[ccp4bb] Fwd: Aussie synchrotron sports sponsorship - very funny

[Ho-Leung Ng]

I hope this helped our Australian friends get more funding!


Ho


The TV show 'Thank God You're Here' goes to the synchrotron!

http://www.youtube.com/watch?v=N_zbySqumaA&feature=related

Re: [ccp4bb] Bfactor as Measure of Presence

[James Holton]

James Stroud wrote:

Hello,

I have a 2.45 A structure with an average b factor of 50.6. A region I 
am particularly interested in has an average b factor of 87. At what 
point do I say that the region is "disordered"? Does it come down to 
maps? If I have reasonable simulated omit maps but the b factor is 87, 
how much confidence can I have about my interpretation of the maps?


James


I think the traditional definition of "disordered" is that you don't see 
it in a map contoured at 1 sigma.  What kind of map seems to depend on 
your particular scientific pedigree and the epoch of crystallographic 
convention in which you live, such as the 2fo-fc era.  The 3fo-2fc clan, 
or the current maximum-likelihood era of 2mFo-DFc.  However, if the 
atoms are particularly important to you, you might not call them 
"disordered" until they don't show up in a Fo-Fc map contoured at 0.5 sigma.


There does not appear to be a clear definition.  In fact, what 
crystallographers call "disordered" might be called "high resolution" in 
some other technique.  After all, you know the atoms are in the unit 
cell somewhere, so your resolution is already ~10x better than the best 
visible light microscopes.  Hence, the B-factor is somehow connected to 
resolution.  I once did a survey of the PDB (Equation 4 in Holton JSR 
2009) plotting the average atomic B-factor of all PDB files with a given 
stated "RESOLUTION" listed in the file.  The plot (which I did not show 
in the paper) is here:

http://bl831.als.lbl.gov/~jamesh/pickup/reso_vs_avgB.png

One possible origin of the average general trend (B = 4*d^2+12 where d 
is the resolution) is because a B-factor Gaussian has a full-width at 
half-max roughly given by B = 14.2*fwhm^2 and if we consider that B ~ 
4*d^2 and sqrt(14.2/4) ~ 2, this implies that 2*fwhm ~ d, or that the 
"feature size" at a d-spacing of d is ~d/2 (picture a sine wave with 
full-cycle period d).
The minimum average B value of 12 for low d could arise because a carbon 
atom at rest has the width of a B-factor Gaussian with B ~ 12 (0.9 A), 
and since the actual B-factor Gaussian is convoluted with this shape, B 
values smaller than 12 have vanishingly small influence on the electron 
density. ... Maybe.


I'm sure there are other explanations for the origins of this trend, and 
I admit there are plenty of PDBs that do not obey it.  (It is an average 
trend)  But, this expression does suggest how one might relate the 
B-factor of an atom to an "effective resolution".  That is, atoms with 
high B-factors do not contribute significantly to high-angle spots, but 
atoms with low B-factors do.  So, one could say that your overall 
average B=50.6 indicates an effective resolution of 3.1 A and your "bad" 
region has an effective resolution of 4.3 A.  Now, your observed 
resolution is 2.45 A, which implies that your data are better than 
average, or perhaps your average B is being pulled up by the high-B 
region? 

Nevertheless, I still find it useful to convert B-factors into 
equivalent d-spacings because it is easier to think about how good 
electron density looks in terms of resolution.  That is, your electron 
density is a sum of a 4.3 A structure (your "bad" region) and a 2.45 A 
structure (your atoms with B ~ 36), which leads to an average overall B 
of 50.6.  Therefore, your "disordered" region has electron density 
similar to that of a typical 4.3 A structure, and the kinds of 
conclusions you can make from that part of the structure will be similar 
to what you could conclude if the overall structure were 4.3 A.  Sorry.  
This is probably not what you wanted to hear.


Interestingly, however, protein crystals that respond well to 
dehydration treatments (such as with the Proteros/Rigaku FMS) tend to be 
cases where one molecule in the ASU has significantly higher B than 
another.  After dehydration, the "loose" molecule orders up (sometimes).


-James Holton
MAD Scientist

Re: [ccp4bb] Refining partially occupied DNA on top of itself

[Ho-Leung Ng]

Hi Bryan,

 Can't you choose an asymmetric unit that includes two protein
molecules and the entire non-palindromic DNA?


ho

Re: [ccp4bb] Lost my protein

[Ho-Leung Ng]

Is your protein sticking to the membrane or crashing/aggregating from
the concentrating?

Try to find a buffer condition in which your protein is more soluble
and less aggregation prone. You can try varying salt concentration,
pH, and inclusion of additives like glycerol or detergent.


Ho
UC Berkeley

Re: [ccp4bb] CCP4BB Digest - 4 May 2009 to 5 May 2009 (#2009-124)

[Dima Klenchin]

I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and 
collected the supernatant, and then refolded the protein over a Ni column, 
as I had a his tag protein. To do this, you load and wash the protein 
normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll 
have to try a few concentrations) and then a detergent (I used Triton 
X-100). Then you can elute. Here's the paper I used as a guide:


Biotechnol Appl Biochem 2006, 43:137-145


Except it's the other way around - the column is washed in the presense of 
detergent which is then slowly removed by b-cyclodextrin washes. If the 
reversal thing worked for you, your protein likely does not require neither 
b-cyclodextrin nor Triton for refolding.


