Re: [ccp4bb] the f' and f'' of heavy cluster

[LISA]

Dear Dr. Matthias Zebisch,

how to do the specific cluster search in phaser sad pipline? Thank you.

Lisa


On Thu, Nov 21, 2013 at 3:37 PM, Matthias Zebisch <
matthias.zebi...@bbz.uni-leipzig.de> wrote:

> Dear Lisa,
>
> if you have proper anomalous data I rather recommend using the Phaser SAD
> pipeline and specify cluster search.
> Worked instantly for me.
>
> Good luck!
>
> -
> Dr. Matthias Zebisch
> Division of Structural Biology,
> Wellcome Trust Centre for Human Genetics,
> University of Oxford,
> Roosevelt Drive,
> Oxford OX3 7BN, UK
>
> Phone (+44) 1865 287549;
> Fax (+44) 1865 287547
> Email matth...@strubi.ox.ac.uk
> Website http://www.strubi.ox.ac.uk
> -
>
>
> On 11/21/2013 6:29 AM, LISA wrote:
>
>> Dear All,
>> I am running autosharp with a single wavelength data soked with Ta6Br12.
>> This data collected at the wavelength of 1.254A. I told the autosharp the
>> f' -20 and f''10.5. The autosharp result said these values are not correct?
>> How can we get the f' and f'' of this cluster? Thank you.
>> Lisa
>>
>
>

Re: [ccp4bb] the f' and f'' of heavy cluster

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Lisa,

the wavelength indicates you collected the data at a synchrotron. Do
you have a fluorescence scan of your crystal? That should tell you f'
and f''.

Other than that you can use Ethan Merritt's excellent server at
http://skuld.bmsc.washington.edu/scatter/ - this, however, tells you
the same values you already mentioned, so unless sharp tells you why
it thinks these values were incorrect, I would carry on redardless.

Best,
Tim

On 11/21/2013 07:29 AM, LISA wrote:
> Dear All,
> 
> I am running autosharp with a single wavelength data soked with
> Ta6Br12. This data collected at the wavelength of 1.254A. I told
> the autosharp the f' -20 and f''10.5. The autosharp result said
> these values are not correct? How can we get the f' and f'' of this
> cluster? Thank you.
> 
> Lisa
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Icedove - http://www.enigmail.net/

iD8DBQFSjcwpUxlJ7aRr7hoRAn27AKDldnStuPdHKyjYedQq2wdJI61eawCeIYLX
zPj1+cptJsiSH8uUnr8SuZo=
=YgjB
-END PGP SIGNATURE-

Re: [ccp4bb] the f' and f'' of heavy cluster

[George M. Sheldrick]

Dear Lisa,

You could try using SHELXD (e.g. via hkl2map) to find the heavy atoms,
it doesn't need f' and f". However you should take into account that
many soaks have not absorbed the intended heavy atoms or clusters, and
that if a tantalum bromide cluster has actually been incorporated it
will probably be disordered.

Best wishes, George


On 11/21/2013 07:29 AM, LISA wrote:
> Dear All,
>  
> I am running autosharp with a single wavelength data soked with Ta6Br12.
> This data collected at the wavelength of 1.254A. I told the autosharp
> the f' -20 and f''10.5. The autosharp result said these values are not
> correct? How can we get the f' and f'' of this cluster? Thank you.
>  
> Lisa

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

Re: [ccp4bb] the f' and f'' of heavy cluster

[Clemens Vonrhein]

Dear Lisa,

[there is a SHARP discussion list at
http://www.globalphasing.com/mailman/listinfo/sharp-discuss]

On Thu, Nov 21, 2013 at 02:29:23PM +0800, LISA wrote:
> Dear All,
> 
> I am running autosharp with a single wavelength data soked with Ta6Br12.
> This data collected at the wavelength of 1.254A. I told the autosharp the
> f' -20 and f''10.5. The autosharp result said these values are not correct?

Are these values from a fluorescence scan? or just theoretical values?

> How can we get the f' and f'' of this cluster?

By far the best: look at the processed fluorescence scan for your
crystal. If you didn't do one (unlikely) or you don't have it any more
(unlucky) you can use calculated values (e.g. with the CCP4 crossec
program).

Remember that for clusters there are two steps you have to be
concerned about: (1) HA detection and (2) HA refinement. SHARP itself
implements a 'spherical cluster' approach for the second tasks
(ie. once you have some sites), which can be very powerful if your
clusters are truly disordered. See

  http://www.globalphasing.com/sharp/manual/appendix3.html#generalSPHCLUSTER
  http://www.globalphasing.com/sharp/manual/chapter4.html#CLISTspecify

Often you will need to use a more manual/traditional approach here:

 * finding the HA positions by running e.g. SHELXD by hand instead of
   as part of an automatic pipeline

 * running the HA parameter refinement and phasing program with the
   correct parametrisation for your cluster (see above), followed by
   manual inspection of LLG residual maps to find minor sites and
   decide if your cluster is ordered/disordered.

 * deciding on the correct hand/enantiomorph

 * doing density modification, averaging, model building etc.

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] Oxford Cryosystems 600 series Cryostream Cooler

[Karen McIntyre]

Oxford Cryosystems 600 series Cryostream Cooler

We would like to offer an Oxford Cryosystems 600 series Cryostream Cooler free 
of charge. The recipient will have to arrange the shipping.

