Re: [ccp4bb] Milch and Minor

[Bernhard Rupp (Hofkristallrat a.D.)]

Thanks to all - got a few!

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Bernhard Rupp (Hofkristallrat a.D.)
Sent: Monday, October 18, 2010 7:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Milch and Minor

Dear All, 

my J Appl Cryst collection does not extend far enough to unearth

J. Appl. Cryst. (1974). 7, 502-505[ doi:10.1107/S0021889874010284  ]
The indexing of single-crystal X-ray rotation photographs
J. R. Milch and T. C. Minor

perhaps someone has a pdf and would certainly not email it to me ;-)

Many thx, BR
-
Bernhard Rupp, Hofkristallrat a.D.
001 (925) 209-7429
+43 (676) 571-0536
b...@ruppweb.org
hofkristall...@gmail.com
http://www.ruppweb.org/
--
Der einzigartige chillout-mix in der Hofkristall-lounge
--

[ccp4bb] JAC (2010), 43,1242-1249

[Jürgen Bosch]

Lesenswert !
Worth reading !
http://journals.iucr.org/j/issues/2010/05/02/issconts.html

Could somebody send me the pdf from Bayes 1763 ?

Jürgen
-
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
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

Re: [ccp4bb] Milch and Minor

[Jürgen Bosch]

Only once ? I doubt that :-)
-
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
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Oct 18, 2010, at 10:15 PM, Thomas Juettemann wrote:

> I certainly did not email it to him!
> 
> On Mon, Oct 18, 2010 at 19:05, Bernhard Rupp (Hofkristallrat a.D.)
>  wrote:
>> Dear All,
>> 
>> my J Appl Cryst collection does not extend far enough to unearth
>> 
>> J. Appl. Cryst. (1974). 7, 502-505[ doi:10.1107/S0021889874010284  ]
>> The indexing of single-crystal X-ray rotation photographs
>> J. R. Milch and T. C. Minor
>> 
>> perhaps someone has a pdf and would certainly not email it to me ;-)
>> 
>> Many thx, BR
>> -
>> Bernhard Rupp, Hofkristallrat a.D.
>> 001 (925) 209-7429
>> +43 (676) 571-0536
>> b...@ruppweb.org
>> hofkristall...@gmail.com
>> http://www.ruppweb.org/
>> --
>> Der einzigartige chillout-mix in der Hofkristall-lounge
>> --
>> 

Re: [ccp4bb] Milch and Minor

[Thomas Juettemann]

I certainly did not email it to him!

On Mon, Oct 18, 2010 at 19:05, Bernhard Rupp (Hofkristallrat a.D.)
 wrote:
> Dear All,
>
> my J Appl Cryst collection does not extend far enough to unearth
>
> J. Appl. Cryst. (1974). 7, 502-505    [ doi:10.1107/S0021889874010284  ]
> The indexing of single-crystal X-ray rotation photographs
> J. R. Milch and T. C. Minor
>
> perhaps someone has a pdf and would certainly not email it to me ;-)
>
> Many thx, BR
> -
> Bernhard Rupp, Hofkristallrat a.D.
> 001 (925) 209-7429
> +43 (676) 571-0536
> b...@ruppweb.org
> hofkristall...@gmail.com
> http://www.ruppweb.org/
> --
> Der einzigartige chillout-mix in der Hofkristall-lounge
> --
>

[ccp4bb] Milch and Minor

[Bernhard Rupp (Hofkristallrat a.D.)]

Dear All, 

my J Appl Cryst collection does not extend far enough to unearth

J. Appl. Cryst. (1974). 7, 502-505[ doi:10.1107/S0021889874010284  ]
The indexing of single-crystal X-ray rotation photographs
J. R. Milch and T. C. Minor

perhaps someone has a pdf and would certainly not email it to me ;-)

Many thx, BR
-
Bernhard Rupp, Hofkristallrat a.D.
001 (925) 209-7429
+43 (676) 571-0536
b...@ruppweb.org
hofkristall...@gmail.com
http://www.ruppweb.org/
--
Der einzigartige chillout-mix in der Hofkristall-lounge
--

Re: [ccp4bb] Refinement

[Jim Fairman]

Did you check your data for twinning and/or pseudo symmetry using
phenix.xtriage or Pointless?  If the space group is incorrect, these
programs will also assist you in selecting the correct one.  What were the
Z-scores for the rotation and translation functions?

On Mon, Oct 18, 2010 at 7:39 PM, Jyotica Batra wrote:

> Hi All
>
> I have a dataset at 1.9A, spacegroup-P212121 (unit cell: 37.7, 39.52,
> 231.72, 90, 90, 90),
> I used MR phaser and got a structure solution with LLG= 320 (1copy/a.u) .
>  During refinement, the R-free (50%) and R-factors (42%) never go down.
> I have tried refining in phenix and refmac both, but still the high R-free
> problem persists.  At this point I'm seeking help to know the possible
> reasons for this high R-free.
>
> Thanks in advance!
>
> Jyotica
>
>
>


-- 
Jim Fairman, Ph D.
Post-Doctoral Fellow
National Institutes of Health - NIDDK
Lab: 1-301-594-9229
E-mail: fairman@gmail.com james.fair...@nih.gov

Re: [ccp4bb] Refinement

[Jürgen Bosch]

1. How does the MR map look like ? Do you see features resembling a real 
solution or just noise ?

2. If the map looks decent, then try running a rigid body refinement using 
Refmac. Does the Rwork/Rfree dro ?

3. At this point you should look at the difference density map in Coot and be 
able to identify "wrong" amino acids e.g. if your MR model had an Ala where 
there is truly a His or Phe, you should see a huge blob of green density 
indicating the missing atoms in your current model.