I've had more luck in on-column refolding by steps of decreasing urea 
concentrations: 2, 1, 0.5, 0 M (3-5 column volumes each with 30 min in 
between at room temperature).


Dima

Re: [ccp4bb] CCP4BB Digest - 4 May 2009 to 5 May 2009 (#2009-124)

[Geoffrey Feld]

Refold protein 8M Urea:


Sanjiv,

The recommendations for rapid dilution and trying 6M GdmCl are great
recommendations. As an alternative, I had a similar problem years ago with a
membrane-associated receptor domain that was mostly found in inclusion
bodies after expression (I assume this is your problem). We tried expressing
at lower temperatures and in the presence of sorbitol, which are common
methods for getting proteins to express in solution, but I found an
alternative method worked. This will work if you have a tag for column
purification (ie his tag). I incubated my pelets in 8M urea for a couple of
hrs at 37C, spun and collected the supernatant, and then refolded the
protein over a Ni column, as I had a his tag protein. To do this, you load
and wash the protein normally, but before eluting, run b-cyclodextrin (i
think 0.1%, you'll have to try a few concentrations) and then a detergent (I
used Triton X-100). Then you can elute. Here's the paper I used as a guide:

Biotechnol Appl Biochem 2006, 43:137-145

Hope that helps!
Geoff
-- 
Geoffrey K. Feld

Krantz Lab
College of Chemistry
492 Stanley Hall
University of California, Berkeley

"Vigilia pretium libertatis"

Re: [ccp4bb] pointless question

[Phil Evans]

I think Pointless 1.2.23 was a broken version which escaped to the  
wild in CCP4 release 6.1.0. The more recent release 6.1.1 has a fixed  
version, and some more recent versions are also available from our ftp  
site


eg ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.3.2...

sorry about this

Phil


On 6 May 2009, at 19:21, Robert Nolte wrote:

I'm hoping someone can help me with a pointless problem.  I am  
trying to reindex
data into an orientation that I used to solve the structure  
initially.  While
I can get pointless to give me the reindexing needed to make the new  
data match
the old data for the project, when I ask it to write the data to  
HKLOUT it does
not carry out the reindexing. I was under the impression from the  
documentation
it would write out the reindexed solution.  Am I doing something  
wrong or have
I found a bug in my particular space group. I seem to recall getting  
this to work
on a different project in the past.  I have also tried a number of  
different
versions of pointless, and all give me the same results. The output  
file is shown below.

Thanks in advance for any help.
Regards,
Bob Nolte


-> pointless hklin input.mtz  hklref reference.mtz hklout  
reindex.mtz >& pointless.log


contents of pointless.log

###
### CCP4 6.1: POINTLESS version 1.2.23 : 26/09/08##
###
User: unknown  Run date: 28/ 4/2009 Run time: 13:48:05


Please reference: Collaborative Computational Project, Number 4. 1994.
"The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst.  
D50, 760-763.

as well as any specific reference in the program write-up.

OS type:  linux
Release Date: 26th September 2008


  **
  **
  * POINTLESS  *
  *   1.2.23   *
  **
  *   Determine Laue group from unmerged intensities   *
  * Phil Evans MRC LMB, Cambridge  *
  * Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
  **
  **


---

Reading reference data set from file reference.mtz
Maximum resolution in file reference.mtz:1.810
Columns for F, sigF (squared to I): F_881  SIGF_881
Number of valid observations read:18733
 Highest resolution: 1.81
 Unit cell:   72.6672.6665.9890.0090.00   120.00
Space group: P 3 2 1

Spacegroup information obtained from library file:
Logical Name: SYMINFO   Filename: /apps/ccp4/ccp4-6.1.0/lib/data/ 
syminfo.lib


Maximum resolution in file input.mtz:1.870
Columns for F, sigF (squared to I): F_880  SIGF_880
Number of valid observations read:17028
 Highest resolution: 1.87
 Unit cell:   72.5872.5866.2090.0090.00   120.00
Space group: P 3 2 1

Possible alternative indexing schemes
 Operators labelled "exact" are exact by symmetry
 For inexact options, deviations are from original cell
(root mean square deviation between base vectors)
 Maximum accepted RMS deviation between test and reference cells  
(TOLERANCE) =  2.0


 [h,k,l]exact
   [-h,-k,l]exact


Normalising reference dataset


Log() fit for intensity normalisation: B (slope) -18.82


Normalising test dataset


Log() fit for intensity normalisation: B (slope) -18.21

Alternative indexing relative to reference file reference.mtz

$TEXT:Result:$$ $$

  Alternative reindexing   CC R(E^2)Number  
Cell_deviation

  [-h,-k,l]  0.9670.092 17012  0.14
   [h,k,l]   0.0670.495 17011  0.14
$$

===

Copying merged MTZ file from input.mtz
to reindex.mtz

 Reindexing operator [h,k,l]

 Real space transformation   (x,y,z)

Reindexed space group : P 3 2 1
 Crystal: HKL_base
 Cell:   72.58  72.58   66.290 90120
 Crystal: unknown
 Cell:   72.58  72.58   66.290 90120

 17080 reflections copied to output file

$TEXT:Reference: $$ Please reference $$
P.R.Evans, 'Scaling and assessment  of data quality' Acta Cryst.  
D62, 72-82  (2005).

$$

Re: [ccp4bb] homology modeling

[Sue Roberts]

A metaserver such as bioinfobank is helpful if your sequence is not  
super-secret, especially if one is not an experienced modeller.


http://meta.bioinfo.pl/submit_wizard.pl

It submits your sequence to a range of sequence-based and threading- 
based servers.  Models (c-alpha, built with modeller) can be  
downloaded, inspected, and compared.  Alignment files for input to  
modeller can also be downloaded, allowing you to easily build all-atom  
models.


