[ccp4bb] AW: [ccp4bb] Tris buffer in cryo protectant

[Herman . Schreuder]

Dear Ursula,

just a stupid question: did you  try freezing a crystal? There are quite a few 
crystal structures in the PDB with (a) bound Tris molecule(s), so quite some 
crystals were not destroyed by a pH shock during freezing.
If you tried freezing and saw no/bad diffraction, you should try to take some 
shots at room temperature to find out whether the diffraction is destroyed by 
the freezing, or whether your crystal diffracts just badly from the very 
beginning. There are sleeves you can put over the loop with your crystal to 
prevent the crystal from drying out.

Good luck,
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Ursula 
Schulze-Gahmen
Gesendet: Freitag, 12. Juni 2015 22:47
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Tris buffer in cryo protectant

Does anyone have experience with Tris buffer in cryo protectants? I would 
expect the pH of the cryosolution to increase a lot during flash freezing which 
could perhaps destroy the diffraction. I rarely use Tris for crystallization 
but the current protein really prefers Tris. I would appreciate any comments.
Ursula

--
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

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Re: [ccp4bb] Tris buffer in cryo protectant

[Enrico Stura]

Ursula,

After extensive testing, I have found out that in most cases pH changes  
during flash freezing does
not pose a problem. In some cases it can  be beneficial. I encourage you  
to try +/- 2 pH units

from your crystallization pH.

Sometimes a sub-optimal pH is chosen just because the crystals were  
obtained the first time at that pH,
sometimes it is for strategic reasons, for example to prevent excessive  
nucleation.
The best pH to obtain BIG crystals is not always the best pH to get the  
best diffraction.

Test various pH if you have enough crystals to do that.

Enrico.


On Fri, 12 Jun 2015 22:47:10 +0200, Ursula Schulze-Gahmen  
 wrote:



Does anyone have experience with Tris buffer in cryo protectants? I would
expect the pH of the cryosolution to increase a lot during flash freezing
which could perhaps destroy the diffraction. I rarely use Tris for
crystallization but the current protein really prefers Tris. I would
appreciate any comments.

Ursula




--
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
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
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e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71

Re: [ccp4bb] Residual density feature

[Ben Bax]

Dear Colin,
  I think the residual blob usually represents a combination of different 
chemical entities.

I think the correct thing to do is to try to dock all the possible chemical 
entities you have in your crystallisation buffer, purification buffers and 
things that have carried through in an unknown manner from the purification 
into the blob and try and estimate their occupancies (if they give a chemically 
reasonable fit).

If it is a big enough blob you could just try and dock glycerol (if this is in 
your cryo) and water and have them at half occupancy, and have a comment in the 
pdb header.

Regards, Ben


Ben Bax
Senior Scientific Investigator
BioMolecular Sciences UK
RD Platform Technology & Science

GSK
Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
Email   benjamin.d@gsk.com
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[cid:image001.png@01D0A754.53FEE6D0]



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Colin Levy
Sent: 14 June 2015 09:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Residual density feature

Dear all,

I am currently working on a structure that contains a residual density feature 
located within the active site. Due to a combination of factors including 
limited occupancy, modest resolution, twinning etc it has not been possible to 
unambiguously identify this feature despite fairly extensive efforts.

What is the best way of dealing with such a feature when depositing the 
structure? Ideally I would like to draw attention to the presence of residual 
density whilst not implying that I have been able to identify it.

Many thanks,

Colin


Manchester
Protein
Structure
Facility

Dr. Colin W. Levy
MIB G034
Tel.  0161 275 5090
Mob.07786 197 554
c.l...@manchester.ac.uk




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Re: [ccp4bb] Residual density feature

[Boaz Shaanan]

Hi Colin,


You may have tried this but just in case you haven't, perhaps you should try put a few water molecules in the blob and refine? After that the geometry of the water arrangement may give you a better idea what the blob represents. It worked for me in quite
 a few cases. 


  Cheers,


              Boaz


 
 
Boaz Shaanan, Ph.D.