Please contact Marty Rajaratnam (Randall division of Cell and Molecular 
Biophysics, King's College London) email 
r.rajarat...@kcl.ac.uk if you are interested in 
acquiring this system.

Ms Marty Rajaratnam
3.19 Randall Division of Cell and Molecular Biophysics
New Hunt's House
Guy's Campus
King's College London
LONDON. SE1 1UL
Tel: 02078486428


-- 
Scanned by iCritical.

[ccp4bb] Stereo monitor

[Tobias Beck]

Dear all,

We are looking into ordering a stereo monitor. I am aware that this
question comes up every so often on this BB (and I read the post by Alice),
but maybe someone has any comments or recent experience with passive vs.
active monitors?

And: Which graphics card can you recommended? The monitor will be used with
Linux and Windows, so support of the correspoding graphics card with both
systems is required.

Thanks and best wishes,

Tobias.

-- 
___

Dr. Tobias Beck
ETH Zurich
Laboratory of Organic Chemistry
Wolfgang-Pauli-Str. 10, HCI F 322
8093 Zurich, Switzerland
phone:   +41 44 632 68 65
fax:+41 44 632 14 86
web:  http://www.protein.ethz.ch/people/tobias
___

[ccp4bb] AW: [ccp4bb] Dealnig with O-linked mannose

[Herman . Schreuder]

Dear Dmitry,

I only work with N-glycosylated proteins and here the people from coot and 
refmac have done a wonderful job in creating all necessary dictionaries. I 
would be very surprised if this would not be true for O-glycosidic bonds. 
Instead of reinventing the wheel myself, I would first try the dictionaries 
available in coot. So what I would do is:

Go to the Ser - Get monomer MAN (to get a monomer with the most recent atom 
names) - delete O4, or which oxygen gets replaced by the OG of the Ser
- create Link (from the extenstions/modeling menu) and see what happens. Doing 
a real-space refinement on the sugar only usually produces a fit to the density 
which is sufficient for refinement with Refmac or Buster.

By me Asn-Nag bond is shown as a dotted line, so once the Ser-Man bond is 
created, it should also be displayed. Alternatively, you could also manually 
add a LINK record to your pdb file with an editor. 

Best regards,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dmitry 
Rodionov
Gesendet: Mittwoch, 20. November 2013 19:49
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Dealnig with O-linked mannose

Good day!

I am refining what appears to be O-mannosylated protein structure.

In my hands Coot (0.7.1) does not form the SER-MAN bond automatically.
I made a SER-MAN.cif with jLigand, which takes care of the glycosidic bond.  
However, now the peptide bonds are not made to this custom residue (same chain, 
consecutive numbering).

Am I doing something wrong? How can I fix this?

Many thanks,

Dmitry

Re: [ccp4bb] Orientation of molecules

[Appu kumar]

Dear All,
   I think i have not explained my problem precisely. This
may be weird one but let me elaborate more. I have have a protein
moleculeA, having N-term, and C-term end. Structurally, it is dimer
with anti-parallel arrangement i.e N-terminal of one copyA of molecule
form dimer in such a way that it copyB would be arranged in
antiparallel fashioned (N-term of copyA is besides C-term of CopyB).
So when i am searching for two copy of molecule in phaser it is giving
me two copy of molecule in parallel arrangement. So my question is,
how to tell phaser that after fixing the orientation of first copy, to
change the orientation of 2nd copy with respect to first one so that
their n-teminal and c-terminal lies beside each other. I am looking
for your valuable suggestion.
Thank you

On 21/11/2013, Matthias Zebisch  wrote:
> i think you need to explain the problem in greater detail to the community
>
> eg. how do you come to know that the orientation of mol2 is 180° flipped
> - perhaps it is correct as phaser puts it?
>
> No one will be able to help you with so little information
>
> Best wishes,
>
> Matthias
>
> -
> Dr. Matthias Zebisch
> Division of Structural Biology,
> Wellcome Trust Centre for Human Genetics,
> University of Oxford,
> Roosevelt Drive,
> Oxford OX3 7BN, UK
>
> Phone (+44) 1865 287549;
> Fax (+44) 1865 287547
> Email matth...@strubi.ox.ac.uk
> Website http://www.strubi.ox.ac.uk
> -
>
> On 11/21/2013 5:38 PM, Appu kumar wrote:
>> Dear All,
>>  I seek your valuable suggestion on a MR problem. I am
>> asking phaser to search for two molecules in ASU, first molecules
>> phaser searched right but when placing the second molecule, it
>> orientation has got flipped by 180 deegree. Is there any way to tell
>> phaser to fix the orientation of second copy of molecules. ? Looking
>> forward for your valuable suggestion
>>
>> Thank you very much in advance
>> Appu
>>
>
>
> --
> -
> Dr. Matthias Zebisch
> Division of Structural Biology,
> Wellcome Trust Centre for Human Genetics,
> University of Oxford,
> Roosevelt Drive,
> Oxford OX3 7BN, UK
>
> Phone (+44) 1865 287549;
> Fax (+44) 1865 287547
> Email matth...@strubi.ox.ac.uk
> Website http://www.strubi.ox.ac.uk
> -
>
>

Re: [ccp4bb] Sokalan cp 42 as a cryoprotectant

[Jim Pflugrath]

Here's an easy experiment to try:

What happens when you flash-cool your reservoir solution in Liquid Nitrogen?  
Does it remains "glass-clear" at 100 K or lower?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of abhimanyu singh 
[abhisingh@gmail.com]
Sent: Thursday, November 21, 2013 10:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Sokalan cp 42 as a cryoprotectant

Dear all,

Recently I got couple of crystallization hits in conditions containing
30-40% sokalan cp 42 provided in MIDAS commercial screen from
molecular dimensions. I tried to look up for information regarding its
probable cryo protection activity but failed to find anything. Could
someone have any clue about this ?



