parts of model is unlikely to improve the map simply because this makes model even more incomplete. The main purpose of computing the omit map is not to improve the overall map but to verify the atoms in question.

- Typically, the density appears weak because the signal is buried in noise. For example, we do not see H atoms in 2A resolution maps not because the information about them is not there (note, H atoms being weak scatterers contribute the most to low resolution reflections - similarly to bulk solvent, and their contribution to high resolution data approaches zero), but because the model quality and therefore noise at that resolution such that it hides hydrogens' signal. However, we do see H atoms at high resolution (say 1A and higher) not because of presence of high resolution reflections, but because the model quality is typically high and the noise level is below the hydrogens' signal.
Having said this, one possible way of improving your "weak density" is to improve the model as much as you can: make sure you modeled all alternative conformations, all solvent, etc.

- B-factor sharpening may help, although keep in mind that it will enhance the noise too.

- Other options: kick maps, omit kick maps, b-factor sharpened kick maps...

- You can try GrowDensity method (Acta  Cryst. (1997). D53, 540-543) which is available as phenix.grow_density. This is still under (slow) development, so if you decide to go this route than I can help you with the details.

Pavel.

HTH,

Shya

Database with protein experimental pKa values, with possibility to search for Cysteines and .pdb structure

From: Thomas Juettemann
Date: 24 August 2011 16:31
Subject: [ccp4bb] Database with protein experimental pKa values, with possibility to search for Cysteines and .pdb structure

Hello all,

this might not be the appropriate place, but I was wondering if anyone
knows of a database with protein experimental pKa values, with
possibility to search for Cysteines and .pdb structures.

"PINT: Protein-protein Interactions Thermodynamic Database" seems to be down.

The question arose on the pymol mailing list, and I  thought I give it
a shot here.

Many thanks for any pointers!

Best,
Thomas

LABELIT with XDS

From: James Whittle
Date: 24 August 2011 13:10

Dear all,

I'm able to index my images in LABELIT. Is it possible to use that
indexing solution in XDS?

James

----------
From: Boaz Shaanan
Date: 2011/8/24

Hi,

I apologise for the empty message. If you take the cell dimensions that you got in LABELIT into XDS it should work. I guess you couldn't get XDS to index from fresh, is that correct?

         Boaz

----------
From: Nicholas Sauter
Date: 24 August 2011 18:12

James,

After indexing the images with LABELIT it is possible to write out an XPARM.XDS file that defines the unit cell, orientation, and beam parameters.  

For example, index an image first:

labelit.index lysozyme_001.img --index_only

This gives several possible Bravais lattice solutions...e.g. triclinic=1; tetragonal=9.  Suppose you want XDS parameters for the tetragonal setting:

labelit.xds_xparm 9

That outputs the XPARM.XDS file.  I'd like to get any feedback from the community...this feature was added to Labelit in March 2011.

Nick Sauter

--
Nicholas K. Sauter, Ph. D.
Computer Staff Scientist/Engineer
Physical BioSciences Division
Lawrence Berkeley National Laboratory
1 Cyclotron Rd., Bldg. 64R0121
Berkeley, CA 94720-8118
(510) 486-5713

PISA question

From: Zhou, Tongqing 
Date: 23 August 2011 17:16
To: CCP4BB@jiscmail.ac.uk

Dear CCP4bb,

We are trying to get some detailed interface information from an antibody:antigen complex, PDBePISA used to have an option to select atomic level details, now the new web interface only gives residue level information. Is there a way to run PISA (in the CCP4 package) with a different configuration to get the detailed information?

Thanks,

Tongqing

Tongqing Zhou, Ph.D.

