Re: [ccp4bb] suggestion on improving sea urchin like crystals

[william lee]

Here is some information regarding to my
crystals. 


Crystallization condition:


0.9M-1.7M Ammonium Tartrate Dibasic


50mM-150mM Bis-Tris Propane (pH7.0 &
6.5) / 50mM-150mM Tris (pH7.5 & 8.5)

 



Protein buffer contains 50mM ADA, pH6.5,
100mM NaCl, 10mM B-ME  


 
Regards
William
 
Date: Fri, 11 Apr 2014 01:50:18 +0100
From: williamlee0...@hotmail.com
Subject: [ccp4bb] suggestion on improving sea urchin like crystals
To: CCP4BB@JISCMAIL.AC.UK






Dear All,

 

I am currently working on a ligand bound
protein complex. From my initial crystallization screens I have identified a
condition which gives me sea urchin like crystals. I managed to repeat these
crystals in my optimization conditions, in fact I can see crystals in all the
wells but there is no significant difference between them. All conditions give
me similar size and amount of crystals. To confirm these are protein crystals
or not, I tried expose the crystals to X-ray beam (in-house). Good news is that
I have no obvious salt diffraction at high resolution, but the bad news is my
low resolution diffractions are not really believable. In addition, these
crystals seem to be quite easy to separate into needles but they are too small
to tell if they do crack like most the protein crystals.

 

I have attached a picture of my crystals
and the diffraction pattern I got at low resolution. 

 

I am hoping if anyone can suggest me on how
to improve or change the shape of these crystals if they are genuine protein
crystals.

 

 

Many thanks Kind regards

William


  

[ccp4bb] 2nd Australian Advanced Methods in Crystallography Workshop

[Sofia Caria]

Dear all,

The Australian Synchrotron is pleased to announce the 2nd Australian Advanced 
Methods in Crystallography Workshop. This workshop will explore the state of 
the art in macromolecular crystallography. It will combine lectures on advanced 
topics in data collection, processing and structure determination with hands-on 
tutorials using both MX beamlines at the Australian Synchrotron. International 
experts will present their experience in solving crystal structures, model and 
ligand building, twinning and refinement.

Associated with this workshop there will also be a day for Lipid Cubic Phase 
Crystallisation Satellite (LCP). Registration as part of the main event or 
separately is possible (see details in programme and registration).

Confirmed speakers:  Kay Diederichs, Harry Powell, Viviane Richter, Santosh 
Panjikar, Navraj Pannu, Daniele de Sanctis, Paul Adams, Joana Pereira, Paul 
Emsley, Tom Caradoc-Davies, David Aragão and Wei Liu.

Workshop dates: 22-25th June 2014
Registration deadline: 22th May 2014
Website: https://events.synchrotron.org.au
Contact: 
xtalworkshop2...@synchrotron.org.au

Best regards,

Sofia Caria
(by the organising committee)

[cid:0132A221-6C9B-4F6B-8A46-5EA169E19418]
Organising committee:
Daniel Eriksson- Australian Synchrotron
David Aragão - Australian Synchrotron
Jade Forwood - Charles Sturt University
Mihwa Lee - La Trobe University
Sofia Caria - La Trobe University
Stephanie Gras - Monash University
<>

Re: [ccp4bb] About crystallization diffraction problem

[Roger Rowlett]

A couple of thoughts:

 * We have actually managed to grow quite a few crystals like this.
   Sometimes they are not single crystals, but stacks of mis-aligned
   plates that come apart easily when handled or subjected to osmotic
   stress. Sometimes these stacks give great-looking diffraction
   patterns, but they turn out to be multiple lattices :(  (You find
   this out when you try to index your beautiful diffraction images and
   it runs off the rails.) We got around this issue by taking our
   crystallization condition and doing a full additive screen
   (everything we had in the reagent drawer that looked like an
   additive, salts, solvents, etc.) at 50-200 mM or 5-10%. We found a
   condition that gave slightly thicker plates that were single
   crystals and not stacks of thin plates, and those crystals
   cryoprotected and diffracted well.
 * If your plates are actually single crystals, but are just fragile or
   have high solvent content and are being torn apart by osmotic
   stresses introduced by your cryoprotectant, I highly recommend
   trying the cryomixes described in Vera & Stura, Cryst. Growth Des.
   (2014) 14, 427-435. For crystals grown in PEGs, one or more of these
   mixes are like magic with fragile crystals. For salt conditions, 2.5
   M lithium sulfate is also very dependable. Saved our bacon for a
   couple of high solvent content crystals recently.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 4/10/2014 1:59 PM, Anamika Singh wrote:

Dear All,

I want to get some help regarding crystallization for one of the 
protein I am working. This is a recombinant protein of molecular 
weight of 17.5 kDa.I am getting crystals shape like thin plates in 5 
days, in different conditions like bis tris pH 6.5-8, HEPES pH 6-8 
with .1M nacl , .01M DTT having PEG 3350, PEG 6000 as precipitant. But 
whenever we used to put crystal in cryoprotectant like 20 % ethylene 
glycol, glycerol, MPD it used to split like layers of some tree barks.
And the crystal which were diffracting not getting diffracted above 
3.0 Angstrom.


Please help me out.

Thanks in advance
--
Anamika

[ccp4bb] About crystallization diffraction problem

[Anamika Singh]

Dear All,

I want to get some help regarding crystallization for one of the protein I
am working. This is a recombinant protein of molecular weight of 17.5 kDa. I
am getting crystals shape like thin plates in 5 days, in different
conditions like bis tris pH 6.5-8, HEPES pH 6-8 with .1M nacl , .01M DTT
having PEG 3350, PEG 6000 as precipitant. But whenever we used to put
crystal in cryoprotectant like 20 % ethylene glycol, glycerol, MPD it used
to split like layers of some tree barks.
And the crystal which were diffracting not getting diffracted above 3.0
Angstrom.

Please help me out.

Thanks in advance
-- 
Anamika

Re: [ccp4bb] About crystallization diffraction problem

[Das, Debanu]

Hi Anamika,

1) What is the conc of PEG3350, PEG6000 in the crystallization drop? If high 
enough, you may not need additional cryoprotectant. Just try freezing intact 
crystals without additional cryo and collecting data.

2) Grow the crystals in presence of ethylene glycol or glycerol (i.e. add cryo 
in crystallization solution). You can also try to rescreen with some commercial 
crystallization kits that include cryoprotectant.

3) I presume you see the cracking during single step of cryoprotectant soaking. 
Have you tried serial soaks in increasing amounts of cryo, say from 1-16 or 18% 
EG, GOL in steps of few % and short soaks at each step? The gradual transition 
may be better in your case.

4) You can try to slow down the crystallization time (conc, temp), which may 
give you larger/thicker crystals (or even try seeding), which may lead to more 
durable crystals that survive the cryoprotection soak.

5) You should probably try some diffraction (maybe just 1-2 images) without any 
additional cryo and/or capillary mount in mother liquor to see what your 
resolution really is and see what is the impact from the cryoprotection step.

Best,
Debanu.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anamika 
Singh
Sent: Thursday, April 10, 2014 11:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] About crystallization diffraction problem

Dear All,



I want to get some help regarding crystallization for one of the protein I am 
working. This is a recombinant protein of molecular weight of 17.5 kDa. I am 
getting crystals shape like thin plates in 5 days, in different conditions like 
bis tris pH 6.5-8, HEPES pH 6-8 with .1M nacl , .01M DTT having PEG 3350, PEG 
6000 as precipitant. But whenever we used to put crystal in cryoprotectant like 
20 % ethylene glycol, glycerol, MPD it used to split like layers of some tree 
barks.
And the crystal which were diffracting not getting diffracted above 3.0 
Angstrom.


Please help me out.