4. Fix the sequence of your MR model to your true sequence

5. WARNING don't continue to read the next option if you want to learn and have 
fun by yourself !

6. at 1.9 Å ARP/wARP will be able to build most of your structure automatically

Jürgen

P.S. It's likely you have the correct space group simply by gaussian 
distribution over P222 pointgroups, but maybe that's what the Rwork/Rfree are 
indicating  Did you run Phaser in P222 + all other possible space groups or did 
you feed it with P212121 and only ran it in this space group ?
-
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
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Oct 18, 2010, at 7:39 PM, Jyotica Batra wrote:

> Hi All
> 
> I have a dataset at 1.9A, spacegroup-P212121 (unit cell: 37.7, 39.52, 231.72, 
> 90, 90, 90), 
> I used MR phaser and got a structure solution with LLG= 320 (1copy/a.u) .  
> During refinement, the R-free (50%) and R-factors (42%) never go down.
> I have tried refining in phenix and refmac both, but still the high R-free 
> problem persists.  At this point I'm seeking help to know the possible 
> reasons for this high R-free.
> 
> Thanks in advance!
> 
> Jyotica
> 
> 

[ccp4bb] Refinement

[Jyotica Batra]

Hi All

I have a dataset at 1.9A, spacegroup-P212121 (unit cell: 37.7, 39.52, 231.72, 
90, 90, 90), 
I used MR phaser and got a structure solution with LLG= 320 (1copy/a.u) .  
During refinement, the R-free (50%) and R-factors (42%) never go down.
I have tried refining in phenix and refmac both, but still the high R-free 
problem persists.  At this point I'm seeking help to know the possible reasons 
for this high R-free.

Thanks in advance!

Jyotica

[ccp4bb] Positions available

[Chavas Leo]

Dear all --

for those interested, we have few good positions opening at the High  
Energy Accelerator Research Organization (KEK).
You don't want to miss the chance to come and work in this wonderful  
country that is Japan!!!

Please have a look at the links below:
http://www.kek.jp/intra-e/jobs/

Kind regards.

-- Leo -- 


---
Chavas Leonard M.G., Ph.D.
Assistant Professor
Structural Biology Research Center
Photon Factory
High Energy Accelerator Research Organization (KEK)
305-0801 Tsukuba Oho 1-1
Japan
---
Phone : +81(0)29-864-5642 (4901)
Fax : +81(0)29-864-2801
E-mail : leonard.cha...@kek.jp
---
Science Advisory Board (BIT Life Sciences)
Editorial Board (JAA)
http://pfweis.kek.jp/~leo
---

Re: [ccp4bb] Cys auxotroph

[M T]

I think you should look at pubmed id 14604526, it works well for me. You should 
obtain the cells if you contact the authors. 

Michel.

Le 18 oct. 2010 à 17:27, Daniel Bonsor  a écrit :

> Have you looked at the Keio collection. Though they are not compatible with 
> T7 promoters, you can use the λDE3 Lysogenization Kit to add the T7 
> polymerase. 
> 
> A possible source is from Yale http://cgsc.biology.yale.edu/Auxotrophs.php 
> though of course there are others.
> 
> 
> Dan

[ccp4bb] Positions available

[Leo Chavas]

Dear all --

for those interested, we have few good positions opening at the High Energy
Accelerator Research Organization (KEK).
You don't want to miss the chance to come and work in this wonderful country
that is Japan!!!
Please have a look at the links below:
http://www.kek.jp/intra-e/jobs/

Kind regards.

-- Leo --

---
Chavas Leonard M.G., Ph.D.
Assistant Professor
Structural Biology Research Center
Photon Factory
High Energy Accelerator Research Organization (KEK)
305-0801 Tsukuba Oho 1-1
Japan
---
Phone : +81(0)29-864-5642 (4901)
Fax : +81(0)29-864-2801
E-mail : leonard.cha...@kek.jp
---
Science Advisory Board (BIT Life Sciences)
Editorial Board (JAA)
http://pfweis.kek.jp/~leo
---

[ccp4bb] Positions available

[naalas horced]

Dear all --

for those interested, we have few good positions opening at the High Energy
Accelerator Research Organization (KEK).
You don't want to miss the chance to come and work in this wonderful country
that is Japan!!!
Please have a look at the links below:
http://www.kek.jp/intra-e/jobs/

Kind regards.