Sue

Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
s...@email.arizona.edu
http://www.biochem.arizona.edu/xray

Re: [ccp4bb] Refolding of Denatured Protein

[Puey Ounjai]

In fact, I agree with Artem that detergent can be very difficult to get rid
of. It totally depend on the property of detergent. In your case that the
protein has no transmembrane region. You probably dont need much detergent
in your solution to keep your protein happy. I dont think having detergent
in solution would cause any problem in crystalization. Frankly, I have never
heard of any body get success trying to purify membrane associated protein
without detergent. Even if your protein get refolded correctly (this is a
very big if here), how can you know that your protein behave nicely after
refolding in your buffer solution. After the process, It can aggregate also.


According to you, the protein is in the cell pellet, Is it still membrane
associated or it express as an inclusion body? Are you sure that it is
correctly folded at the first place or it is already misfolded even before
you start purifying it? To my understanding, Refolding is a very difficult
process and it is so difficult to control so it is better to avoid the
process especially if you want to work with protein structure. You will
never know how heterogeneous your sample is or how well did it get refolded.
You can only check if the refolding can regain any activity or not.

My suggestion is that if you want to crystalize this protein, you should
find the way to avoid refolding if possible. If protein express as an
inclusion body then changing host might be a better way to go.

Good luck,
Puey

On Wed, May 6, 2009 at 5:27 AM,  wrote:

> I hate to be the Negative Nancy here - but removal of detergent can be a
> significantly harder task than removal of urea. Here's why:
>
> Urea is a small molecule which diffuses freely and does not normally stick
> to proteins - so removal of urea is no harder than removal of salt (we
> ignore the considerations of protein stability here). On the other hand,
> detergents can be easy or very hard to get rid of, depending on which
> particular detergent and what particular protein are brought together.
> Some of the nastier polymeric detergents are quite different to remove
> 'completely' because they stick to proteins, stick to plastic and/or resin
> beads, form micelles (thus refusing to dialyze) and so on and so forth.
>
> There are numerous acceptable ways to remove sticky detergents, and some
> of them include treatment with hydrophobic matrices that 'suck up'
> detergent molecules, exchange with a 'milder' detergent, and so forth.
>
> Good luck!
>
> Artem
> >
> > dear sanjiv
> > i dont think that the removal of detergent is more difficult than
> > urea,since u are purifying ur protein over Ni-NTA column,so after a few
> > wash over there u can remove the detergent completely.
> >
> > best
> > atul kumar
> >
> > -Original Message-
> > From: CCP4 bulletin board on behalf of David Cobessi
> > Sent: Tue 5/5/2009 6:18 PM
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] Refolding of Denatured Protein
> >
> > Sanjiv Kumar wrote:
> >>  I have not tried purifying this protein either with detergent or with
> >> /Guanidine Hydrochloride. I can try with both /Guanidine HCl and
> >> detergent. Since I got good purification with the 8M urea, I was
> >> thinking if this protein could be refolded and used further. Isn't
> >> removal of detergent more difficult then Urea?
> >> Sanjiv Kumar
> >> Lab. No. 411,
> >> Functional Genomics Unit,
> >> Institute of Genomics and Integrative Biology,
> >> New Delhi-110007
> >> India
> > Is it a transmembrane protein or not? or just a protein associated to
> > the membrane (with a GPI anchor for example)?
> > If it is a transmembrane protein, you should try to use detergents for
> > extraction and during the purification.
> > David
> >
> > --
> > David Cobessi
> > Institut de Biologie Structurale
> > 41, Rue Jules Horowitz
> > 38027 Grenoble Cedex-1, France
> > Tel:33(0)438789613
> > 33(0)608164340
> > Fax:33(0)438785122
> >
> >
>

Re: [ccp4bb] Bfactor as Measure of Presence

[Mark A. White]

James,

That is a highly subjective question.  However, given that your average
B-factor is 51, a B-factor of ~87 for the loop is not exceptionally
high.  It is less than twice the average.  If your average B was 10 and
your loop had a B-factor of 17 what would you think?  I would imagine
that if you applied TLS the residual isotropic B-factors might seem very
reasonable.  The real test is the quality of your maps.  I always look
as simulated omit maps and even RESOLVE solvent-flipped (reduced bias)
maps.  Others might use Arp-Warp, DM, Bucaneer/Pirate, or SHARP for
density modification/phase improvement.  What do Fo-Fc maps tell you?
Do you have excess electron density?  Are the carbonyls and Cb positions
visible in the map?  Is this a tube of wispy density which could be a
polypeptide or just noise?

