[ccp4bb] PhD pattern-recognition-for-protein-crystallisation-strategies

[Hargreaves, David]

Dear CCP4bb,

Slightly off topic but perhaps of interest to students.

Synopsis:

The MARCO (MAchine Recognition of Crystallization Outcomes) project showed that 
automated image analysis can be used to classify experimental results, having a 
94% correct classification rate. Work on a custom image classifier using 
convolutional neural networks is already underway at AstraZeneca. In this 
project, the student will incorporate Active Learning into the training process 
to improve performance and use additional information from UV images to achieve 
higher accuracy. As the main aim of the project, the student will then use the 
annotated crystallisation images to connect compounds or proteins with 
experimental conditions using novel graph-based networks.

This is a 4-year iCASE studentship based in the Mathematics Department at the 
University of York fully funded by EPSRC and AstraZeneca.

Link to details:

https://www.findaphd.com/phds/project/pattern-recognition-for-protein-crystallisation-strategies/?p120098


Dr. David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure & Biophysics
Dr David Hargreaves.
Office R1-09/Lab FL54
AstraZeneca Darwin Building
Unit 310
Cambridge Science Park
Milton Road
Cambridge
CB4 0WG

telephone: +441223223546
mobile: 07525 742054

David.Hargreaves @astrazeneca.com

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Re: [ccp4bb] challenges in structural biology

[Hargreaves, David]

Dear CCP4bb,

From my industrial perspective: The crystallisation bottleneck has probably 
been fairly well addressed. High throughput screening using robotics and around 
20ul of protein per screen is common. There are lots of screens to try even if 
they are rather redundant in their content. However, I feel there are two 
things missing: high throughput protein production at microscale and specific 
tools for construct design. Generally, industry is interested in the druggable 
site which perhaps is only one domain of a multidomain target protein. Getting 
a suitably robust crystal system often requires more than one iteration of 
multiple construct design which in my experience is something of a lottery. 
Some helpful design tools and an order of magnitude increase in the number of 
constructs that can be delivered from protein supply would help.

Best wishes,

David


Dr. David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure & Biophysics
Dr David Hargreaves.
Office R1-09/Lab FL54
AstraZeneca Darwin Building
Unit 310
Cambridge Science Park
Milton Road
Cambridge
CB4 0WG

telephone: +441223223546

David.Hargreaves @astrazeneca.com

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Re: [ccp4bb] suggestions for cryoprotectant

[Hargreaves, David]

Hi Firdous,

I had success last week with a condition containing 1.5M MgCl2 which I’d tried 
cryo-ing with high salt before and got no diffraction.
Adding dimethylsulphoxide to a final concentration of 10% v/v in the well 
solution worked well for me. DMSO was bound in the structure which can be a 
pest. Maybe other low mwt organics e.g. butane 2,3 diol, at around 10% might 
also work.
Good luck!

David


Dr. David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure & Biophysics
Dr David Hargreaves.
Office R1-09/Lab FL54
AstraZeneca Darwin Building
Unit 310
Cambridge Science Park
Milton Road
Cambridge
CB4 0WG

telephone: +441223223546
mobile: 07525 742054

David.Hargreaves @astrazeneca.com

Please consider the environment before printing this e-mail



From: CCP4 bulletin board  On Behalf Of Firdous Tarique
Sent: 19 October 2018 22:58
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] suggestions for cryoprotectant

Dear members

I have got beautiful crystal hits in SaltRx screens which are not diffracting 
to a good resoultion. All of them are salt based condition and I am not able to 
formulate a good cryoprotectant for these crystals. I also think that in my 
case the poor resolution is due to a poor cryoprotectant selection.

The conditions are as follows:

1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
2>0.5M KCN 100mM Tris pH8.5
3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
4>4M Sodium Nitrate 100mM Tris pH8.5
5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6

There are few more conditions but so far not able to see good diffraction with 
using lower peg and glycerol based cryoprotectants.

Can anybody suggest me good cryos conditions for salt based crystallization 
conditions or anything good for SaltRx crystallization hits.