Thank you.


Greetings,

--
Abhimanyu Kumar Singh
Ph.D. Student
Department of Macromolecular Structures
National Center for Biotechnology (CNB-CSIC)
C/ Darwin 3, Campus de Cantoblanco,
28049 Madrid, Spain.
E-Mail: abhimanyu.si...@cnb.csic.es

Re: [ccp4bb] Sokalan cp 42 as a cryoprotectant

[Clemens Grimm]

Dear Abhimanyu,

Sokalan CP42 is a modified polycarboxylate with 'medium' molecular  
weight. In terms of cryoprotective properties it is likely to behave  
similar to PEG 2. Try adding 25-35% glycerol as a starting point.


Best,
Clemens


Zitat von abhimanyu singh :


Dear all,

Recently I got couple of crystallization hits in conditions containing
30-40% sokalan cp 42 provided in MIDAS commercial screen from
molecular dimensions. I tried to look up for information regarding its
probable cryo protection activity but failed to find anything. Could
someone have any clue about this ?



Thank you.


Greetings,

--
Abhimanyu Kumar Singh
Ph.D. Student
Department of Macromolecular Structures
National Center for Biotechnology (CNB-CSIC)
C/ Darwin 3, Campus de Cantoblanco,
28049 Madrid, Spain.
E-Mail: abhimanyu.si...@cnb.csic.es






--
Dr. Clemens Grimm
Institut für Biochemie
Biozentrum der Universität Würzburg
Am Hubland
D-97074 Würzburg
Germany
e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de
phone : +49 0931 31 84031
-

Re: [ccp4bb] Dealnig with O-linked mannose

[Dmitry Rodionov]

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and real-space 
refine the result. My apologies for not being clear enough.

Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 5.41)
It does not automatically make the bond between MAN C1 and OG of SER either.

Here is the way I finally made the connection followed by refinement it in Coot:

1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions->Modelling ...->make Link (Click 2 atoms) ... Click on C1 of MAN 
and OG of SER
dashed bond appears
5) Sphere refine 

A whole different issue that was touched in the replies is how this model will 
be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect the 
described link without massaging by means of either pdb.edits or apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so step 4 
might have to be repeated.

Best regards,
Dmitry




smime.p7s
Description: S/MIME cryptographic signature

Re: [ccp4bb] Dealnig with O-linked mannose

[Engin Özkan]

Hi,

On 11/20/13, 4:17 PM, Zhijie Li wrote:
If you need to refine the structure with phenix.refine then you need 
to make an edit file to specify that the mannose C1 is linked to the 
ser OG by a covalent bond. 
I am not sure if this is what was meant, but you don't need to define 
bonds explicitly in phenix. Phenix.refine has the same (or a forked 
version, I am not sure) monomer library as refmac's, and it contains the 
MAN-SER and MAN-THR linkages, which includes bond distance and angle 
restraints, but no torsion angles (see below). You can invoke a MAN-SER 
or MAN-THR linkage from the phenix.refine GUI, or add a parameter file 
to define it. You shouldn't need to specify distances and angles. I am 
guessing the not-fully mature linkage-detection function in phenix, 
which works well for N-linked sugars, might also work for O-linked, but 
Nigel Moriarty from the PHENIX team would know (looking forward to this 
being automatic).


By the way, this is how MAN-SER looks in the latest phenix monomer library:

#
data_link_MAN-SER
#
loop_
_chem_link_bond.link_id
_chem_link_bond.atom_1_comp_id
_chem_link_bond.atom_id_1
_chem_link_bond.atom_2_comp_id
_chem_link_bond.atom_id_2
_chem_link_bond.type
_chem_link_bond.value_dist
_chem_link_bond.value_dist_esd
 MAN-SER  1 C1  2 OGsingle   1.4390.020
loop_
_chem_link_angle.link_id
_chem_link_angle.atom_1_comp_id
_chem_link_angle.atom_id_1
_chem_link_angle.atom_2_comp_id
_chem_link_angle.atom_id_2
_chem_link_angle.atom_3_comp_id
_chem_link_angle.atom_id_3
_chem_link_angle.value_angle
_chem_link_angle.value_angle_esd
 MAN-SER  1 C1  2 OG  2 CB  108.7003.000
 MAN-SER  1 O5  1 C1  2 OG  112.3003.000

Engin

[ccp4bb] Sokalan cp 42 as a cryoprotectant

[abhimanyu singh]

Dear all,

Recently I got couple of crystallization hits in conditions containing
30-40% sokalan cp 42 provided in MIDAS commercial screen from
molecular dimensions. I tried to look up for information regarding its
probable cryo protection activity but failed to find anything. Could
someone have any clue about this ?



Thank you.