-- Leo --

---
Chavas Leonard M.G., Ph.D.
Assistant Professor
Structural Biology Research Center
Photon Factory
High Energy Accelerator Research Organization (KEK)
305-0801 Tsukuba Oho 1-1
Japan
---
Phone : +81(0)29-864-5642 (4901)
Fax : +81(0)29-864-2801
E-mail : leonard.cha...@kek.jp
---
Science Advisory Board (BIT Life Sciences)
Editorial Board (JAA)
http://pfweis.kek.jp/~leo
---

[ccp4bb] Takeda San Diego, Scientist, Membrane Protein Chemistry

[Tatone, Josephine (TPNA)]

Takeda San Diego, Scientist Membrane Protein Chemistry

We are seeking outstanding Membrane Protein Scientist to processes,
express and then highly purify integral membrane proteins (e.g. GPCRs,
ion channels and membrane-associated proteins) and their complexes with
agonists or antagonists for crystallographic analysis to support further
design as drug candidates. 
We are looking to hire at the Principal or Sr. Scientist level however
will consider those with strong post doc work at the Scientist I/II
level.  
Minimum Requirements include: 

  Industry experience strongly preferred. 
*Ph.D. in life science with thesis, publication(s), OR
*MS in life science with minimum 9+ years of relevant experience and
distinguished publication record, OR 
*BS in life science with minimum12+ years of relevant experience and
distinguished publication record
*Demonstrated knowledge and experience with biochemistry assays, protein
purification, protein expression. 


To apply online for this, or any other position at Takeda, please go to
www.takedasd.com/careers. If you know someone else who may be a fit for
this opportunity, please feel free to pass this message along. 

Best wishes, 

Talent Acquisition 
Takeda Pharmaceuticals North America, Inc.


*Please note that applying does not guarantee that you will be
interviewed or hired for a position at Takeda.


###
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[ccp4bb] CNIO International PhD and Postdoctoral Programmes_2011 call

[Marco Mazzorana]

On behalf of Mar Pérez (Head of CNIO Training Office)


Dear colleague,

I would like to draw your attention to the 2010 calls for the 
International Ph.D. and Postdoctoral Programmes at the Spanish National 
Cancer Research Centre (CNIO). We would greatly appreciate if you could 
forward the information about these exciting programmes to potential 
candidates.


The *CNIO* offers excellent training and research opportunities in 
cutting edge basic and applied cancer research. The programmes are open 
to outstanding young graduates from all over the world who want to 
pursue ambitious research projects. Highly motivated individuals are 
encouraged to apply for the following of CNIO's research programmes: 
Molecular Oncology, Cancer Cell Biology, Structural Biology and 
Biocomputing, Molecular Pathology, Human Cancer Genetics and Clinical 
Research.


The CNIO *International Postdoctoral Programme* offers up to 4 very 
competitively funded two-year postdoc positions to outstanding junior 
scientists. Applications are considered at regular intervals; the 2010 
call closes on *December 31^st , 2010*.


The CNIO *International Ph.D. Programme* each year supports 10 
candidates for a 4-year Ph.D.  We have one selection per year. The 
deadline for applications is *March 15th, 2011*.


Our webpage provides further information on both programmes and the 
application procedure:


- Postdoc Programme: www.cnio.es/postdoc 

To print a copy of the poster: 
http://www.cnio.es/es/cursos/descargas/postdoctorado/Poster_postdoc_2010.pdf


- Ph.D. Programme: www.cnio.es/phd 

To print a copy of the poster: 
http://www.cnio.es/es/cursos/descargas/doctorado/Poster_PhD_2011.pdf


Please, feel free to distribute this message on your local institute 
network and thank you very much in advance for advertising our 
programmes in your institution.


Best regards,

Mar Pérez, PhD

Head of Training Office

Fax: +34 912246980

E-mail: post...@cnio.es  or p...@cnio.es 



Websites: www.cnio.es/postdoc  and 
www.cnio.es/phd 




--

---
Marco Mazzorana, PhD

Structural Biology and Biocomputing Programme

CNIO - Centro Nacional de Investigaciones Oncológicas
C/ Melchor Fernández Almagro, 3, E-28029 Madrid (España)

Phone: +34 91 2246900 ext. 3033
Fax: +34 91 2246976
www.cnio.es
---




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Re: [ccp4bb] Cys auxotroph

[Daniel Bonsor]

Have you looked at the Keio collection. Though they are not compatible with T7 
promoters, you can use the λDE3 Lysogenization Kit to add the T7 polymerase. 

A possible source is from Yale http://cgsc.biology.yale.edu/Auxotrophs.php 
though of course there are others.


Dan

[ccp4bb] Cys auxotroph

[Yogesh Gupta]

Does anyone know a commercial source of  Cys-auxotroph strain of E. coli to
prepare a doubly (Se-Cys + Se-Met) labeled protein? I would appreciate if
you can share your experience if you have labeled your protein in this way
to improve Se signal for phasing.

Thanks,
-Yogesh

-- 
--
Yogesh K. Gupta, Ph.D.
Dept of Structural & Chemical Biology
Mount Sinai School of Medicine
Icahn Medical Institute
1 Gustave L. Levy Place, Box 1677
New York, NY, USA 10029

Tel:+1 212-659-8639
--

Re: [ccp4bb] Big difference between anomalous map and density modification map

[Clemens Vonrhein]

Hi Mousheng,

[if it gets too SHARP-centric we can also move this discussion also to
sharp-discuss or our developers mailing list sharp-develop]

On Mon, Oct 18, 2010 at 08:33:47AM -0500, Wu, Mousheng wrote:
> my solvent content is supposed to be 65% (one molecule per
> ASU).

which seems to match your lowish resolution limit (as a general rule).

> Sharp automatically did density modification using 37.5% solvent
> content.

I see. Did you get a clear and significant difference in score for the
two hands? It not only tells you which is the correct hand, but a
large difference also tells you that one set of phases is
significantly better than the other and that there is real phasing
information in those.