Thanks in advance

-- 
Anamika 

Re: [ccp4bb] Renumbering of water molecules during deposition

[MARTYN SYMMONS]

I had thought that after discussion on this bulletin board we could find no 
rational scientific reason why the PDB renumbers waters from the N to the C 
term of the polypeptides (and 5' to 3' of nucleic chains?). 
https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1311&L=CCP4BB&D=0&P=12314

I think that provided you follow the agreed rules on chain ids (see below) then 
you have a 'right of veto' on the water renumbering. You also have a 'right of 
veto' for the water movement to symmetry-related positions - which would 
involve chain id change. 

So probably the best way is to assign the chain ids yourself and just say you 
don't want any of your waters renumbered. Then keep track of them yourself as 
Ed suggested using documented community tools.

One related question is how waters with alternate positions are dealt with. I 
cannot find clear documentation on this. Often sidechains with alternate 
positions motivate alternate waters during refinement. But I believe these are 
removed during the deposition water renumbering step. So if you have particular 
waters in alternative positions - say owing to a Michaelis complex at the 
active site - then the renumbering will likely split your water in alternate 
positions to produce two apparently unrelated, differently-numbered, solvent 
atoms. It could be argued how can you know these are alt pos of the same water? 
But if you have refined them like that then it is good for users of your 
coordinates to see that documented.  

It may be that the new PDB Deposition System is more flexible in this regard - 
I have not used it yet.

Below is the current agreed procedure from the wwPDB website.

Hope that helps. 
 regards
  Martyn 
How are chain IDs assigned to chemical groups and waters?
All chemical groups and waters within 5 Ångstroms of any atom of a polymeric 
chain will be considered to be associated with that chain and will be assigned 
the same chain ID as that polymer. If a particular chemical group/water is 
equidistant from more than one chain, then the chain ID is randomly assigned to 
be that of any one of these polymers.
Waters further than 5 Ångstroms away from the polymer that can be moved by 
symmetry to within 5 Ångstroms, will then be automatically moved and the author 
will be notified. If there are objections, the waters will be moved back to 
their original positions.
Waters further than 5 Ångstroms away from any polymer, which cannot be brought 
closer to a polymer chain by application of symmetry, will be brought to the 
attention of the depositor. The waters will be listed in REMARK 525 with the 
distance to the nearest macromolecule listed. Authors will be given the 
opportunity to remove the waters or update the coordinates. Authors may choose 
to retain the distant waters in the entry.



 From: Edward A. Berry 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Monday, 7 April 2014, 15:25
Subject: Re: [ccp4bb] Renumbering of water molecules during deposition
 

After deposition (my experience is with RCSB) you will get back a processed
file to verify.  See how the waters have been renumbered, and renumber
in such a a way that your original scheme is easily identifiable but in
keeping with whatever the renumbering was trying to achieve- e.g. your
waters X1,X2,X3 could become A1001, A1002, A1003 and the symmetry related
waters B1001, B1002, B1003. Discuss with the annotator to see how your
waters can be renumbered in a recognizable way in keeping with requirements
of the PDB conventions. Then in future depositions keep this same scheme
(you may still need to renumber the original processed file each time).
eab

Eckhard Hofmann wrote:
> Hi all,
> during deposition of structures the water numbering is frequently changed.
> I would love to keep specific numbers for critical water molecules e.g.
> in an active site in related proteins (mutants, ligand soaks), to
> facilitate easier discussion in a manuscript.
>
> Is there a general agreement how to treat these?
>
> One way to deal with this is would be to use an arbitrary offset:
> A1-A233 protein
> A301 ligand
> A302 another ligand
> A401-404 4 special waters
> A500- general solvent
>
> Any input welcome ;-)
> All the best,
> Eckhard
> --
> Prof. Dr. Eckhard Hofmann 
> Ruhr-Uni Bochum
> AG Proteinkristallographie, LS Biophysik, ND04/318
> 44780 Bochum
> Tel: +49-(0)234/32-24463, Sekr. -24461, FAX: -14762
>