Dept. of Life Sciences  
Ben-Gurion University of the Negev  
Beer-Sheva 84105    
Israel  
    
E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan  
Fax:   972-8-647-2992 or 972-8-646-1710
 
 








From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Colin Levy [c.l...@manchester.ac.uk]
Sent: Sunday, June 14, 2015 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Residual density feature



Dear all,


I am currently working on a structure that contains a residual density feature located within the active site. Due to a combination of factors including limited occupancy, modest resolution, twinning etc it has not been possible to unambiguously identify
 this feature despite fairly extensive efforts. 


What is the best way of dealing with such a feature when depositing the structure? Ideally I would like to draw attention to the presence of residual density whilst not implying that I have been able to identify it.


Many thanks,


Colin







Manchester

Protein
Structure
Facility

Dr. Colin W. Levy
MIB G034
Tel.  0161 275 5090
Mob.07786 197 554
c.l...@manchester.ac.uk

[ccp4bb] workable data from PDB sf-cif

[StrBio]

Hi All,

How one can convert pdb-sf.cif file downloaded from PDB to a workable
format for further refinement (may be by phenix or Refmac) of the deposited
structure?

I need help.

Re: [ccp4bb] workable data from PDB sf-cif

[Schubert, Carsten [JRDUS]]

Phenix has a menu item for this: Reflection Tools -> Import CIF structure 
factors

HTH

Carsten

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of StrBio
Sent: Monday, June 15, 2015 1:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] workable data from PDB sf-cif

Hi All,
How one can convert pdb-sf.cif file downloaded from PDB to a workable format 
for further refinement (may be by phenix or Refmac) of the deposited structure?
I need help.

Re: [ccp4bb] workable data from PDB sf-cif

[Hattne, Johan]

Or "Convert to/modify/extend MTZ" in "Reflection Data Utilities”.  You can 
“Import reflection file in mmCIF format and create MTZ file”.

// Johan

> On Jun 15, 2015, at 13:58, Schubert, Carsten [JRDUS]  
> wrote:
> 
> Phenix has a menu item for this: Reflection Tools -> Import CIF structure 
> factors
>  
> HTH
>  
> Carsten
>  
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of StrBio
> Sent: Monday, June 15, 2015 1:53 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] workable data from PDB sf-cif
>  
> Hi All,
> 
> How one can convert pdb-sf.cif file downloaded from PDB to a workable format 
> for further refinement (may be by phenix or Refmac) of the deposited 
> structure?
> 
> I need help.

  Research Specialist @ Gonen Lab

Janelia Research Campus * 19700 Helix Drive
Ashburn, VA 20147 * +1 (571) 209-4000 extension 3376

Re: [ccp4bb] workable data from PDB sf-cif

[Tim Gruene]

Dear StrBio,

you can use the command line tool cif2mtz. If the file is rather recent,
and you are only interested in the first block, you can type

 cif2mtz hklin yourciffile.sf hklout yourmtzfile.mtz << eof
 end
 eof

There are some subtleties that you may only convert correctly after
looking into the mmCIF-file, e.g. when more than 1 block is present and
you want the data from but the first block. In most cases the above
should work.

Regards,
Tim

On 06/15/2015 07:52 PM, StrBio wrote:
> Hi All,
> 
> How one can convert pdb-sf.cif file downloaded from PDB to a workable
> format for further refinement (may be by phenix or Refmac) of the deposited
> structure?
> 
> I need help.
> 

-- 
--
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
phone: +49 (0)551 39 22149

GPG Key ID = A46BEE1A




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Re: [ccp4bb] workable data from PDB sf-cif

[Pavel Afonine]

Why not just use CIF files directly? Note, most Phenix tools accept input
model and data in cif format (and can write them back in cif too).
Pavel

On Mon, Jun 15, 2015 at 10:52 AM, StrBio  wrote:

> Hi All,
>
> How one can convert pdb-sf.cif file downloaded from PDB to a workable
> format for further refinement (may be by phenix or Refmac) of the deposited
> structure?
>
> I need help.
>
>

Re: [ccp4bb] workable data from PDB sf-cif

[Shibom Basu]

Well, you can also try sf-convert program from PDB to convert sf.cif file
to any other format mtz, or cns whatever you want.