Greetings,

-- 
Abhimanyu Kumar Singh
Ph.D. Student
Department of Macromolecular Structures
National Center for Biotechnology (CNB-CSIC)
C/ Darwin 3, Campus de Cantoblanco,
28049 Madrid, Spain.
E-Mail: abhimanyu.si...@cnb.csic.es

Re: [ccp4bb] the f' and f'' of heavy cluster

[Jason Busby]

If you set the atom type to "TX", phaser will use the built-in scattering 
factors for the tantalum bromide cluster.  This will be represented as a point 
scatterer rather than the actual cluster - if you have very good data you may 
be able to actually position the tantalum atoms in the cluster and get better 
phase information that way.

Jason.

--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 84155
fx:  +64 9 3737414

On 21/11/2013, at 9:32 PM, LISA wrote:

Dear Dr. Matthias Zebisch,

how to do the specific cluster search in phaser sad pipline? Thank you.

Lisa


On Thu, Nov 21, 2013 at 3:37 PM, Matthias Zebisch 
mailto:matthias.zebi...@bbz.uni-leipzig.de>>
 wrote:
Dear Lisa,

if you have proper anomalous data I rather recommend using the Phaser SAD 
pipeline and specify cluster search.
Worked instantly for me.

Good luck!

-
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK

Phone (+44) 1865 287549;
Fax (+44) 1865 287547
Email matth...@strubi.ox.ac.uk
Website http://www.strubi.ox.ac.uk
-


On 11/21/2013 6:29 AM, LISA wrote:
Dear All,
I am running autosharp with a single wavelength data soked with Ta6Br12. This 
data collected at the wavelength of 1.254A. I told the autosharp the f' -20 and 
f''10.5. The autosharp result said these values are not correct? How can we get 
the f' and f'' of this cluster? Thank you.
Lisa

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

Hi Engin,

Thanks for the information! I didn't realize that O-glycans can also be 
specified as specific linkages like N-glycans. Those two extra angle 
restraints should be helpful.


Zhijie


-Original Message- 
From: Engin Özkan

Sent: Thursday, November 21, 2013 1:00 PM
To: Zhijie Li
Subject: Re: [ccp4bb] Dealnig with O-linked mannose

Hi,

On 11/20/13, 4:17 PM, Zhijie Li wrote:


If you need to refine the structure with phenix.refine then you need to 
make an edit file to specify that the mannose C1 is linked to the ser OG 
by a covalent bond.

I am not sure if this is what was meant, but you don't need to define
bonds explicitly in phenix. Phenix.refine has the same (or a forked
version, I am not sure) monomer library as refmac's, and it contains the
MAN-SER and MAN-THR linkages, which includes bond distance and angle
restraints, but no torsion angles (see below). You can invoke a MAN-SER
or MAN-THR linkage from the phenix.refine GUI, or add a parameter file
to define it. You shouldn't need to specify distances and angles. I am
guessing the not-fully mature linkage-detection function in phenix,
which works well for N-linked sugars, might also work for O-linked, but
Nigel Moriarty from the PHENIX team would know (looking forward to this
being automatic).

By the way, this is how MAN-SER looks in the latest phenix monomer library:

#
data_link_MAN-SER
#
loop_
_chem_link_bond.link_id
_chem_link_bond.atom_1_comp_id
_chem_link_bond.atom_id_1
_chem_link_bond.atom_2_comp_id
_chem_link_bond.atom_id_2
_chem_link_bond.type
_chem_link_bond.value_dist
_chem_link_bond.value_dist_esd
 MAN-SER  1 C1  2 OGsingle   1.4390.020
loop_
_chem_link_angle.link_id
_chem_link_angle.atom_1_comp_id
_chem_link_angle.atom_id_1
_chem_link_angle.atom_2_comp_id
_chem_link_angle.atom_id_2
_chem_link_angle.atom_3_comp_id
_chem_link_angle.atom_id_3
_chem_link_angle.value_angle
_chem_link_angle.value_angle_esd
 MAN-SER  1 C1  2 OG  2 CB  108.7003.000
 MAN-SER  1 O5  1 C1  2 OG  112.3003.000 

Re: [ccp4bb] Stereo monitor

[mesters]

  
  
Sorry, I have to correct a wrong
  statement in my previous email..
  
  I own a CLUB 3D mini displayport to active dvi dual link adaptor
  330 mhz stereo 3d gaming cable (the 270 mhz model is the wrong
  model so do not buy the 270 mhz one!!!)
  
  - J. -
  
  Am 21.11.13 13:50, schrieb mesters:


  
  Hi Tobias,

besides several advantages (cheaper hardware, cheaper glasses,
in general less headaches), the drawbacks of passive monitors
are:
1) fine lines running across the screen that you will notice on
a white bachground if the viewing distance is not that big
2) reduced viewing angle because of the polarisation sheet
placed in front of the display
3) half the resolution in the vertical plane with stereo "on"
that in addition may cause problems with the menus of certain
programs

For the combination "professional stereo", "afordable" and
"linux" the only sensible option (my humble opinion) is to buy a
nvidia quadro card (the k2000d) and the

Asus VG278HR (not HE!) with build-in emitter and one pair of
nvidia glasses. This combination works under linux and windows.
The price of the monitor plus one pair of glasses is ±500€ and
of the quadro k2000d card ±450€ (both handle the dual link DVI-D
standard). This combination is still cheaper than having to buy
a professional card with a 3-pin stereo exit (nvidia k4000 @
>900€), a good stereo monitor (±350€ Asus VG278HE 144 Hz) and
a seperate set of glasses with IR-emitter (±130€). The stereo of
the k2000d-VG278HR combination is great (fantastic actually and
much much better than a good old CRT because of the resolution
and brightness) as the monitor drives the switching of the
glasses... better than the latest 27 inch AOC passive stereo
monitor (280€) I recently bought and operate at home..