Thanks

Firdous



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Re: [ccp4bb] Precipitation issue during refolding

[Hargreaves, David]

Maybe this has already been mentioned in which case apologies:
I’d try rapid dilution. My only experience of having to refold worked both by 
dialysis and the dilution method but dilution had the advantage of allowing 
various conditions to be tested quickly and more controllably at small scale. I 
started with 100mgml-1 (1ml) protein in 5M GuHCl and did a 100 fold dilution 
with very rapid stirring. I followed this up years later by trying to refold 
using the Mosquito, tiny drops and a 96 well refolding screen. The plates set 
up set up as if they were crystallisation experiments and were sealed and 
imaged. The readout was visible precipitation in the drops. Those that stayed 
clear were perhaps the better conditions although I couldn’t categorically say 
they were; it was just a guide to follow up on. A better readout would be 
activity and the Mosquito could be replaced by a multichannel pipette. I guess 
it hinges on whether you are getting enough protein to start experimenting with.

Good luck!

David

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar 
Manna
Sent: 21 September 2017 11:12
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Precipitation issue during refolding

Hi,

I am working with a serine protease. As the protein is not soluble I am 
purifying it from the inclusion bodies followed by refolding. The protein shows 
good activity after refolding but the major concern is the 'precipitation'. 
Though the protein express quite well, but I loose almost 50-60% protein during 
refolding (Buffer: 100 mM Tris pH 8.0, 125 mM MgCl2, 100 mM KCL, 5% Glycerol, 2 
mM GSH, 1 mM GSSG and 1 M NDSB) as it precipitates. Refolding is done in room 
temperature. I tried refolding at 4 degree, but it even end up with more 
precipitant. It also precipitates during concentrating, so in general I almost 
loose most of the protein during this refolding and concentration steps. I 
start with 6 lit culture that give around 1-2 mg protein in the final step, 
which I am not happy with.
​
Any suggestion to deal this issue would be highly appreciated.

Thank you in advance.

Best,

Dipankar​

--
Dipankar Manna, Ph.D
Postdoctoral Researcher
Department of Molecular Medicine
Institute of Basic Medical Sciences
University of Oslo, Domus Medica
Oslo, Norway

Mob   : +47 451 66 517
E-mail: dipankar.ma...@medisin.uio.no
   dipankar.biot...@gmail.com
http://www.med.uio.no/imb/english/people/aca/dipankam/


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Re: [ccp4bb] Sliconizing of cover-slips

[Hargreaves, David]

Hi Praveen,

20 years ago I seemed to spend more time siliconising cover slips than setting 
up the Magic 50. We had special jigs to hold a couple of dozen slips for 
washing, drying and finally immersion in silane solution. This method was very, 
very tedious. An alternative method was to pull a vacuum over the slips 
scattered on a glass Petri dish in a desiccator containing around 50mls of 
silane solution in a beaker. The dish sat on top of the beaker and the vacuum 
from a water tap pump would make the silane solvent boil. This was marginally 
quicker and used less silane solution. Either way, each slip was then 
individually polished using lens tissue and the dust blown away using 
compressed air before storage.
My own “quick and dirty” method was to fill a tall plastic measuring cylinder 
with solution and drop the slips in serially and let them tumble through the 
solution as they sank. The cylinder should be tall and have a diameter 
comparable to that of the cover slip. I’d do around 300 at a time like this. 
All methods require scattering the slips onto aluminium foil and giving them a 
quick bake at around 100DegC to get rid of excess silane solution and HCl 
immediately after treatment. I gave up polishing them and just used to wash the 
slips in HPLC grade ethanol before drying in the oven and storing (hiding) 
them.  Whichever way you choose, you do need to do the siliconising in a fume 
cupboard.

Best wishes,

David


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Praveen 
Kumar Tripathi
Sent: 27 April 2017 10:26
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Sliconizing of cover-slips

Dear all,

sorry for off topic question.

May i know if anybody uses homemade silinization of coverslips for protein 
crystallization purposes?

I have purchased Sigmacote SL2-100 ml for silanizing coverslips for hanging 
drop protein crystallization setup.

Please share your methods to siliconize coverslip using Sigmacote SL2-100 ml if 
anybody uses.

Thanks in advance

regards
Praveen



--
Praveen Kumar Tripathi
PhD Research Scholar
Kusuma School of Biological Sciences
Indian Institute of Technology Delhi-110016
+91-9873625228


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Re: [ccp4bb] Crystals grow at the bottom of the tube.

[Hargreaves, David]

I have been presented with crystals grown in nmr tubes on two separate 
occasions. Both diffracted very well and thankfully, neither required a 
magnetic field for optimisation. On the first occasion I did use the initial 
sample as a seed stock.

Best wishes,

David

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of kiki
Sent: 18 October 2016 11:42
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystals grow at the bottom of the tube.