> that is why I run solvent flattening directly from Sharp.

Did you use the tools within our interface? Probably ...

> >More confusion: what do you mean with "anomalous map"? One might call a map 
> >with anomalous differences and some phases such an "anomalous map" ... but I 
> >guess you rather mean an electron density map with phases from some 
> >procedure based on anomalous differences, right?
> 
> you are right. the anomalous map I calculated is anomalous differences map 
> using FFT 

Ok - so you are comparing an anomalous difference map with an electron
density map? I'm not sure why, but:

 * the anomalous difference map (using phases from SHARP or your
   density modified phases) should only show the substructure,
   i.e. only the Se atoms and no light atoms

 * the electron density map after density modification shows
   everything: light and heavy atoms.

Why are you comparing those two maps? To check if your heavy atom
sites are where they should be or if all are correct?

If you trust your density modified phases (and you should, since they
show nice helical density): just calculate such an anomalous
difference map using your density modified phases ... it's a two
mouse-click operation in the SHARP/autoSHARP interface and should show
you peaks and their distance to your initial heavy-atom sites.

Are those 6 'poor' sites in any way different after SHARP refinement?
Much higher B-factor or lower occupancy? Do you have additional peaks
in your log-likelihood residual maps (showing the true position of
those potentially wrong sites or if they are split)? Do you have peaks
in the ANO residual maps exactly at the sites - showing wrong f"
values (similar for f' values in ISO residual maps)? I.e.: is your HA
model as good as it could be?

Cheers

Clemens

-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

[ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?

[Marcus Winter]

Dear Klaus,





With extensive travel in the meantime, we're sorry that it took us so long to 
get

back to you on this.



Well, the true answer is that any resolution might be expected on the PX 
Scanner.

Thus, sometimes we have observed higher resolutions on the PX Scanner than

observed from the 'same' protein crystals using a third generation synchrotron 
source.

The point is that as well as determining the 'native' diffraction qualities 
(resolution limit,

mosaicity and unit cell, etc.) of the chosen crystal, using the PX Scanner one 
can also

establish the efficacy of the cryo-protection and the further 'handling' stages 
of the

crystal workflow, etc.  (So, in the cases above, perhaps the cryo-conditions 
were

'wrong', etc...).



Moreover, using the PX Scanner, one can unambiguously identify the 
best-diffracting

individual crystal in any droplet across the whole crystallisation plate: most 
importantly

in situ without any kind of 'disturbance'...  And even if the crystals do indeed

diffract - on the PX Scanner, to lower resolution than they do on a more 
powerful

source, of course you can still rank them and then pick up the clearly 'best' 
ones for further

analysis.  As experienced by many researchers: there can be very significant 
differences

between crystals, even grown in the same drop.



You, all, are most welcome to visit our labs, bringing your plates, in order to 
collect

in situ crystal diffraction data: as an evaluation both of your crystals and the

PX Scanner instrument.  We look forward to this !





Yours,



Tadeusz Skarzynski

Marcus Winter



(Oxford Diffraction Ltd. - now Agilent Technologies)







-Original Message-
From: Klaus Fütterer [mailto:k.futte...@bham.ac.uk]
Sent: 30 September 2010 11:00
To: WINTER,MARCUS (A-Varian,ex1)
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the 
synchrotron?



Marcus,



May I ask the following: assuming 8 A is obtained from a single

crystal on the home source, what diffraction limit would one expect

on the PX scanner?



Best regards,



Klaus





===



 Klaus Fütterer, Ph.D.

 Reader in Structural Biology

   Undergraduate Admissions



School of Biosciences   P: +44-(0)-121-414 5895

University of Birmingham   F: +44-(0)-121-414 5925

Edgbaston E: k.futte...@bham.ac.uk

Birmingham, B15 2TT, UK   W: www.biochemistry.bham.ac.uk/klaus/

===











On 30 Sep 2010, at 10:44, Marcus Winter wrote:



>

>

> This recent discussion does tend towards the ideal scenario: of

> identifying ones

> best-diffracting crystals... before embarking on the synchrotron trip.

>

> The established Oxford Diffraction PX Scanner home laboratory

> instrument can

> therefore be most useful.  This enables the direct X-ray screening

> of individual

> (putative) single crystal objects, in situ, in the (any SBS format)

> crystallisation plate.

>

>

> Yours sincerely,

>

> Marcus Winter (Oxford Diffraction Ltd. - now Agilent Technologies)

>

>

>

> -Original Message-

> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf

> Of Phil Jeffrey

> Sent: 28 September 2010 19:20

> To: CCP4BB@JISCMAIL.AC.UK

> Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at

> the synchrotron?

>

> Often this reflect crystal size - a small crystal in a big beam (or

> one

> with a long path in air) on a home source would see the small

> diffraction signal drop below the noise level quite quickly - often at

> the low resolution intensity dip that sits very approximately around 6

> Angstrom.  On a synchrotron source with a tight low-divergence beam

> that

> matches more closely the crystal dimensions that same crystal will

> appear to do rather better.

>

> Also one is more likely to expose the crystal longer (in terms of

> total

> photon numbers) at a synchrotron, which itself begets better signal/

> noise.