On Mon, Jun 15, 2015 at 10:52 AM, StrBio  wrote:

> Hi All,
>
> How one can convert pdb-sf.cif file downloaded from PDB to a workable
> format for further refinement (may be by phenix or Refmac) of the deposited
> structure?
>
> I need help.
>
>

Re: [ccp4bb] workable data from PDB sf-cif

[StrBio]

Thank you all.
I got several options, and I am using phenix pdb sf-cif to mtz protocol.

Thanks once again for your help.

On Mon, Jun 15, 2015 at 1:38 PM, Shibom Basu  wrote:

> Well, you can also try sf-convert program from PDB to convert sf.cif file
> to any other format mtz, or cns whatever you want.
>
>
>
> On Mon, Jun 15, 2015 at 10:52 AM, StrBio 
> wrote:
>
>> Hi All,
>>
>> How one can convert pdb-sf.cif file downloaded from PDB to a workable
>> format for further refinement (may be by phenix or Refmac) of the deposited
>> structure?
>>
>> I need help.
>>
>>
>
>

Re: [ccp4bb] workable data from PDB sf-cif

[Donald Damian Raymond]

Hi,

I've written a bash script that does just what you want.

Find it here -->  
https://github.com/donaldraymond/crystallography-scripts/blob/master/PDB_data.sh

Run the script with a PDB ID. Like this "PDB_data 1yks"

The script downloads the PDB and sf.mmCIF files from the PDB. It then converts 
the cif file to an MTZ  you can use in refmac, phenix, buster, COOT, etc.

Best,
Donald

.
Donald Damian Raymond, Ph.D.
Postdoctoral Research Fellow
Stephen C. Harrison Lab
Harvard Medical School
CLSB 3082
email: raym...@crystal.harvard.edu
http://www.linkedin.com/in/donalddraymond/


On Jun 15, 2015, at 1:52 PM, StrBio  wrote:

> Hi All,
> 
> How one can convert pdb-sf.cif file downloaded from PDB to a workable format 
> for further refinement (may be by phenix or Refmac) of the deposited 
> structure?
> 
> I need help.
>  

Re: [ccp4bb] workable data from PDB sf-cif

[Huw Jenkins]

> On 15 Jun 2015, at 20:00, Donald Damian Raymond  wrote:
> 
> Hi,
> 
> I've written a bash script that does just what you want.

and potentially deletes a lot of files it didn’t create...

#cleanup
rm temp* "$cif_file" 2> /dev/null



Huw

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Re: [ccp4bb] workable data from PDB sf-cif

[Donald Damian Raymond]

Hi Huw,

$cif_file is a variable that holds the sf-cif file downloaded from www.RCSB.org

see line 126 of the script 

>>   cif_file="$pdb_id-sf.cif"

After converting the cif to an MTZ, the cif is no longer necessary. That is why 
the script deletes it (clean up).

Best,
Donald


On Jun 15, 2015, at 3:56 PM, Huw Jenkins  wrote:

> 
>> On 15 Jun 2015, at 20:00, Donald Damian Raymond  wrote:
>> 
>> Hi,
>> 
>> I've written a bash script that does just what you want.
> 
> and potentially deletes a lot of files it didn’t create...
> 
> #cleanup
> rm temp* "$cif_file" 2> /dev/null
> 
> 
> 
> Huw
> 
> 

Re: [ccp4bb] workable data from PDB sf-cif

[Donald Damian Raymond]

Hi all,

Huw was correct, there is potential to delete temp files that the script did 
not create that I had not considered. I've changed "rm temp* "$cif_file" 2> 
/dev/null" to "rm temp.mtz temp.pdb "$cif_file" 2> /dev/null"

Best,
Donald


On Jun 15, 2015, at 3:56 PM, Huw Jenkins  wrote:

> 
>> On 15 Jun 2015, at 20:00, Donald Damian Raymond  wrote:
>> 
>> Hi,
>> 
>> I've written a bash script that does just what you want.
> 
> and potentially deletes a lot of files it didn’t create...
> 
> #cleanup
> rm temp* "$cif_file" 2> /dev/null
> 
> 
> 
> Huw
> 
>