Dear all users,

 I got my crystals at the bottom of the tube on ice just before I started to 
screen crystals. Those crystals' shapes were good but easy to melt when I 
harvested them. I shot these crystals. They only diffracted to 7A to 8A. Has 
anyone ever met this situation before? How to improve?

Thanks,
Lingyuan


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Re: [ccp4bb] DNA RNA annealing problem

[Hargreaves, David]

Hi Weifei,

When I was looking at Holliday junction annealing I found that the salt 
concentration was important. NaCl around 300-500mM improved yield as did MgCl2 
at lower (100-200mM) concentrations. So, it might be worth trying higher salt.

Dave

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ChenWeiFei
Sent: 03 July 2015 16:14
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] DNA RNA annealing problem

Dear all,
I want to get a complex of DNA-RNA-protein. But I have a big problem of 
annealing DNA-RNA.
The length of DNA is 19nt and RNA is 17nt.
Annealing protocol:
2uM DNA
2uM RNA
10mM Tris-Hcl
100mMNaCl
1mMEDTA

Heated to 95 for 5min, cooling down slowly for nearly 2h to room temperature.

I can just get a result of two single strand DNA/RNA. PAGE analysis.

No double helix was founded.

Does anyone have the same problem or know how to fix it.

Thank you for your answering.

Best regards,
Weifei



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Re: [ccp4bb] Tev cleavage

[Hargreaves, David]

You could try adding TEV to your protein just prior to crystallisation. I 
produced 2 equivalent structures this way; one with the  tag clearly visible 
packing between molecules in an I centred space group and a 2nd (with TEV 
added) in P21 where the tag was no longer visible (the packing suggested it 
would not be accommodated and therefore cleaved). I don’t think many people try 
detagging their protein in conditions as extreme as those in a crystallisation 
trial but in the end that’s where the protein needs to be happy. Yes, there are 
lots of caveats with this technique, not least sub optimal cleavage conditions, 
but it’s a very simple experiment to do.

Dave

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana 
Rangel
Sent: 02 May 2015 18:57
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tev cleavage


Dear all,

I'd like some help about my protein cause I've a lot of problems in cleavage 
moment. In this step after aproximadately 30 minutes (37C) occur precipitation 
almost 50% .
I tried control it:
- Protein diluition (no results)
- Cleavage 4C ( no cleavage)
-Modifying buffers: add 10% glycerol and 5% glucose (no crystallization)
- Add salt (1M - no results)
- Add serial tev (500ul in the first time and more 500ul in second time- 37C) 
total precipitation
- Crystallization with 7 histag ( poor crystallization, no diffraction)

Now I need to produce this protein with semet that became the protein more 
hidrofobic, probably.

So, If anyone could help me...

Thanks in advance

Giulliana Rangel



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Re: [ccp4bb] Thin plate crystals

[Hargreaves, David]

The crystals don't look that bad in my opinion. Maybe the cryo step is sub 
optimal? Try using large loops (reduces surface tension forces during 
transfer). Butane 2,3 diol is a good cryo to try. Maybe shoot them at room temp 
(in situ) to get an idea of how they diffract before you manipulate them.
Good luck!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prerana G.
Sent: 24 April 2015 04:01
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Thin plate crystals

Dear all,
I am working on a protein (40kDa) which forms very thin plate shaped crystals 
which diffracts at very low resolution. Protein concentration that i have used 
for crystallisation is approx. 8mg/ml. I have attached the picture of the 
protein crystal.

How can I improve upon the shape of the crystal?


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Re: [ccp4bb] Crystallization issue

[Hargreaves, David]

On the face of it, I wouldn’t get overly excited by the drop. However, I’ve 
been underwhelmed and wrong before. If you’ve got access to a synchrotron 
beamline that can shoot the material in-situ it might be a good experiment to 
try.
Best of luck,
Dave

Dr. David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Dr David Hargreaves.
OfficeR1-09/Lab FL54
AstraZeneca Darwin Building
Unit 310
Cambridge Science Park
Milton Road
Cambridge
CB4 0WG

telephone: +441223223546
mobile: 07525 742054

David.Hargreaves @astrazeneca.commailto:name.surn...@astrazeneca.com

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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of P Silva
Sent: 05 January 2015 19:12
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallization issue

Hi everyone,

Sorry for the non related crystallization question. In a crystallization 
screening (20ºC) I got in 70% of the drops some fibrils/gel (see figure below) 
which I do not know how to interpret. Has anyone got the same kind of behavior 
in crystallization screenings? It could be related with the stability of the 
protein (salt concentration, pH,...)?
The buffer of the protein is 20mM Tris pH 7.5, 150mM NaCl.