>

> Alternatively: everyone tries harder before synchrotron trips

>

> Phil Jeffrey

> Princeton

>

> On 9/28/10 1:27 PM, Francis E Reyes wrote:

> > Hi all

> >

> > I'm interested in the scenario where crystals were screened at

> home and

> > gave lousy (say < 8-10A) but when illuminated with synchrotron

> radiation

> > gave reasonable diffraction ( > 3A) ? Why the discrepancy?

> >

> > Thanks

> >

> > F

Re: [ccp4bb] Big difference between anomalous map and density modification map

[Wu, Mousheng]

Hi, Clemens,

sorry for the confusing and unclear expression of my case.

>Confused ... what program/procedure did you use for what you call "density 
>modification" and what for "solvent flattening"?

 my solvent content is supposed to be 65% (one molecule per ASU). Sharp 
automatically did density modification using 37.5% solvent content. that is why 
I run solvent flattening directly from Sharp.

>More confusion: what do you mean with "anomalous map"? One might call a map 
>with anomalous differences and some phases such an "anomalous map" ... but I 
>guess you rather mean an electron density map with phases from some procedure 
>based on anomalous differences, right?

you are right. the anomalous map I calculated is anomalous differences map 
using FFT 

>   fft hklin eden_flat_53.2pc.mtz mapout eden_flat_53.2pc.map < LABI F1=FBshasol PHI=PHIBshasol
>  fft hklin eden_flat_53.2pc.mtz mapout eden_flat_53.2pc.map < LABI F1=FBshasol PHI=PHIBshasol e

yes, I calculated both density maps. the density around all 6 selenium sites is 
poor.


Thank you very much.

mousheng





Mousheng Wu, PhD
Center for Membrane Biology
Department of Biochemistry and Molecular Biology
The University of Texas Medical School at Houston
6431 Fannin Street, Houston, TX, USA, 77030

From: Clemens Vonrhein [vonrh...@globalphasing.com]
Sent: Monday, October 18, 2010 8:05 AM
To: Wu, Mousheng
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Big difference between anomalous map and density  
modification map

Dear Mousheng,

as others, I'm also a bit confused about the steps you did:

On Sun, Oct 17, 2010 at 02:29:12PM -0500, Wu, Mousheng wrote:
> Dear All,
>
> I have a MAD dataset at 4Å and shelxD can find clear-cut 10
> solutions (my protein has 12 methionines).

Good.

> I ran autosharp to refine them followed by density modification.

Yes.

> After density modification, I ran solvent flattening.

Confused ... what program/procedure did you use for what you call
"density modification" and what for "solvent flattening"?

> Then I calculated the anomalous map using the phase from sharp and
> the electron density map from solvent flattening.

More confusion: what do you mean with "anomalous map"? One might call
a map with anomalous differences and some phases such an "anomalous
map" ... but I guess you rather mean an electron density map with
phases from some procedure based on anomalous differences, right?

So you calculated an electron density map from SHARP: which map
coefficients did you use? It should be:

  fft hklin eden.mtz mapout eden.map < The anomalous map shows the density around these 10 selenium sites
> are clear and round.

Good.

> However, the density map from solvent flattening showed that only 4
> selenium sites have clear and round density.

Is the map after "density modifcation" or the one after "density
modification plus solvent flattening"? It is very unclear ...

> The density around these 4 sites clearly showed beautiful
> helices. surprisingly, other 6 selenium sites have poor density or
> no density at all.  The electron density around them is not very
> good and the predicted helical density is flat. I check the electron
> density before I ran solvent flattening. The result is same.  I am
> quite confused about the big difference from these two maps.

We first need to establish what steps you performed and how to see if
these are the actually correct maps to look at.

> I also try SnB to find the selenium sites. The solutions are same as
> those from ShelxD. But How to explain the poor density around the
> selenium sites in the density modification map?  Is there any
> problem with my selenium sites?

Remember that the purely HA-phased maps (i.e. directly after SHARP)
will be dominated by those heavy atoms ... so no surprise they are
nice and round Se density.

Se-MET (or S-Met for that matter) often has alternate conformations in
the side-chain: maybe your phases are actually better and therefore
model those side-chains now more accurately, i.e. as disordered
side-chains. If your environment around those sites is also disorderd
('flattened' helices) this is all not surprising.

Cheers

Clemens

--

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] Big difference between anomalous map and density modification map

[Clemens Vonrhein]

Dear Mousheng,

as others, I'm also a bit confused about the steps you did:

On Sun, Oct 17, 2010 at 02:29:12PM -0500, Wu, Mousheng wrote:
> Dear All,
> 
> I have a MAD dataset at 4Å and shelxD can find clear-cut 10
> solutions (my protein has 12 methionines).

Good.

> I ran autosharp to refine them followed by density modification.

Yes.

> After density modification, I ran solvent flattening.

Confused ... what program/procedure did you use for what you call
"density modification" and what for "solvent flattening"?

> Then I calculated the anomalous map using the phase from sharp and
> the electron density map from solvent flattening.

More confusion: what do you mean with "anomalous map"? One might call
a map with anomalous differences and some phases such an "anomalous
map" ... but I guess you rather mean an electron density map with
phases from some procedure based on anomalous differences, right?

So you calculated an electron density map from SHARP: which map
coefficients did you use? It should be:

  fft hklin eden.mtz mapout eden.map < The anomalous map shows the density around these 10 selenium sites
> are clear and round.