Mark


On Wed, 2009-05-06 at 03:45 -0500, James Stroud wrote:

> Hello,
> 
> I have a 2.45 A structure with an average b factor of 50.6. A region I  
> am particularly interested in has an average b factor of 87. At what  
> point do I say that the region is "disordered"? Does it come down to  
> maps? If I have reasonable simulated omit maps but the b factor is 87,  
> how much confidence can I have about my interpretation of the maps?
> 
> James

Yours sincerely,

Mark A. White, Ph.D.
Assistant Professor, Dept. Biochemistry and Molecular Biology, 
Manager, Sealy Center for Structural Biology and Molecular Biophysics
X-ray Crystallography Laboratory,
Basic Science Building, Room 6.660 C
University of Texas Medical Branch
Galveston, TX 77555-0647
Tel. (409) 747-4747
Cell. (409) 539-9138
Fax. (409) 747-4745
mailto://wh...@xray.utmb.edu
http://xray.utmb.edu
http://xray.utmb.edu/~white

Re: [ccp4bb] pointless question

[Robert Nolte]

An output file is created on hklout with or without the -copy flag.   The problem is why is it picking the unity matrix (solution #2) for the reindexing operator rather than the  first matrix (-h -k l) that it identifies under reindexing (which is clearly the correct answer). -Original Message-
From: Jan Abendroth 
Sent: May 6, 2009 2:45 PM
To: Robert Nolte 
Subject: Re: [ccp4bb] pointless question

Hi Bob,including a -copy flag might not be totally pointless:pointless -copy hklin ...Jan2009/5/6 Robert Nolte 
I'm hoping someone can help me with a pointless problem.  I am trying to reindex
data into an orientation that I used to solve the structure initially.  While
I can get pointless to give me the reindexing needed to make the new data match
the old data for the project, when I ask it to write the data to HKLOUT it does
not carry out the reindexing. I was under the impression from the documentation
it would write out the reindexed solution.  Am I doing something wrong or have
I found a bug in my particular space group. I seem to recall getting this to work
on a different project in the past.  I have also tried a number of different
versions of pointless, and all give me the same results. The output file is shown below.
  Thanks in advance for any help.
        Regards,
        Bob Nolte


-> pointless hklin input.mtz  hklref reference.mtz hklout reindex.mtz >& pointless.log

contents of pointless.log

 ###
 ### CCP4 6.1: POINTLESS             version 1.2.23 : 26/09/08##
 ###
 User: unknown  Run date: 28/ 4/2009 Run time: 13:48:05


 Please reference: Collaborative Computational Project, Number 4. 1994.
 "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763.
 as well as any specific reference in the program write-up.

OS type:      linux
Release Date: 26th September 2008


        **
        *                                                    *
        *                     POINTLESS                      *
        *                       1.2.23                       *
        *                                                    *
        *   Determine Laue group from unmerged intensities   *
        *     Phil Evans MRC LMB, Cambridge                  *
        * Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
        *                                                    *
        **


---

Reading reference data set from file reference.mtz
Maximum resolution in file reference.mtz:    1.810
Columns for F, sigF (squared to I): F_881  SIGF_881
Number of valid observations read:    18733
   Highest resolution:     1.81
   Unit cell:   72.66    72.66    65.98    90.00    90.00   120.00
      Space group: P 3 2 1

 Spacegroup information obtained from library file:
 Logical Name: SYMINFO   Filename: /apps/ccp4/ccp4-6.1.0/lib/data/syminfo.lib

Maximum resolution in file input.mtz:    1.870
Columns for F, sigF (squared to I): F_880  SIGF_880
Number of valid observations read:    17028
   Highest resolution:     1.87
   Unit cell:   72.58    72.58    66.20    90.00    90.00   120.00
      Space group: P 3 2 1

Possible alternative indexing schemes
   Operators labelled "exact" are exact by symmetry
   For inexact options, deviations are from original cell
      (root mean square deviation between base vectors)
   Maximum accepted RMS deviation between test and reference cells (TOLERANCE) =  2.0

                               [h,k,l]    exact
                             [-h,-k,l]    exact

 Normalising reference dataset

Log() fit for intensity normalisation: B (slope)     -18.82

 Normalising test dataset

Log() fit for intensity normalisation: B (slope)     -18.21

Alternative indexing relative to reference file reference.mtz

$TEXT:Result:$$ $$

    Alternative reindexing           CC     R(E^2)    Number Cell_deviation
            [-h,-k,l]              0.967    0.092     17012      0.14
             [h,k,l]               0.067    0.495     17011      0.14
$$

===

Copying merged MTZ file from input.mtz
                          to reindex.mtz

   Reindexing operator         [h,k,l]

   Real space transformation   (x,y,z)

Reindexed space group : P 3 2 1
   Crystal: HKL_base
   Cell:   72.58  72.58   66.2        90     90    120
   Crystal: unknown
   Cell:   72.58  72.58   66.2        90     90    120

   17080 reflections copied to output file

$TEXT:Reference: $$ Please reference $$
P.R.Evans, 'Scaling and assessment  of data quality' Acta Cryst. D62, 72-82  (2005).
$$
-- --Jan AbendrothdeCODE biostructuresSeattle / Bainbridge Island, WA, USAwork: JAbendroth_at_decode.comhome: Jan.Abendroth_at_gmai

[ccp4bb] pointless question

[Robert Nolte]