Staff Scientist

Structural Biology Section

Vaccine Research Center, NIAID/NIH

Building 40, Room 4607B

40 Convent Drive, MSC3027

Bethesda, MD 20892

(301) 594-8710 (Tel)

(301) 793-0794 (Cell)

(301) 480-2658 (Fax)

----------
From: <eugene.krissinel
Date: 23 August 2011 17:50
To: CCP4BB@jiscmail.ac.uk


Dear Tongqing

No there is no such option in CCP4's PISA, but I put this on the list for further developments. Regrettably PDBe changed the interface after I moved out and I cannot help you with that. However, you may ask them to put that option back.

Sorry,

Eugene.

----------
From: MARTYN SYMMONS
Date: 24 August 2011 11:35
To: CCP4BB@jiscmail.ac.uk


Dear All

I may not have understood but if I run PDBe PISA
and go Interface>Details
then I get a table of the form eg for H-bonds

Hydrogen bonds
 1       A:GLN 213[ NE2]         3.52    A:SER   3[ O  ]
 2       A:ASN 112[ ND2]         2.77    A:SER   3[ O  ]
 3       A:GLN 213[ NE2]         2.99    A:SER   4[ O  ]
 4       A:LYS 154[ NZ ]         2.74    A:ILE   5[ O  ]
 5       A:ARG 214[ NH2]         2.75    A:GLU  16[ OE2]
 6       A:TYR 168[ N  ]         2.86    A:LYS  31[ O  ]
 7       A:TYR 168[ OH ]         3.00    A:ASN  34[ OD1]
 8       A:LYS 175[ NZ ]         2.95    A:ASP  36[ OD2]
 9       A:ARG 129[ NH1]         3.22    A:ASP 127[ O  ]
 10      A:ARG 129[ NH2]         2.90    A:ASP 127[ O  ]
 11      A:HIS 363[ ND1]         3.47    A:GLN 132[ O  ]
 12      A:HIS 363[ ND1]         3.41    A:GLN 132[ OE1]
 13      A:TYR 350[ OH ]         2.71    A:LEU 179[ O  ]

which seems to include atom details (not Hs however).

In the new pages there is no longer a pulldown for atomic detail but this now is the default.

Hope that helps
       Martyn



----------
From: Tom Oldfield
Date: 24 August 2011 14:22
To: CCP4BB@jiscmail.ac.uk


Tongqing

Thank you for highlighting an issue with a recent release of the PDBePisa service.  The  option to return the atom detail as well as the residue detail within the PISA service was accidentally missed from the production service during a major update of features.  I am sorry that this has effected your work.

We are have identified and fixed the problem and are currently testing the change and should have the  fix in production  by the end of the week.

Regards
Tom Oldfield
PDBe

Immediate opeining for a Post-DOC fellowship based at Diamond & the Research Complex at Harwell

From: Martin Walsh
Date: 24 August 2011 14:21
Subject: [ccp4bb] Immediate opeining for a Post-DOC fellowship based at Diamond & the Research Complex at Harwell



--------------------------------------------------------------------------------------
RESEARCH ASSOCIATE: A Multidisciplinary Approach to Protein Function and Exploitation
--------------------------------------------------------------------------------------

Department of Physics/Strathclyde Institute of Pharmacy and Biomedical Sciences
&
DIAMOND LIGHT SOURCE

FIXED TERM (1 yr)

Salary £29,099-£35,788

=======================================================================================

               Applications are invited for this joint post between the Department of Physics and the Strathclyde Institute of Pharmacy and Biomedical Sciences at the University of Strathclyde, and Diamond Light Source Ltd (the UK national synchrotron science facility situated at Harwell in Oxfordshire).  The post holder will be based at Diamond, with work being directed by Dr's Neil Hunt, Nicholas Tucker, Paul Hoskisson at Strathclyde and Martin Walsh at Diamond.

               The appointment is initially for one year. The aim of the project is to combine X-ray crystallography, time resolved 2D-IR spectroscopy and biochemical techniques to study the structure and function of the E coli Hmp NO dioxygenase enzyme system and to work towards integration of ultrafast spectroscopy with the Diamond MX beamlines for future exploitation in biological research. The project will involve elements of biochemistry, structural biology and infrared spectroscopy, including time resolved and multidimensional methods. Expertise in all areas is not required though a strong background in structural biology, protein biochemistry, biophysics, infrared spectroscopy or time resolved/ultrafast spectroscopy would be advantageous.