Good.

> However, the density map from solvent flattening showed that only 4
> selenium sites have clear and round density.

Is the map after "density modifcation" or the one after "density
modification plus solvent flattening"? It is very unclear ...

> The density around these 4 sites clearly showed beautiful
> helices. surprisingly, other 6 selenium sites have poor density or
> no density at all.  The electron density around them is not very
> good and the predicted helical density is flat. I check the electron
> density before I ran solvent flattening. The result is same.  I am
> quite confused about the big difference from these two maps.

We first need to establish what steps you performed and how to see if
these are the actually correct maps to look at.

> I also try SnB to find the selenium sites. The solutions are same as
> those from ShelxD. But How to explain the poor density around the
> selenium sites in the density modification map?  Is there any
> problem with my selenium sites?

Remember that the purely HA-phased maps (i.e. directly after SHARP)
will be dominated by those heavy atoms ... so no surprise they are
nice and round Se density.

Se-MET (or S-Met for that matter) often has alternate conformations in
the side-chain: maybe your phases are actually better and therefore
model those side-chains now more accurately, i.e. as disordered
side-chains. If your environment around those sites is also disorderd
('flattened' helices) this is all not surprising.

Cheers

Clemens

-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] Big difference between anomalous map and density modification map

[David Schuller]

 On 10/17/10 15:29, Wu, Mousheng wrote:

Dear All,

I have a MAD dataset at 4Å and shelxD can find clear-cut 10 solutions (my 
protein has 12 methionines). I ran autosharp to refine them followed by density 
modification. After density modification, I ran solvent flattening. Then I 
calculated the anomalous map using the phase from sharp and the electron 
density map from solvent flattening. The anomalous map  shows the density 
around these 10 selenium sites are clear and round. However, the density map 
from solvent flattening showed that only 4 selenium sites have clear and round 
density.The density around these 4 sites clearly showed beautiful helices. 
surprisingly, other 6 selenium sites have poor density or no density at all.  
The electron density around them is not very good and the predicted helical 
density is flat. I check the electron density before I ran solvent flattening. 
The result is same.  I am quite confused about the big difference from these 
two maps.  I also try SnB to find the selenium sites. The solutions are same as 
those from ShelxD. But How to explain the poor density around the selenium 
sites in the density modification map?  Is there any problem with my selenium 
sites? Any suggestion from crystallographic experts?  Thanks.

Solvent flattening is one form of density modification, so your 
description of what you did is a bit confusing. Did you use the 
appropriate solvent content for the solvent flattening?



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

Re: [ccp4bb] Question regarding S-SAD

[Clemens Vonrhein]

Hi Kornelius,

On Mon, Oct 18, 2010 at 02:03:53PM +0200, Kornelius Zeth wrote:
> What I noticed was that the spot profiles in XDS looked not as the
> are supposed to, presumably because the mosaicity of the spots are
> 0.4-0.6 and spot integration may be hampered. Still, merging
> Rfactors are brilliant.

Be careful about the merging R-factors: they measure internal
consistency but not directly data quality! Yes, if everything is fine,
then the fact that symmetry-related reflections are very similar tells
you something about data quality. But if something goes wrong ...

Given that your data looks slightly twinned and your reflection
profiles are not ideal, this does look to me a bit like a problem with
the spots and potential overlap: either from a second lattice (split
crystal or non-merohedral twinning) or from the same lattice
(overlapped reflection).

As far as I understand XDS it will assign each pixel to the nearest
predicted hkl (experts: please correct me if I'm wrong). So if you
have overlapped reflections (because mosaicity, oscillation angle and
distance are not ideal) you will get systematically truncated
reflections. Since symmetry-related reflections will most likely be
truncated in a very simlar way you will get poor data but a low
Rmerge.

Similar for split crystals or non-merohedral twinning:
symmetry-related reflections could be affected in a very similar and
systematic way (giving low Rmerge) but the spots are just poor and
badly integrated.

I don't think this is a particular XDS problem - rather that other
programs might fail in processing those datasets at all and XDS is
just very good in getting as much out of it as possible.

The only thing that really helps here is to visually inspect
the images and the predictions: are all spots predicted or do you have
signs or split spots or multiple lattices? Don't forget to also look
at 30, 45, 60, 90 degree from image 1 ... Murphy's law dictates that
the first image always looks perfect ;-)

But maybe it is something different after all ... 3.8A SHARP phaser
going to 2.5A should give you something to work with: do you have NCS?
Do you see a significant difference in score for the two hands?

Cheers

Clemens




> 
> Any hints + ideas are welcome!
> 
> Best wishes
> 
> Kornelius
> 
>  --
>  Kornelius Zeth
>  Max Planck Institute for Developmental Biology
>  Dept. Protein Evolution
>  Spemannstr. 35
>  72076 Tuebingen, Germany
>  kornelius.z...@tuebingen.mpg.de
>  Tel -49 7071 601 323
>  Fax -49 7071 601 349

-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] Question regarding S-SAD

[Dirk Kostrewa]

 Dear Kornelius,

a few thoughts that come to my mind:

- Are you sure about the space group P6? For proteins, P6_1, P6_2, ..., 
P6_5 are more usual.


- Did you try both hands of the Se-sites? In case of space group P6_n, 
you have to invert both the handedness of the sites _and_ of the space 
group, resulting in P6_(6-n) (i.e. P6_5 instead of P6_1).