If you already own an older quadro card with a DVI-D exit, you
only need to buy the VG278HR. If you own an older quadro with a
mini display port, you must also buy a CLUB 3D active mini
displayport-dual link dvi adaptor 270 mhz (±115€; we own one and
it works in contrast to cheaper variants) to link-up the monitor
as a DVI-I exit will not handle the amount of data in true
stereo mode, that is, 1080p @ 120Hz. 

The k2000d (85% performance compared to older quadro 4000) is
more than fast enough and the nvidia driver support for the
quadro card under windows and linux is at a professional level,
so no problems there.

- J. -

Am 21.11.13 11:49, schrieb Tobias Beck:
  
  

  

  
Dear all,
  

We are looking into ordering a stereo monitor. I am
aware that this question comes up every so often on this
BB (and I read the post by Alice), but maybe someone has
any comments or recent experience with passive vs.
active monitors? 

And: Which graphics card can you recommended? The
monitor will be used with Linux and Windows, so support
of the correspoding graphics card with both systems is
required.
  
  

Thanks and best wishes,

  
  Tobias.
  

  

  -- 
___

Dr. Tobias Beck
ETH Zurich
Laboratory of Organic Chemistry
Wolfgang-Pauli-Str. 10, HCI F 322
8093 Zurich, Switzerland
phone:   +41 44 632 68 65
fax:        +41 44 632 14 86
web:      http://www.protein.ethz.ch/people/tobias
___ 

  

  

  
  
  
  -- 

Dr. Jeroen R. Mesters
  Deputy and Group Leader
  
  Institute of Biochemistry, University of Lübeck
  Ratzeburger Allee 160, 23538 Lübeck, Germany
  
  phone: +49-451-5004065 (secretariate 5004061)
  fax: +49-451-5004068
  
  http://www.biochem.uni-luebeck.de
  http://www.iobcr.org

      
 --
  If you can look into the seeds of time and tell which
  grain will grow and which will not, speak then to me who
  neither beg nor fear (Shakes

Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?

[Bosch, Juergen]

What is the solution to this?
Hi Meisam,
you have it, it is just three molecules in the asu. Look at the overall crystal 
lattice packing and see if you have contacts supporting each molecule. Generate 
a large representation of your symmetry mates, I suspect you have a channel in 
your crystal lattice, explaining why your Matthews coefficient leads you in a 
wrong direction.
If you are not convinced, calculate a selfrotation function with Molrep and 
look at the Chi=120 section and compare it to the Chi=90.

At least judging from your R-factors, if I understand you correctly you have 
not done much of a refinement yet. Have you performed a real space refinement 
and adjusted side chains etc. ?

Jürgen

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

On Nov 21, 2013, at 12:35 PM, Meisam 
mailto:meisam.nosr...@gmail.com>> wrote:

Dear CCP4ers

I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% 
linear Rfactor.
The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric 
unit, 53% for 4 molecules, and 36% for 5 molecules.
Molecular replacement just finds 3 molecules in the asymmetric unit. Running 
Phaser also gives a partial solution with 3 molecules.
When I refine just the back bone of the protein for the 3 molecules the 
Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular 
replacement on the refined structure again, and I fix it as a model to search 
for another molecule, it still does not find it.

I have attached a photo to show the density for the fourth molecule in the 
asymmetric unit.

What is the solution to this?

Thanks in advance for your help

Meisam

Re: [ccp4bb] the f' and f'' of heavy cluster

[Dyda]

Dear Lisa,

You are not saying how far your data extends in resolution.

In my experience, finding the cluster(s) can work better if you limit your data 
used in the
search ~5A, as scattering goes down beyond this due to interference between 
atoms of the clusters.

It does come back at higher scattering angles, but I rarely have data there in 
these days...

Fred
***
Fred Dyda, Ph.D.   Phone:301-402-4496
Laboratory of Molecular BiologyFax: 301-496-0201
DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov  
Bldg. 5. Room 303 
Bethesda, MD 20892-0560  URGENT message e-mail: 2022476...@mms.att.net
Google maps coords: 39.000597, -77.102102
http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred
***

Re: [ccp4bb] the f' and f'' of heavy cluster

[Christian Roth]

Hi,

one could  indeed get much better phases with the correct positions of 
the HA cluster. If there is sufficient anomalous signal one could use 
the cluster as search model for Molecular replacement using the 
anomalous data as described in Dahms et al.  to get the exact position 
of the cluster in the crystal.


"Localization and orientation of heavy-atom cluster compounds in protein 
crystals using molecular replacement."


Acta Crystallogr D Biol Crystallogr. 2013 Feb;69(Pt 2):284-9

Cheers

Christian

Am 21.11.2013 19:36, schrieb Jason Busby:

If you set the atom type to "TX", phaser will use the built-in
scattering factors for the tantalum bromide cluster.  This will be
represented as a point scatterer rather than the actual cluster - if you
have very good data you may be able to actually position the tantalum
atoms in the cluster and get better phase information that way.