               The successful candidate will join ongoing collaborative projects in place between Strathclyde, DLS and the Research Complex at Harwell (http://www.rc-harwell.ac.uk) that aim to investigate the mechanisms of protein systems from a multidisciplinary perspective. Regular interactions with Strathclyde and the Research Complex are envisaged. As such the project will suit an ambitious and motivated candidate with a strong commitment to multidisciplinary research.

               For informal enquiries please contact martin.walsh@diamond.ac.uk, neil.hunt@strath.ac.uk, nick.tucker@strath.ac.uk or paul.hoskisson@strath.ac.uk.


For an application pack visit http://vacancies.strath.ac.uk or contact Human Resources, University of Strathclyde, Glasgow G1 1XQ. Tel. 0141 553 4133, quoting ref: JA/R70/2011.

Closing date: 22 September 2011

We value diversity and welcome applications from all sections of the community
The University of Strathclyde is a Registered Scottish Charity, No SCO15263

How to handle b-factors at low resolution and very incomplete model?

From: Francis E Reyes
Date: 23 August 2011 18:30


Hi all

What's a way to determining a B-factor to set for all residues of a model at low resolution (4A)? How valid is the wilson B at this resolution? (better than nothing?)

I'm in the process of fitting whole domains into some rasty experimental density maps and was thinking of doing a rigid body or very restrained  ( secondary structure | reference structure | etc ) refinement of the coordinates for helping the fit.

Thanks!

F



---------------------------------------------
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

----------
From: Ed Pozharski
Date: 23 August 2011 18:43



Why not refine the overall B-factor?  Even at 4A it should still be
legitimate.

--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
                                               Julian, King of Lemurs

----------
From: Engin Özkan
Date: 23 August 2011 19:15



I distinctly remember reading a paper where B factors were predicted based on the location of the atoms (core vs exposed, main chain vs side chain) and the predicted values were not that far off. I wonder if B factors could actually be restrained at low resolution towards values reasonable/expected/similar to model values, just as how we put in model restraints for coordinate refinement these days, since many of us working at low resolution have at some point or another experienced catastrophe with B factor refinement. I wonder if this has been tested...

Engin

--
Engin Özkan
Post-doctoral Scholar
Howard Hughes Medical Institute
Dept of Molecular and Cellular Physiology
279 Campus Drive, Beckman Center B173
Stanford School of Medicine
Stanford, CA 94305
ph: (650)-498-7111

----------
From: Ethan Merritt
Date: 23 August 2011 20:03



I believe that's called "TLS refinement" :-) :-) :-)

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

On 11:15 Tue 23 Aug     , Engin Özkan wrote:

> I distinctly remember reading a paper where B factors were predicted

> based on the location of the atoms (core vs exposed, main chain vs

> side chain) and the predicted values were not that far off.

Here's one along those lines:

Flexibility and packing in proteins

Bertil Halle

PNAS (2002) 99: 1274 –1279

--

Thanks,

Donnie

Donald S. Berkholz, Ph.D.

Research Fellow

James R. Thompson lab, Physiology & Biomedical Engineering

Grazia Isaya lab, Pediatric & Adolescent Medicine

Mayo Clinic

______________

PDRA job opportunity

From: Murray, James W
Date: 23 August 2011 15:48
Subject: [ccp4bb] PDRA job opportunity
To: CCP4BB@jiscmail.ac.uk


Dear all,

see below for a PDRA opportunity at Imperial college to work on the structural basis of Photosystem II assembly and repair.