I'm hoping someone can help me with a pointless problem.  I am trying to 
reindex 
data into an orientation that I used to solve the structure initially.  While 
I can get pointless to give me the reindexing needed to make the new data match
the old data for the project, when I ask it to write the data to HKLOUT it does 
not carry out the reindexing. I was under the impression from the documentation 
it would write out the reindexed solution.  Am I doing something wrong or have 
I found a bug in my particular space group. I seem to recall getting this to 
work 
on a different project in the past.  I have also tried a number of different 
versions of pointless, and all give me the same results. The output file is 
shown below.
  Thanks in advance for any help. 
Regards,
Bob Nolte

 
-> pointless hklin input.mtz  hklref reference.mtz hklout reindex.mtz >& 
pointless.log
 
contents of pointless.log

 ###
 ### CCP4 6.1: POINTLESS version 1.2.23 : 26/09/08##
 ###
 User: unknown  Run date: 28/ 4/2009 Run time: 13:48:05


 Please reference: Collaborative Computational Project, Number 4. 1994.
 "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 
760-763.
 as well as any specific reference in the program write-up.

OS type:  linux
Release Date: 26th September 2008


**
**
* POINTLESS  *
*   1.2.23   *
**
*   Determine Laue group from unmerged intensities   *
* Phil Evans MRC LMB, Cambridge  *
* Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
**
**


---

Reading reference data set from file reference.mtz
Maximum resolution in file reference.mtz:1.810
Columns for F, sigF (squared to I): F_881  SIGF_881
Number of valid observations read:18733
   Highest resolution: 1.81
   Unit cell:   72.6672.6665.9890.0090.00   120.00
  Space group: P 3 2 1

 Spacegroup information obtained from library file:
 Logical Name: SYMINFO   Filename: /apps/ccp4/ccp4-6.1.0/lib/data/syminfo.lib

Maximum resolution in file input.mtz:1.870
Columns for F, sigF (squared to I): F_880  SIGF_880
Number of valid observations read:17028
   Highest resolution: 1.87
   Unit cell:   72.5872.5866.2090.0090.00   120.00
  Space group: P 3 2 1

Possible alternative indexing schemes
   Operators labelled "exact" are exact by symmetry
   For inexact options, deviations are from original cell
  (root mean square deviation between base vectors)
   Maximum accepted RMS deviation between test and reference cells (TOLERANCE) 
=  2.0

   [h,k,l]exact
 [-h,-k,l]exact

 Normalising reference dataset

Log() fit for intensity normalisation: B (slope) -18.82

 Normalising test dataset

Log() fit for intensity normalisation: B (slope) -18.21

Alternative indexing relative to reference file reference.mtz

$TEXT:Result:$$ $$

Alternative reindexing   CC R(E^2)Number Cell_deviation
[-h,-k,l]  0.9670.092 17012  0.14
 [h,k,l]   0.0670.495 17011  0.14
$$

===

Copying merged MTZ file from input.mtz
  to reindex.mtz

   Reindexing operator [h,k,l]

   Real space transformation   (x,y,z)

Reindexed space group : P 3 2 1
   Crystal: HKL_base
   Cell:   72.58  72.58   66.290 90120
   Crystal: unknown
   Cell:   72.58  72.58   66.290 90120

   17080 reflections copied to output file

$TEXT:Reference: $$ Please reference $$
P.R.Evans, 'Scaling and assessment  of data quality' Acta Cryst. D62, 72-82  
(2005).
$$

Re: [ccp4bb] Refmac Question

[Eleanor Dodson]

It is a bit hard to understand why without looking at the structure..
One reason things get disordered is if the occupancies get misset so 
they add up to a number > 1.0. In that case REFMAC decided this a a 
"clash situation" and tries to remedy it ..


Could that have happened?

 I build alternate conformations ifI can see them. Obviously they 
exiist at every resolution, but like waters, it helps to position them 
if the density is sharp.


 Eleanor

Leonard Thomas wrote:
I have been going over some structure for a group here.  When the 
post-doc did the originally modeling  a number of residues looked to 
have alternate conformations.  The resolution of the structures are 
between 2.0 and 1.9 angstroms.  My first question is when re-refining 
in with an updated version of Refmac the residues with the alternate 
conformations become highly disordered.  The original version Refmac 
used was 5.2.0019 and the new version Refmac is 5.5.0088.  I think the 
original ccp4 version was 6.0.2 and the latest version of ccp4 is now 
being used.  All refine runs were done under OS X though on different 
processors.  As far as I can tell all the input parameters are the 
same.  Any ideas as to what is going on.


As a followup question, at what point do you actually consider 
alternate conformations?  The current resolution of these structures 
seem  to be an a gray area from my perspective, the the density seems 
to indicate alternate conformations.


Cheers,

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
620 Parrington Oval
Norman, OK 73019

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

[ccp4bb] Refmac Question

[Leonard Thomas]

I have been going over some structure for a group here.  When the post- 
doc did the originally modeling  a number of residues looked to have  
alternate conformations.  The resolution of the structures are between  
2.0 and 1.9 angstroms.  My first question is when re-refining in with  
an updated version of Refmac the residues with the alternate  
conformations become highly disordered.  The original version Refmac  
used was 5.2.0019 and the new version Refmac is 5.5.0088.  I think the  
original ccp4 version was 6.0.2 and the latest version of ccp4 is now  
being used.  All refine runs were done under OS X though on different  
processors.  As far as I can tell all the input parameters are the  
same.  Any ideas as to what is going on.