- I wouldn't worry about the mosaicity.

Best regards,

Dirk.

Am 18.10.10 14:03, schrieb Kornelius Zeth:

Dear all,

we have collected S-SAD data (2x4000 Frames, 0.1 degrees, wedges of 40 Frames, 
Rf between 3-5% overall) and received a dataset to 2.5 A which is complete with 
a redundancy of 30-40. Space group is P6 and the AU 120 residues, 6 Mets. 
Native data are up to 1.9 A Sharp and Phenix both return the expected number of 
sites and given the phasing power to approx. 3.8 A after HA refinement I was 
hoping to get some maps which I could use for tracing the structure. However 
DM, Solomon and Resolve all fail to significantly improve the phases and 
densities look not as if they can be traced. There is some partial twinning of 
15%. What I noticed was that the spot profiles in XDS looked not as the are 
supposed to, presumably because the mosaicity of the spots are 0.4-0.6 and spot 
integration may be hampered. Still, merging Rfactors are brilliant.

Any hints + ideas are welcome!

Best wishes

Kornelius

  --
  Kornelius Zeth
  Max Planck Institute for Developmental Biology
  Dept. Protein Evolution
  Spemannstr. 35
  72076 Tuebingen, Germany
  kornelius.z...@tuebingen.mpg.de
  Tel -49 7071 601 323
  Fax -49 7071 601 349


--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***

[ccp4bb] Question regarding S-SAD

[Kornelius Zeth]

Dear all,

we have collected S-SAD data (2x4000 Frames, 0.1 degrees, wedges of 40 Frames, 
Rf between 3-5% overall) and received a dataset to 2.5 A which is complete with 
a redundancy of 30-40. Space group is P6 and the AU 120 residues, 6 Mets. 
Native data are up to 1.9 A Sharp and Phenix both return the expected number of 
sites and given the phasing power to approx. 3.8 A after HA refinement I was 
hoping to get some maps which I could use for tracing the structure. However 
DM, Solomon and Resolve all fail to significantly improve the phases and 
densities look not as if they can be traced. There is some partial twinning of 
15%. What I noticed was that the spot profiles in XDS looked not as the are 
supposed to, presumably because the mosaicity of the spots are 0.4-0.6 and spot 
integration may be hampered. Still, merging Rfactors are brilliant.

Any hints + ideas are welcome!

Best wishes

Kornelius

 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 kornelius.z...@tuebingen.mpg.de
 Tel -49 7071 601 323
 Fax -49 7071 601 349

Re: [ccp4bb] Pseudo-symmetry/refinement

[Esko Oksanen]

  Seema,

  The problem actually originates from the scaling of the data that  
assumes a monomodal distritbution of the intensities. This is not the  
case with pseudotranslation and there are two sets of reflections: the  
weak, pseudo-absent reflections and the strong reflections. The  
scaling that assumes errors to be proportional to the intensity  
therefore underestimates the error of the strong reflections and  
overestimates the error of the weak ones. What I did was to write a  
script that selected the weak and the strong reflections from the  
output of XDS and rescale each set. I would then just run Refmac using  
each set in turn; the weak ones for rigid body and the strong ones for  
restrained refinement. The procedure is described in Oksanen et. al  
(2006) Acta Cryst. D62,  1369-1374.


 HTH,
 Esko

On 17.10.2010, at 19.23, Seema Nath wrote:

Would you please explain how to proceed for "rigid body refinement  
against weak reflection" & "normal restrained refinement against  
strong reflection".

Re: [ccp4bb] que fem sobre això miquel? es veu que l'Esther Cid en el seu dia ja ho va enviar.

[Esther Fernandez]

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>>>
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>>> Secretary Structural &Computational Biology Programme
>>> Institute for Research in Biomedicine
>>> Parc Científic de Barcelona
>>> c/ Baldiri Reixac 10-12
>>> 08028 Barcelona - Spain
>>> Tel. +34 93 403 99 53
>>> Fax. +34 93 403 99 76
>>>
>> esther.fernandez.rodrig...@irbbarcelona.org
>> http://www.irbbarcelona.org
>>
>>
>>
>> --
>>
>
>
>
> --




-- 

*Esther Fernández*
Secretary Structural &Computational Biology Programme
Institute for Research in Biomedicine
Parc Científic de Barcelona
c/ Baldiri Reixac 10-12
08028 Barcelona - Spain
Tel. +34 93 403 99 53
Fax. +34 93 403 99 76
esther.fernandez.rodrig...@irbbarcelona.org
http://www.irbbarcelona.org

Re: [ccp4bb] Reindexing scaled data

[Eleanor Dodson]

You dont need to reprocess - just reindex k,l,h or whatever you want to do..
Reprocessing is not necessary if you are keeping the same pointgroup. 
There is no change in the expected geometry of the diffraction.If for 
example you were changing from pontgroup P222 where all angles are 
restrained to 90 to PG P2 where alpha ad gama could vary away from 90, 
then you need to reprocess..




Eleanor


On 10/17/2010 08:45 PM, Jürgen Bosch wrote:

Try pointless first with your P222 data and as output option it will provide 
you the most likely space group. The warning in the GUI is just a warning, 
maybe P21 21 21 is not the right space group that's why you get the warning :-)

In terms of reprocessing your data, you are not dependent on HKL2000, take 
Mosflm or XDS no license required, hassle free reprocessing of your data.