James


http://www.jobs.ac.uk/job/ADC906/research-associate/


Research Associate
Imperial College London - Faculty of Natural Sciences, Division of Molecular Biosciences

Salary: £31,300 - £39,920 per annum

We are seeking to appoint a Research Associate to investigate the roles of assembly factors involved in the assembly and repair of Photosystem II using biochemical and structural biology techniques. The successful applicant will be based within the Nixon group in the Department of Life Sciences at the South Kensington Campus of Imperial College London. The post holder will assist in scientific research into the mechanisms of assembly and repair of Photosystem II. You will be expected to produce independent and original research; submit publications to journals and assist with teaching and administration of the research group.

The successful applicant will hold a PhD degree in biochemistry, microbiology, structural biology or a related discipline, or an equivalent level of professional qualifications and experience. You will have experience of experimental design and statistical analysis. You must also be able to demonstrate research experience in a molecular biology/cyanobacterial microbiology environment and experience of standard molecular biology techniques. A successful track record in academic publications is essential. Experience of culturing cyanobacteria, protein purification, synchrotron X-ray data collection, X-ray crystallography and biological NMR are desirable.

You must be willing to travel to synchrotrons for X-ray data collection and be able to follow protocols and learn new techniques in a concise and skilled manner. You must also work autonomously and show initiative with research and be able to prioritise work in response to deadlines. The willingness to travel within the UK and abroad to conduct research and attend conferences is essential. You must be prepared to work unsociable hours as the work demands from time to time.

You must have strong written communication skills and the ability to write clearly and succinctly for publication. Advanced computer skills, including word-processing, spreadsheets, Internet are also essential. A flexible attitude towards work and the willingness to cooperate as part of a team as well as the ability to work independently and be open-minded and cooperative are essential. You must also have an enthusiastic approach to research, the ability to develop and apply new concepts, techniques and methods and a creative approach to problem solving. You must show initiative, be committed to meeting deadlines, maintaining and enhancing facilities and training others in their use.

This is a fixed term position and is funded by the BBSRC for up to three years.

Our preferred method of application is online via our website http://www3.imperial.ac.uk/employment (please select "Job Search" then enter the job title or vacancy reference number including spaces - NS 2011 147 KT into "Keywords"). Please complete and upload an application form as directed, attaching a curriculum vitae, a cover letter and the name and contact details of two referees. For informal enquiries please contact Professor Peter Nixon: p.nixon@imperial.ac.uk

Alternatively, If you are unable to apply online, please contact Richard Bowman by email r.bowman@imperial.ac.uk to request an application form.

Closing date: 19 September 2011

Committed to equality and valuing diversity. We are also an Athena Silver SWAN Award winner and a Stonewall Diversity Champion.


--
Dr. James W. Murray
David Phillips Research  Fellow
Division of Molecular Biosciences
Imperial College, LONDON
Tel: +44 (0)20 759 48895

Check PDB file for missing atoms

From: Tiago Barros
Date: 23 August 2011 20:19:05 GMT+01:00

Dear all,

Does anyone know a program that will check a PDB file for missing atoms and output a list of the corresponding residues?

Many thanks in advance,

Tiago

> ***********************************************************************
>
> Tiago Barros, PhD
> Kuriyan lab - Molecular and Cellular Biology
> University of California, Berkeley
> 527 Stanley Hall, QB3
> Berkeley, CA 94720-3220
> USA
>
> ***********************************************************************
>


> From: Ed Pozharski
> Date: 23 August 2011 20:39:57 GMT+01:00
 >
> REFMAC reports a (truncated?) list in the log file.  Coot can also
> identify missing atoms.  I would be pretty sure there is something like
> phinix.which_atoms_are_missing or such :)
>
> --
> Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
>                                                Julian, King of Lemurs