As a followup question, at what point do you actually consider  
alternate conformations?  The current resolution of these structures  
seem  to be an a gray area from my perspective, the the density seems  
to indicate alternate conformations.


Cheers,

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
620 Parrington Oval
Norman, OK 73019

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

Re: [ccp4bb] desktop models- summary

[Christopher Rife]

Thanks very much for the replies. Here is a list of resources:
http://www.bathsheba.com/
http://www.rpc.msoe.edu/cbm/
http://www.crystalprotein.com/
http://www.bioetch.com/
http://www.luminorum.com/
http://bioetch.com/

And if you want to roll your own:
http://www.rap-man.com/
http://homemade3dprinter.blogspot.com/

Chris


> -Original Message-
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
> Christopher Rife
> Sent: Tuesday, May 05, 2009 2:42 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] desktop models
> 
> Hi,
> 
> I am looking to have a model produced from a PDB, i.e. something that
> we
> can put on the desk for everyone to admire :)
> 
> Googling for such a thing is rather challenging, so I'm curious if
> anyone
> has suggestions as to where I might get something like that made. So
> far,
> I have found two options:
> 1. Molecular Dimensions:
> http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223&step=2
> (etching in a glass block)
> 
> 2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/
> (nylon models)
> 
> I think #2 is more what we have in mind. Does anyone have any
> experience
> with their final product in terms of quality and durability?
> 
> Thanks very much.
> 
> Chris
> 
>

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[ccp4bb] Refining partially occupied DNA on top of itself

[Bryan Schmidt]

Hello,
I am having difficulty using Phenix to build and refine a DNA duplex. The
issue is that my asymmetric unit consists of one protein monomer bound to
DNA, but the protein is a dimer and the DNA is not palindromic, so each
monomer is bound to a different sequence of DNA. As such, the density for
the two different DNA half-sites is averaged out in the asymmetric unit. I
have tried to place two duplexes directly on top of one another, each with
0.5 occupancy, and then refine. But I have noticed two problems. The first
is that when xyz refinement is off, and I look at the output files, the
density for DNA is awfully green, as if there were only *one* helix with
0.5 occupancy there. The other problem I noticed is that when I turn xyz
refinement on, and look at the output files, one of the two half-sites
gets moved several angtroms, so that it is in a region that generally has
no density. I expect that, if done properly, the backbone of both
half-site DNAs ought not to move. Any advice would be greatly appreciated.
Thanks,
Bryan Schmidt

Re: [ccp4bb] Bfactor as Measure of Presence

[Herman . Schreuder]

Dear James,

I had a similar case with an active site loop which could have an open
and closed conformation. This loop had extremely high B-factors but
good, albeit low electron density. I believe this was because the loop
had this (well-defined) conformation only in say 50% of the protein
molecules, having another or disordered conformation in the rest of the
molecules. The high B-factors are thus a refinement artifact of this
conformation having partial occupancy. In the end, it comes down to
(SA-omit) maps.

Best regards,
Herman 

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
James Stroud
Sent: Wednesday, May 06, 2009 10:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Bfactor as Measure of Presence

Hello,

I have a 2.45 A structure with an average b factor of 50.6. A region I
am particularly interested in has an average b factor of 87. At what
point do I say that the region is "disordered"? Does it come down to
maps? If I have reasonable simulated omit maps but the b factor is 87,
how much confidence can I have about my interpretation of the maps?

James

Re: [ccp4bb] problem in transformation of pqe 30 clone

[artem]

Hello

In order to know *exactly* what the reason for poor transformation outcome
was one has to do all sorts of experiments. This is not likely to be your
goal, right? Leaky expression, overload of DNA, somehow compromised cells,
or even plain old user error - and numerous other reasons can be proposed
(including some esoteric ones like incompatibility of your gene product
with a specific strain, for reasons going beyond protocol errors and
simple considerations).

This can take a couple of years to sort out.

If you're talking about using M15[pREP4] cells then the reason why you
failed earlier is likely to be toxicity - pREP4 plasmid supplies lacI
in-trans, which represses 'stray' transcription reasonably well. The cells
that you used before and did not succeed with must have had low intrinsic
levels of lacI and thus permitted leakage.

To test this theory you could streak a couple of colonies of the
M15[pREP4] cells on a plate that has a small amount of IPTG in the agar
(say 0.1 mM) - if the colonies don't grow at all, or appear to be very
tiny then you know it's your protein toxicity that's the issue here. Keep
in mind that bacteria are extremely survival-oriented and therefore you
will eventually generate 'safe' mutants and recombinants that will be able
to grow even on IPTG. Likely those mutants will be useless to you from
practical standpoint.