Jason.

--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 84155
fx:  +64 9 3737414

On 21/11/2013, at 9:32 PM, LISA wrote:


Dear Dr. Matthias Zebisch,
how to do the specific cluster search in phaser sad pipline? Thank you.
Lisa


On Thu, Nov 21, 2013 at 3:37 PM, Matthias Zebisch
mailto:matthias.zebi...@bbz.uni-leipzig.de>> wrote:

Dear Lisa,

if you have proper anomalous data I rather recommend using the
Phaser SAD pipeline and specify cluster search.
Worked instantly for me.

Good luck!

--__---
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK

Phone (+44) 1865 287549;
Fax (+44) 1865 287547
Email matth...@strubi.ox.ac.uk 
Website http://www.strubi.ox.ac.uk 
--__---


On 11/21/2013 6:29 AM, LISA wrote:

Dear All,
I am running autosharp with a single wavelength data soked
with Ta6Br12. This data collected at the wavelength of 1.254A.
I told the autosharp the f' -20 and f''10.5. The autosharp
result said these values are not correct? How can we get the
f' and f'' of this cluster? Thank you.
Lisa

Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?

[Mark J van Raaij]

half-seriously...old-school method:
- apart from the excellent suggestions by Juergen and Phil, you could 
experimentally determine the density of the crystals and calculate their 
protein content from that. This looks like a fun method to try:
http://journals.iucr.org/j/issues/1999/05/00/wb0070/wb0070.pdf
(some work, but would be an independent way to estimate the solvent content)

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 21 Nov 2013, at 18:35, Meisam wrote:

> Dear CCP4ers
> 
> I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% 
> linear Rfactor.
> The Mattew coefficient gives 10% probability for 3 molecules in the 
> asymmetric unit, 53% for 4 molecules, and 36% for 5 molecules. 
> Molecular replacement just finds 3 molecules in the asymmetric unit. Running 
> Phaser also gives a partial solution with 3 molecules. 
> When I refine just the back bone of the protein for the 3 molecules the 
> Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular 
> replacement on the refined structure again, and I fix it as a model to search 
> for another molecule, it still does not find it.
> 
> I have attached a photo to show the density for the fourth molecule in the 
> asymmetric unit.
> 
> What is the solution to this?
> 
> Thanks in advance for your help
> 
> Meisam
> 

Re: [ccp4bb] How can I find the other molecule in the asymmetric unit?

[Phil Jeffrey]

Meisam:

Probabilities are just that: many of us have had structures with large 
solvent contents that are statistically unlikely.


Pedantic quibble:  "It scales in P21 Space group with 7% linear 
Rfactor." really means that it scales in primitive monoclinic with a 
reasonable Rsymm, and I hope you also checked P2 as well as P21 when 
doing molecular replacement.  P2 is rare, but not unprecedented.


When you say "refine just the back bone" do you mean you're refining 
just a poly-ALA model or a non-mutated one ?  Because if so, absent the 
side-chains and any waters, an R-factor of 31% quite good.  If so, add 
side-chains and then waters and continue refining and see how things 
look.  3-fold averaging across the current monomers plus your decent 
resolution should make the sequence interpretation straightforward.


But:

* if you can see interpretable density for the fourth molecule, build in 
secondary structure elements, refine those, repeat until you can see a 
substructure you recognize, then place the monomer manually.


* use Arp/wArp to autobuild your structure - this has the benefit that 
often the map you'll get out will be a very good one even if you build 
the remaining monomer manually.  If you're lucky it'll build it all for you.


* Autobuild in Phenix can also do much the same

(I would do the first two, and perhaps all three, in parallel until one 
emerges a clear winner)


* if the above still doesn't resolve things, consider the possibility 
that the fourth molecule is not what you think it is, or may be 
statistically disordered


Phil Jeffrey
Princeton

On 11/21/13 12:35 PM, Meisam wrote:

Dear CCP4ers

I have a data set that diffracts 1.96 Å. It scales in P21 Space group with 7% 
linear Rfactor.
The Mattew coefficient gives 10% probability for 3 molecules in the asymmetric 
unit, 53% for 4 molecules, and 36% for 5 molecules.
Molecular replacement just finds 3 molecules in the asymmetric unit. Running 
Phaser also gives a partial solution with 3 molecules.
When I refine just the back bone of the protein for the 3 molecules the 
Rfree/Rwork does not go better than 34% / 31%, and when I run the molecular 
replacement on the refined structure again, and I fix it as a model to search 
for another molecule, it still does not find it.

I have attached a photo to show the density for the fourth molecule in the 
asymmetric unit.

What is the solution to this?

Thanks in advance for your help

Meisam

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

Hi Dmitry,

COOT does have the MAN-SER linkage record in its monomer lib, but it won't 
detect the bond for you. It also haven't provided an interface for the user 
to specify the bond type yet.
The COOT procedure you described is perfectly fine for generating a generic 
covalent bond record for a PDB file. However without specifying the linkage 
to be "MAN-SER" you do lose some restraints for the bond angles (as Engin 
pointed out in his post regarding phenix.refine).