> From: Pavel Afonine
> Date: 23 August 2011 21:01:41 GMT+01:00
>
> Hi Tiago,
>
> easy, two steps:
>
> 1) Save the lines between *** into a file say called run.py:
>
> ***
> import os,sys
> from mmtbx.monomer_library import pdb_interpretation
> from mmtbx import monomer_library
> import mmtbx.monomer_library.server
> from cStringIO import StringIO
>  
> def exercise(args):
>   file_name = args[0]
>   mon_lib_srv = monomer_library.server.server()
>   ener_lib = monomer_library.server.ener_lib()
>   processed = monomer_library.pdb_interpretation.process(
>     mon_lib_srv           = mon_lib_srv,
>     ener_lib              = ener_lib,
>     file_name             = file_name,
>     keep_monomer_mappings = True,
>     log                   = StringIO())
>   for monomer_mapping in processed.all_chain_proxies.all_monomer_mappings:
>     ma = monomer_mapping.missing_non_hydrogen_atoms.keys()
>     if(len(ma)>0):
>       print monomer_mapping.pdb_residue_id_str, "Missing atoms:", ma
>
> if (__name__ == "__main__"):
>   exercise(sys.argv[1:])
> ***
>
> 2) Run it from the command line as:
>
> phenix.python run.py model.pdb
>
> and it will list you incomplete residues and missing atoms, just like this:
>
> pdbres="ASP A   1 " segid="A   " Missing atoms: ['C', 'OD1', 'CA', 'CG', 'O', 'N', 'OD2']
> pdbres="THR A   5 " segid="A   " Missing atoms: ['C', 'CB', 'CA', 'OG1', 'O', 'N', 'CG2']
> pdbres="THR A  10 " segid="A   " Missing atoms: ['C', 'CG2', 'OG1', 'O']
> pdbres="SER A  12 " segid="A   " Missing atoms: ['CA', 'N']
>
> Let me know if you need any help with this.
> Pavel.
>
> P.S.: You need to have PHENIX installed.
>
>



> From: Pavel Afonine
> Date: 23 August 2011 21:14:49 GMT+01:00
>
> Hi Ed,
>
> On Tue, Aug 23, 2011 at 12:39 PM, Ed Pozharski wrote:
>  I would be pretty sure there is something like
> phinix.which_atoms_are_missing or such :)
>
> good idea! I can turn the script that I just sent into this command so it is available in a couple of days -:)
>
> Pavel.


> From: "Frances C. Bernstein"
> Date: 23 August 2011 21:52:28 GMT+01:00

> According to the PDB Format document, entries with missing
> atoms should have a parsable REMARK 470.  You might want
> to write some quick code to look for that REMARK.
>
> The PDB staff could comment about the accuracy of REMARK
> 470 in its entries.
>
>                           Frances Bernstein
>
> =====================================================
> ****                Bernstein + Sons
> *   *       Information Systems Consultants
> ****    5 Brewster Lane, Bellport, NY 11713-2803


> From: Diana Tomchick
> Date: 23 August 2011 23:12:10 GMT+01:00
>
> The RCSB PDB Validation server will also report this info for you.
>
> Diana
>
> **************************************************
> Diana R. Tomchick
> Associate Professor
> Department of Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214B
> Dallas, TX 75390-8816

------------------------------

Date: Wed, 24 Aug 2011 12:10:11 +0200
From: "Bernhard C. Lohkamp" 

Subject: Re: Check PDB file for missing atoms

Coot:

Extension -> Modelling -> Residues with Missing Atoms...

Or watch out for the blue bars in the rotamer validation graph in Coot.

B

Is there a protein that it could interact with different proteins by the same part?