Once you induce the M15[pREP4] cells - the toxicity will manifest itself
again. You will likely observe total cessation of growth (for a long
period of time) and possibly even lysis of the culture. Therefore you may
have to adjust your fermentation protocol to take this into account. One
of the common adjustments is deliberate induction at higher OD, another is
to include 0.8% glucose in the growth medium; there are other options
available to you. Hopefully once you induce transcription, the gene
products do not shut down protein synthesis - which would be a disaster
since it would likely shut down its own synthesis as well. Since I don't
know what you're growing, this is just one of the possibilities :)

Artem

>
> now i have transformed pqe 30 clone into the m15 host successfully, can
> someone let me know exactly what was reason behind the problem into
> transformation??is it leaky expression into the other host that is toxic
> for the cells,if it so then will i would be  able to get good expression
> into this host???
>
> atul
> -Original Message-
> From: CCP4 bulletin board on behalf of ar...@xtals.org
> Sent: Tue 5/5/2009 6:24 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone
>
> To clarify: I am not implying that I've worked with many proteins that
> express better in XL1-blue cells than they would express in BL21(DE3) or
> such if cloned into a pET vector or similar. In fact I can probably recall
> only one or two that expressed *better* in XL1 cells. However in the good
> old days pQE series of vectors was quite commonly used and I had things in
> that were inherited from others - these 'things' were fairly simple to
> express in XL1-blue whereas they gave me loads of trouble in other strains
> and I was too busy/lazy to re-clone them.
>
> Artem
>
>> Hello Artem,
>>
>>  We express almost all our proteins in BL21 derivatives. It sounds
>> like you've worked with many proteins that express/behave better in
>> XL1-Blue?
>>
>>
>> ho
>> UC Berkeley
>>
>> -
>> XL1-Blue is a strain of E. coli. Whether it is or isn't an expression
>> host
>> depends on the definition, and I am not going to argue semantics.
>>
>> The T5 promoter works with regular garden variety RNApol of E. coli.
>> Therefore ANY E. coli strain is an 'expression host' for vectors that
>> contain this promoter.
>>
>> I've expressed many proteins in XL1-Blue and I see no reason why you
>> can't
>> express yours, either.
>>
>> Artem
>>
>
>

Re: [ccp4bb] Refolding of Denatured Protein

[artem]

I hate to be the Negative Nancy here - but removal of detergent can be a
significantly harder task than removal of urea. Here's why:

Urea is a small molecule which diffuses freely and does not normally stick
to proteins - so removal of urea is no harder than removal of salt (we
ignore the considerations of protein stability here). On the other hand,
detergents can be easy or very hard to get rid of, depending on which
particular detergent and what particular protein are brought together.
Some of the nastier polymeric detergents are quite different to remove
'completely' because they stick to proteins, stick to plastic and/or resin
beads, form micelles (thus refusing to dialyze) and so on and so forth.

There are numerous acceptable ways to remove sticky detergents, and some
of them include treatment with hydrophobic matrices that 'suck up'
detergent molecules, exchange with a 'milder' detergent, and so forth.

Good luck!

Artem
>
> dear sanjiv
> i dont think that the removal of detergent is more difficult than
> urea,since u are purifying ur protein over Ni-NTA column,so after a few
> wash over there u can remove the detergent completely.
>
> best
> atul kumar
>
> -Original Message-
> From: CCP4 bulletin board on behalf of David Cobessi
> Sent: Tue 5/5/2009 6:18 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Refolding of Denatured Protein
>
> Sanjiv Kumar wrote:
>>  I have not tried purifying this protein either with detergent or with
>> /Guanidine Hydrochloride. I can try with both /Guanidine HCl and
>> detergent. Since I got good purification with the 8M urea, I was
>> thinking if this protein could be refolded and used further. Isn't
>> removal of detergent more difficult then Urea?
>> Sanjiv Kumar
>> Lab. No. 411,
>> Functional Genomics Unit,
>> Institute of Genomics and Integrative Biology,
>> New Delhi-110007
>> India
> Is it a transmembrane protein or not? or just a protein associated to
> the membrane (with a GPI anchor for example)?
> If it is a transmembrane protein, you should try to use detergents for
> extraction and during the purification.
> David
>
> --
> David Cobessi
> Institut de Biologie Structurale
> 41, Rue Jules Horowitz
> 38027 Grenoble Cedex-1, France
> Tel:33(0)438789613
> 33(0)608164340
> Fax:33(0)438785122
>
>

[ccp4bb] Postdoctoral Position in Structural Biology, Brisbane, Australia

[Justine Hill]

Postdoctoral Position in Structural Biology

A postdoctoral position is available immediately in a team working to  
understand the structure and assembly of signalling complexes involved  
in apoptosis and inflammation.  The successful applicant will make  
extensive use of x-ray crystallography and protein NMR spectroscopy in  
combination with other biochemical and biophysical methods to  
characterise the structure and mechanisms of protein interactions that  
underlie caspase activation and regulation.


Location:  The University of Queensland, Brisbane, Australia
The University of Queensland is one of Australia's top universities,  
located at the beautiful St. Lucia campus on the Brisbane river.  The  
School of Chemistry and Molecular Biosciences (SCMB) is a  
distinguished multidisciplinary school for teaching and research that  
encompasses the traditional academic disciplines of Chemistry,  
Biochemistry, Molecular Biology, Microbiology and Parasitology.  The  
School is well equipped for contemporary molecular biology and protein  
biochemistry research, and is supported by the University’s  
outstanding structural biology infrastructure including 750 MHz and  
900 MHz NMR spectrometers, and facilities for x-ray crystallography  
and electron microscopy.  State-of-the-art equipment is available for  
all aspects of the work including FR-E x-ray generator /R-axis IV++  
and Saturn 944 CCD x-ray detectors, Mosquito crystallization robot and  
the Rock Imager imaging system.  Access is also available to the  
Australian Synchrotron in Melbourne.