It seems that refmac is the only program (that I've used so far) that will 
automatically detect the N- and O-glycan linkages and put that information 
into the header of a PDB file. This seems to be the easiest way of 
generating a proper "LINKRMAN-SER" line in the PDB header. Note that 
refmac writes a LINKR record for its own use, not "LINK" which is the PDB 
standard. You can simply manually change the "LINKR" to "LINK " to turn it 
into a PDB standard record, but LINKR works fine for newer versions of COOT 
too.  The advantage of taking this path instead of writing a LINK line 
manually (or modifying the LINK line you get from COOT) is that you can be 
sure that the "MAN-SER" is put in the right columns - PDB format is 
position-sensitive.


Alternatively, here is a LINK line that you can modify to fit your sequence 
and paste to your PDB file. Just make sure you only replace characters, not 
inserting/deleting any:
LINK C1  MAN B1001 OG  SER B 912 
MAN-SER


For phenix.refine, yes, it does not write any linkage information to the 
header or use those information stored in PDB headers. But you can always 
paste that LINK line you get from other places to the PDB files generated by 
phenix.refine. There is no need to redo the building  in COOT every time. 
The LINK line only specifies the bonding between two atoms. So as long as 
you haven't renumbered the residues or changed the chain IDs, you can 
transfer the same LINK line among your PDB files of the same protein (i.e. 
you do not even have to remake the link in COOT for every structure of the 
same protein: just paste the LINK line and make sure the residue numbers are 
right).


Zhijie



-Original Message- 
From: Dmitry Rodionov

Sent: Thursday, November 21, 2013 4:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dealnig with O-linked mannose

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and real-space 
refine the result. My apologies for not being clear enough.


Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 
5.41)

It does not automatically make the bond between MAN C1 and OG of SER either.

Here is the way I finally made the connection followed by refinement it in 
Coot:


1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions->Modelling ...->make Link (Click 2 atoms) ... Click on C1 of 
MAN and OG of SER

dashed bond appears
5) Sphere refine

A whole different issue that was touched in the replies is how this model 
will be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect the 
described link without massaging by means of either pdb.edits or 
apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so step 
4 might have to be repeated.


Best regards,
Dmitry

Re: [ccp4bb] Stereo monitor

[Andreas Förster]

I second everything Jeroen said except the need for a €450 graphics 
card.  We have a Quadro 600, which was only £105 or something like that. 
 The slightly cheaper Quadro 400 would have worked as well.  There are 
now successors to these cards.  Unless something has changed in their 
specification, they should work with the monitor with built-in emitter. 
 Others can comment on that.


Best regards.


Andreas



On 21/11/2013 10:49, Tobias Beck wrote:

Dear all,

We are looking into ordering a stereo monitor. I am aware that this
question comes up every so often on this BB (and I read the post by
Alice), but maybe someone has any comments or recent experience with
passive vs. active monitors?

And: Which graphics card can you recommended? The monitor will be used
with Linux and Windows, so support of the correspoding graphics card
with both systems is required.

Thanks and best wishes,

Tobias.

--
___

Dr. Tobias Beck
ETH Zurich
Laboratory of Organic Chemistry
Wolfgang-Pauli-Str. 10, HCI F 322
8093 Zurich, Switzerland
phone:   +41 44 632 68 65
fax:+41 44 632 14 86
web: http://www.protein.ethz.ch/people/tobias
___


--
  Andreas Förster
 Crystallization and Xray Facility Manager
   Centre for Structural Biology
  Imperial College London

Re: [ccp4bb] Stereo monitor

[David Schuller]

On 11/21/13 07:50, mesters wrote:

...(both handle the dual link DVI-D standard)...


Are there any monitors on the market yet which can produce stereo 3D 
from a Displayport 1.2 input? With or without a built-in emitter.



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

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

It seem the LINK line I provided eariler was chopped by the email system.

Here it is again:
LINKRC1**MAN*C***1*OG**SER*B*912MAN-SER
Simply replace each * with a space and change the residue IDs.

Also to clarify the procedure of using refmac to generate the MAN-SER 
linkage line:
1 In COOT, get monomer->MAN, delete hydrogens, delete O1. Drag the MAN to 
the proper position. C1 has better be ~1.4A away  from OG and mind the 
anomer conformation.
It seems that refmac detects covalent bonds mainly based on the distances. 
If you accidentaly put the ligand too close to something it should not be 
linked to (happens when you have poor electron density for positioning the 
ligand), refmac might complain that a new ligands is found but the geometry 
information is not provided. In such case the error message is a little 
misleading to the user: it is a new covalent bond, not a new monoer that's 
not described.
2 Merge molecules, Save PDB. For linkages that can be found in the standard 
monomer lib, you do not need to make the bond in COOT. It is refmac that 
will add this record.

3 Send the saved PDB to refmac. Do restrained refinement.
4 Check the resulting PDB to see if you now have the LINKR...MAN-SER line.

And in the refmac log you can find this line:
WARNING : link:MAN-SER  is found dist = 1.178 ideal_dist= 1.439
  ch:BB   res: 912  SER  at:OG  .->CC   res:   1  MAN 
at:C1  .



Zhijie

-Original Message- 
From: Dmitry Rodionov

Sent: Thursday, November 21, 2013 4:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dealnig with O-linked mannose

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and real-space 
refine the result. My apologies for not being clear enough.


Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 
5.41)

It does not automatically make the bond between MAN C1 and OG of SER either.