> 2011/8/23 Yamei Yu
> HI all,
>
> We know one protein can interact with different partners by different
> domains or different parts. Is there a protein that it could interact
> with different proteins by the same part (maybe the same part but in
> different conformations?)? Thank you in advance!!
>
> yamei
>
>

> From: Petr Kolenko
> What about polyclonal antibody? I am sure that you find some
> structures in the PDB.
>
> Petr
>

> From: "Brett, Thomas"
> Look at AP-2 alpha-appendage. And for that matter, the beta appendage too. Alpha appendage binds ~30 different proteins using two sites on one domain.
>
> J Biol Chem. 2004 Oct 29;279(44):46191-203. Epub 2004 Aug 2.
> Dual engagement regulation of protein interactions with the AP-2 adaptor alpha appendage.
> Mishra SK, Hawryluk MJ, Brett TJ, Keyel PA, Dupin AL, Jha A, Heuser JE, Fremont DH, Traub LM.
>
> Structure. 2002 Jun;10(6):797-809.
> Accessory protein recruitment motifs in clathrin-mediated endocytosis.
> Brett TJ, Traub LM, Fremont DH.
>
>
> Mol Biol Cell. 2008 Dec;19(12):5309-26. Epub 2008 Oct 8.
> The AP-2 adaptor beta2 appendage scaffolds alternate cargo endocytosis.
> Keyel PA, Thieman JR, Roth R, Erkan E, Everett ET, Watkins SC, Heuser JE, Traub LM.
>
> -Tom
> Tom J. Brett, PhD
> Assistant Professor of Medicine
> Division of Pulmonary and Critical Care
> Washington University School of Medicine
> Campus Box 8052, 660 S. Euclid
> Saint Louis, MO 63110


_______________________________________
Radisky, Evette S., Ph.D.
> This is very common among the small GTP-binding proteins of the Ras
> superfamily.  For an interesting analysis (although doubtless there are
> lots of more recent examples) you might look at:
> Corbett and Alber, TIBS 26 (12) 710-716 (2001)
>
> Evette S. Radisky, Ph.D.
> Assistant Professor
> Mayo Clinic Cancer Center
> Griffin Cancer Research Building, Rm 310
> 4500 San Pablo Road
> Jacksonville, FL 32224
> (904) 953-6372
> 

> From: Poul Nissen

> Calmodulin
>
> Poul

> The RNA Polymerase II rpb1 CTD interacts with numerous other proteins - it's disordered in isolation, and adopts different conformations with different binding partners (at least in the available structures).

> From: Ed Pozharski
> Lysozyme interacts with two antibodies, HyHel-5 and D44.1 via the same
> epitope but different set of interactions.
>
> --
> "Hurry up before we all come back to our senses!"
>                           Julian, King of Lemurs

Programme for the 18th CCP4 Northern Protein Structure Workshop

The programme for the 18th CCP4 Northern Protein Structure Workshop is now online

http://www.chem.gla.ac.uk/research/groups/protein/gala/Program_2011.htm

B hairpin related.

> On 08/22/2011 03:01 PM, Anuradha Balasubramanian wrote:

> Dear all,

>

> I have a problem . i crystallized same protein in two different pH and when

> viewing the results an important beta hairpin seems extended in PyMOL .

> (image link below).

> https://picasaweb.google.com/anu4884/August222011?authuser=0&feat=directlink

> But analysis of hairpin in promotif shows that they are similar with same

> H-bonds.

> What can be the reason for such ambiguity?

>

> Regards

>

> ANU

>

>

> Dear ANU,
>
> I cannot follow the link to your picture, but one reason might be
> different algorithms in the two different programs for the secondary
> structure prediction. I am usually not too impressed by the suggestions
> that pymol make and you should always check the secondary structure
> prediction against your biochemical knowledge, at least to confirm the
> boundaries.
>
> Cheers, Tim
>

MR problem

> On 08/22/11 03:12, intekhab alam wrote:

> Hi there

> I have a data of heterotrimer protein complex  at 2.9A resolution.

> One protein consists of two domains. I tried phaser as well as molrep which gives a solution with only one domain.

> Rotation function and translation function were found to be fine for these solutions.

> I tried to find missing domain of the protein after fixing one of the domain or the other partner proteins using whole part or various truncations of missing domain.

> I also tried to find and build missing domain using Rosetta with the solutions of Molrep or Phaser as template.