Jürgen

-
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
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Oct 17, 2010, at 3:34 PM, Daniel Bonsor wrote:


Hi all,

This maybe a simple/stupid question. I collected data at the synchrotron, 
integrated and scaled the data in P222 using HKL2000, though I should of scaled 
the data as P212121.

When I try to reindex using Reindex I get a message of

 You are changing the symmetry of merged data  are you SURE you know what 
you are doing
WARNING: ** Symmetry change of merged data **

I am unsure of what I am doing in this case. Currently I cannot reprocess the 
data as I do not have a working version of HKL2000. So my question is can I 
reindex scaled data and what I should be doing with the GUI of reindex?

Thanks in advance for advice.

Just in case it is important my cell dimensions are 109.9280  160.3030  
186.2200   90.   90.   90. and the resolution is 2.7 Angstrom.

Re: [ccp4bb] Big difference between anomalous map and density modification map

[Eleanor Dodson]

Most density modification programs include aspects of solvent flattening 
- it is unnecessary and apparently counter-productive to do it again..


Eleanor



On 10/17/2010 08:29 PM, Wu, Mousheng wrote:

Dear All,

I have a MAD dataset at 4Å and shelxD can find clear-cut 10 solutions (my 
protein has 12 methionines). I ran autosharp to refine them followed by density 
modification. After density modification, I ran solvent flattening. Then I 
calculated the anomalous map using the phase from sharp and the electron 
density map from solvent flattening. The anomalous map  shows the density 
around these 10 selenium sites are clear and round. However, the density map 
from solvent flattening showed that only 4 selenium sites have clear and round 
density.The density around these 4 sites clearly showed beautiful helices. 
surprisingly, other 6 selenium sites have poor density or no density at all.  
The electron density around them is not very good and the predicted helical 
density is flat. I check the electron density before I ran solvent flattening. 
The result is same.  I am quite confused about the big difference from these 
two maps.  I also try SnB to find the selenium sites. The solutions

are same as those from ShelxD. But How to explain the poor density around the 
selenium sites in the density modification map?  Is there any problem with my 
selenium sites? Any suggestion from crystallographic experts?  Thanks.



Mousheng Wu, PhD
Center for Membrane Biology
Department of Biochemistry and Molecular Biology
The University of Texas Medical School at Houston
6431 Fannin Street, Houston, TX, USA, 77030

Re: [ccp4bb] crystal growth

[Enrico Stura]

ODD INDEED!
When you get crystals with the same morphology for three different  
proteins in same condition
you should start suspecting that they are not protein crystals but  
something to do with
a combination of the common buffer for each of the proteins or/and the  
precipitant used


Enrico.

On Sat, 16 Oct 2010 05:56:23 +0200, Seema Nath   
wrote:


All the crystals I got for three different proteins in same condition  
looked similar. I think crystal morphology may vary with the  
crystallizing conditions.





--
Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Pseudo-symmetry/refinement

[Eleanor Dodson]

How can you have 3 copies and one strong pseudo translation?You probably 
need an even number of molecules with pairs related by the translation 
vector.

Eleanor

..

 On 10/17/2010 05:48 AM, Mike John wrote:









Hi all,
The data is 2.2A, p41212, unit cell has c axis twice longer
than a or b. Data processed by HKL2000, the diffraction pattern shows
Strong-weak-weak in C direction, indicating translational
pseudo-symmetry.
Xtriage analysis confirmed the pseudo-symmetry (large Patterson off-origin 
peak).
The model is 100% identical searching prob.
Phaser MR solved the structure easily with good Z-score and LLG(3 copies/a.u). 
The problem comes during the refinement:
The R-free converged around 50% (R-factor 38%) and never go down even though I 
tried hard including, twin possibility.
Seeking help to lower the R-free. Thank you very much
Mike

Re: [ccp4bb] Fill map/mask with dummy atoms?

[Dirk Kostrewa]

 Dear colleagues,

many thanks for all of your kind support, following my request about 
placing dummy atoms into en EM-map! Here is a short summary:
Frederic Vellieux kindly offered me to use his programs with clever 
positioning of atoms in a density.
Paul Emsley pointed to a program "findwaters", that comes with Coot, 
with option "--flood" to fill a density with waters.
John Tanner pointed to a USF program FLOOD from the VOIDOO packages for 
filling cavities with water.
Victor Lamzin sent me a script for ARP/wARP for placing dummy atoms into 
a map.
Alex Shkumatov pointed to two SAXS programs, em2dam and vol2pdb, to 
convert low resolution maps into dummy atoms.


Now, I have plenty of options to play with!

Best regards,

Dirk.

Am 13.10.10 13:49, schrieb Dirk Kostrewa:
 ... maybe, to clarifiy my question a little bit: I want to fill an 
essentially flat cryo-EM-map with dummy atoms. So, a peak search 
doesn't work. I would need a program that fills this map with dummy 
atoms for a few things that I want to try with this "atomic" 
representation.


Best regards,

Dirk.

Am 13.10.10 13:00, schrieb Dirk Kostrewa:

 Dear CCP4ers,

is there a program around that allows to fill an input map or mask 
with dummy atoms?


Best regards,

Dirk.





--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***