Here is the way I finally made the connection followed by refinement it in 
Coot:


1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions->Modelling ...->make Link (Click 2 atoms) ... Click on C1 of 
MAN and OG of SER

dashed bond appears
5) Sphere refine

A whole different issue that was touched in the replies is how this model 
will be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect the 
described link without massaging by means of either pdb.edits or 
apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so step 
4 might have to be repeated.


Best regards,
Dmitry

[ccp4bb] Orientation of molecules

[Appu kumar]

Dear All,
I seek your valuable suggestion on a MR problem. I am
asking phaser to search for two molecules in ASU, first molecules
phaser searched right but when placing the second molecule, it
orientation has got flipped by 180 deegree. Is there any way to tell
phaser to fix the orientation of second copy of molecules. ? Looking
forward for your valuable suggestion

Thank you very much in advance
Appu

Re: [ccp4bb] Stereo monitor

[Sabuj Pattanayek]

The quadro 600 or k600 works fine, been using it for more than a year with
that Asus with the built in emitter. In fact the absolute oldest and
cheapest option which works is the quadro 370, but you'll want the 600 at
least.


On Thu, Nov 21, 2013 at 9:19 AM, mesters wrote:

>  Hi Andreas,
>
> the older quadro 600 (with 96 CUDA cores) features a dual Link DVI-I (do
> not know why they did not call it DVI-D) but if I look at the specs, NVIDIA
> tries to suggest (?) the k600 features a DVI-I only. No mentioning it is
> dual-link. It would not make sense being single-link in the first place but
> make sure before you buy the new k600 (with 196 CUDA cores) it is really a
> dual-link DVI-I *i.e.* DVI-D. Some vendors state for the k600, 1x
> DisplayPort, 1x *Dual Link DVI-I* (Software)... Do not know what
> "software" exactly means at this point because I have never seen that
> before. For the more expensive cards (2000 and up) NVIDIA state they have a
> DVI-D and a DVI-I. Probably they are trying to confuse us to make us buy
> the more expensive models..
>
> - J. -
>

Re: [ccp4bb] Stereo monitor

[Eric Bennett]

Hi David,

We have had success with the ASUS VG248QE connected with DisplayPort, on an HP 
Z620 running RHEL 6.4.

We like to have dual stereo displays on Linux, and Nvidia is discontinuing many 
of the cheaper multi-DVI-port cards.  We had no success trying to take two Acer 
DVI monitors and connect them to DP on a Quadro 4000 (not K4000) using various 
DP/DVI adapters.  So we decided we had to switch entirely to native DP 
connections and chose the VG248QE.No built in emitter on this monitor, 
however.  So I think if you want to use this on Linux, you will need the K4000 
or higher.  We bought a K5000 to test it with, assuming we could fall back to 
the K5000's dual DVI ports and our old DVI monitors if DP didn't work.  But 
since we got DP to work, we will probably go with the K4000 for our next batch 
of Linux workstations, since it appears to be the minimum card that works on 
Linux with the 3-pin emitter, and also provides two DP connectors.

We had problems getting the correct stereo 3-pin bracket adapter for these 
cards; our reseller initially sent us the wrong one that is apparently for 
older Nvidia cards (very nice of them to change the plug periodically!) but we 
were able to get the correct one by talking directly to HP.

The card will drive the monitor at 144 Hz when stereo is disabled in the X 
server config file, but it drops to 120 Hz in stereo mode, which I assume is 
probably the maximum shutter speed on the 3D Vision Pro glasses.

Cheers,
Eric





On Nov 21, 2013, at 9:52 AM, David Schuller wrote:

> On 11/21/13 07:50, mesters wrote:
>> ...(both handle the dual link DVI-D standard)...
> 
> Are there any monitors on the market yet which can produce stereo 3D from a 
> Displayport 1.2 input? With or without a built-in emitter.
> 
> 
> -- 
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu

--
Eric Bennett, er...@pobox.com

Always try to associate yourself with and learn as much as you can from those 
who know more than you do, who do better than you, who see more clearly than 
you.
- Dwight Eisenhower

Re: [ccp4bb] Orientation of molecules

[Phil Jeffrey]

* Open molecular replacement solution in Coot
* Display crystal packing (Draw>Cell & Symmetry), perhaps as Calphas only
* Find the symmetry-related instance of copyB that is in the correct 
position relative to copyA according to your preferences
* Use File>Save Symmetry Coordinates to write the structure transposed 
by that operator (note: select the menu option, then click on the copyB 
instance)
* Since Coot will write the entire structure transposed by that symop, 
assemble the desired solution from copyA from the mol.rep. solution and 
copyB from the transposed solution.  I'm a Luddite so I use emacs and/or 
grep for this.


Phil Jeffrey
Princeton


11/21/13 1:11 PM, Appu kumar wrote:

Dear All,
I think i have not explained my problem precisely. This
may be weird one but let me elaborate more. I have have a protein
moleculeA, having N-term, and C-term end. Structurally, it is dimer
with anti-parallel arrangement i.e N-terminal of one copyA of molecule
form dimer in such a way that it copyB would be arranged in
antiparallel fashioned (N-term of copyA is besides C-term of CopyB).
So when i am searching for two copy of molecule in phaser it is giving
me two copy of molecule in parallel arrangement. So my question is,
how to tell phaser that after fixing the orientation of first copy, to
change the orientation of 2nd copy with respect to first one so that
their n-teminal and c-terminal lies beside each other. I am looking
for your valuable suggestion.
Thank you