> But, there were no solutions, or the solutions are clashed with other domain or proteins.

> Furthermore, R and Rfree is 30 and 40, respectively, and I could not reduce them further.

> There was almost no electron density map in the empty space so that I could not model manually.

>

> Plz guide me ,how can i look for the missing domain in the protein.

>

> regards

>

>

> --

> INTEKHAB ALAM

> LABORATORY OF STRUCTURAL BIOINFORMATICS

> KOREA UNIVERSITY, SEOUL

>

> Dear Intekhab,
> 
> Before putting a lot of effort in trying to find the missing domain, you first should make sure it is there. It may be that only two proteins of the heterotrimer were packed in the crystal, or that one domain got clipped off during crystallization. The way to do it, is to run an sds page gel of a crystal, or a number of crystals if they are too small.
> 
> Best regards,
> Herman
> 
>




> As already noted, it is possible that the other domain is not present, or not ordered. Try refining a bit without it, and see what your stats look like.
>
> It is also possible that it is present, but minor clashes are causing good solutions to be rejected. Go into the output file and examine the TF scores before overlap rejection.
>
> The R and Rfree sound pretty good for an MR solution. About the failure of these numbers to come down, I'm not sure what you tried. Refinement with REFMAC5 should bring them down. You should see indications in the 2fo-fc and fo-fc maps that they are accurate - i.e. that there are real features showing up that are not in your model. You can test that also by deleting some prominent side chains.
>



>

Protein crosslinking before crystallization

From:

Opher Gileadi

Hi all Does anyone have experience or insights on intramolecular crosslinking of proteins before crystallization? The idea is to lock a felxible (multidomain) protein into a restricted conformation. Thanks, Opher

Hi Opher, I seem to recall some old papers in which glutarldehyde was used (in the RESERVOIR only) in order to promote crystallization of proteins with suspected inter-domain flexibility. Unfortunately, I don't have specific references in mind. Cheers, Boaz I found this paper to be very helpful for a similar experiment. "Elucidation of the mechanism and end products of glutaraldehyde cross-linking reaction by x-ray structure analysis". Wine et al. Biotechnology & Engineering, Vol. 98, No. 3, October 15, 2007. Hi Opher, I have a project where a protein was made 'cysless' and selective cysteines were introduced to allow for directed crosslinking using MTS and aldrithiol activated reagents (see Toronto Research Company and Pierce for a range of reagents of different lengths). A side product of these crosslinks was a dramatic improvement in crystal diffraction. Katherine Rank Rayment Lab Thanks all for the comments. I was thinking of crosslinking a protein that hasn't crystallized. Cystein engineering seems a good idea but depends on the availability of a good model. We'll be trying mild crosslinking using bifunctional reagents of various lengths (I suspect glutaraldehyde will not be the best option). Opher

HOLE & CHANNEL

Mon, 15 Aug 2011

Dear CCP4,

I’m looking for the authors of the programs (listed below). I want to analyse our crystal systems and would like to use these algorithms. However, I’m not convinced the addresses on the papers are current.

Does anybody know how to contact the authors or of any other programs that exist for these calculations?

Is there anything in the CCP4 arsenal for this purpose?

I’m happy to be contacted directly.

Thanks,

Dave

“HOLE: A program for the analysis of the pore dimensions of ion channel structural models”

Oliver S. Smart, Joseph G. Neduvelil, Xiaonan Wang, B. A. Wallace, Mark S. P. Sansomt

J. Mol. Graphics, 1996, vol14

“An algorithm to find channels and cavities within protein crystals (CHANNEL)”: Oleg S. Kisljuk, Galina S. Kachalova and Nadejda Ph. Lanina

J. Mol. Graphics, 1994, vol12

David Hargreaves

-----------------------

From:

Andrew Purkiss-Trew

Oliver Smart is now with Global Phasing and HOLE is supported by/available from the Sansom lab at Oxford: http://hole.biop.ox.ac.uk/hole