Brisbane has been voted Australia's most liveable city and has a great  
subtropical climate.  It is only a short drive from some of  
Australia's best beaches and provides opportunities for all sorts of  
outdoors and cultural activities.


The Person:
Applicants should possess a PhD in a relevant field (chemistry,  
biochemistry or biophysics) with experience in structural  
characterisation using x-ray crystallography and/or NMR spectroscopy.   
Experience in protein biochemistry and a range of biophysical  
techniques for characterising protein interactions, including ITC and  
Biacore, is highly desirable.  Excellent communication skills,  
motivation and the ability to work as part of a team are also required.


Salary:
The position is a full-time, fixed term appointment at Research  
Academic Level A for one year in the first instance, with the  
possibility of renewal subject to funding.  The remuneration package  
will be in the range $58,154 to $78,881 AUD per annum, which includes  
employer superannuation contributions of 17%.


Applications:  closing date 25 May 2009
Obtain the position description and selection criteria online (http://www.uq.edu.au/jobs/2009documents/Sciences/3020534.doc 
 ) or contact Dr Justine Hill (j.h...@uq.edu.au) to discuss the role.


Please quote the Reference Number (3020534) and include a covering  
letter, your contact address and telephone number; and a curriculum  
vitae, that includes details of education and qualifications and the  
names and contact details of three referees; a statement addressing  
how each of the selection criteria has been met must also be  
included.  Send applications to Dr Justine Hill (j.h...@uq.edu.au).


Additional information:
The following websites provide information about The University of  
Queensland (http://www.uq.edu.au), our department and lab (http://www.scmb.uq.edu.au 
 and http://profiles.bacs.uq.edu.au/Justine.Hill.html ) and living in  
Brisbane (http://www.ourbrisbane.com).




Justine M. Hill
Group Leader / NHMRC RD Wright Fellow
School of Chemistry and Molecular Biosciences
The University of Queensland
Brisbane QLD 4072, Australia

Ph:  +61 7 3365 4638 (office) or 3365 4875 (lab)
Fax:+61 7 3365 4699
Email:  j.h...@uq.edu.au
Website:  http://profiles.bacs.uq.edu.au/Justine.Hill.html

[ccp4bb] post-doc position available

[R. J. Lewis]

dear BBers

there is a post-doc position available in my lab, funded
by the BBSRC for 3 years in the first instance, to study
the biosynthesis of the cell wall in the model bacterium,
Bacillus subtilis. the cell wall, apart from being the
target of penicillin and vancomycin, defines the shape
of the bacterial cell and provides it with a protective
outer layer. there are a host of proteins involved in
the formation of the wall, some - but not all - of which
are membrane proteins. consequently, there is an
opportunity in newcastle for a talented and dedicated
post-doc with demonstrable previous experience of the
structural biology of membrane proteins. further
particulars of this post can be found on newcastle's
vacancies website: http://www.ncl.ac.uk/vacancies/jobs/
with reference code A397R (ICAMB). please apply for
the post by following the instructions on the website,
the closing date for which is 25th may 2009.

please also note that in addition to the post in my lab,
there is a second post available, funded on the same
grant, in the lab of richard daniel for someone with
a more biological focus. applicants to me should
specify that they are applying for 'post 2'.

cheers


rick

--
R. J. Lewis
Professor of Structural Biology
Institute for Cell and Molecular Biosciences
Faculty of Medical Sciences   Tel: +44 (0)191 222 5482
University of Newcastle   Fax: +44 (0)191 222 7424
Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk

[ccp4bb] Symposium: Light Microscopy meets Structural Biology, EMBL Heidelberg, June 22-23, 2009

[Christoph Mueller]

Dear Colleagues,

We writing to publicise the programme for the Symposium: Light 
Microscopy meets Structural Biology at the European Molecular Biology 
Laboratory (EMBL Heidelberg, Germany) from 22-23 June, 2009.


The symposium aims to bring together structural biologists, cell 
biologists and light microscopy specialists to explore opportunities and 
requirements for structural biologists in using different light 
microscopy techniques and to foster interactions at the interface 
between structural biology and cell biology. The meeting is sponsored by 
the FP6 project SPINE2-COMPLEXES and INSTRUCT.


Please visit the web site for a copy of the programme and 
registration:   http://www-db.embl.de/jss/EmblGroupsOrg/conf_130. 
Deadline for registration: 19 May, 2009.
Please circulate this announcement to interested members and groups 
within your institute.


Best wishes,
Christoph Muller

-
Dr. Christoph W. Muller
Joint Head of Structural and Computational Biology Unit

EMBL
Meyerhofstrasse 1
69117 Heidelberg, Germany

email: cmuel...@embl.de
phone: 0049-6221-387-8320
fax: 0049-6221-387-519
http://www.embl.de
-

[ccp4bb] Bfactor as Measure of Presence

[James Stroud]

Hello,

I have a 2.45 A structure with an average b factor of 50.6. A region I  
am particularly interested in has an average b factor of 87. At what  
point do I say that the region is "disordered"? Does it come down to  
maps? If I have reasonable simulated omit maps but the b factor is 87,  
how much confidence can I have about my interpretation of the maps?


James