Thanks in advance


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Re: [ccp4bb] Solvent channels

[Hargreaves, David]

Hi Reza,

I can recommend the work of Marc-Olivier Coppens and Kourosh Malek and the 
references therein for an interesting journey through solvent channels.

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
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--
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza 
Khayat
Sent: 27 June 2014 12:01
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Solvent channels

Hi,

I'd like to do some soaking experiments with a relatively large molecule. Can 
someone suggest a program/method to display the solvent channels of a crystal? 
We have the crystal structure. I'd like to see if the channels are large enough 
to allow the molecule to travel to the hypothesized binding site.
Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
The City College of New York
Department of Chemistry, MR-1135
160 Convent Avenue
New York, NY  10031
Tel. (212) 650-6070
www.khayatlab.org

Re: [ccp4bb] Invisible atoms in ligands

[Hargreaves, David]

Hi All,

I had an interesting case recently where a Cl atom of a chlorophenyl moiety 
went missing in a structure (primary X-ray damage was the suspected culprit). 
Small molecule Mass Spec suggested the atom was there to start with but it was 
quite obviously missing in the maps (1.9Ang) and the des-chloro refined much 
better. I was asked to replace the missing atom as it was seen as misleading 
because all the assay and biophysical data was generated with the chlorophenyl 
compound; this after all, is what the X-ray model was supporting. I did wonder 
where the Cl atom ended up and in what state.

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.commailto:name.surn...@astrazeneca.com

Please consider the environment before printing this e-mail

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von 
Delft
Sent: 13 June 2014 10:45
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Invisible atoms in ligands

Hi all - talking about ligands, a quick question on that old conundrum, of what 
to do about invisible atoms -- build them with occ=0, or omit them?

For bits of protein, I know all the arguments;  personally I prefer omitting 
atoms because:

  *   for amino acid sidechains, their presence is implied in the residue name.
  *   for whole residues, their presence is implied in the sequence numbering
However:  what about ligands?  Nowhere else in the PDB file is their presence 
implied - or have I missed something?

(Certainly disorder in a ligand is important information that needs to be 
captured!)

Cheers
phx

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[ccp4bb] Kabat, insertion codes refinement

[Hargreaves, David]

Dear CCP4bb,

I'm refining an antibody structure which requires Kabat residue numbering with 
insertion codes. My setup of Refmac5 and Buster both break peptide bonds 
between some (not all) of the residues with insertion codes. I was wondering 
whether there is a special way of handling these residues in refinement?

Thanks,

David

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
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[ccp4bb] pdb insertion codes

[Hargreaves, David]

Dear CCP4bb,

Does anyone know a simple way to get rid of residue number insertion codes and 
then renumber residues normally in pdb files?

Help much appreciated,

David


David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
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[ccp4bb] Crymon

[Hargreaves, David]

Dear CCP4bb,

Does anyone know who (if anyone) supports Crystal Miner (Emerald / Decode).

Kind Regards,

David

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
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[ccp4bb] Calculated vs Measured pI

[Hargreaves, David]

Dear CCP4bbers,



I was wondering whether anyone knew of a program that would calculate
the pI of a protein based on its structure rather than its sequence and
whether such a calculation might correlate better with the measured pI



Dave





David Hargreaves

Associate Principal Scientist

_

AstraZeneca

Discovery Sciences, Structure  Biophysics

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



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Re: [ccp4bb] salt or not?

[Hargreaves, David]

Dear Careina,



I would be cautious of using dyes. Much better to 1) try in-situ
diffraction if possible as this is least invasive or 2) pick a sensible
cryo and just freeze the crystal(s). I would try to same something from
the experiment for seed stock  possibly sequencing.



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

Discovery Sciences, Structure  Biophysics

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Careina Edgooms
Sent: 15 April 2013 11:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] salt or not?



Dear ccp4



I have been performing trials on a protein DNA complex for a while now
and have not seen any crystals form. Today I checked an old plate (over
a month old) and I see 4 large crystals. *excitement* Three of them look
tetragonal in shape (like a pyramid) and one of them looks hexagonal. I
do not know if they are salt or protein. There is calcium chloride in
the buffer. They feel quite soft to touch. They do not cause much
birefringence. One of them does not seem to absorb much izit. It did go
a bit blue but not entirely.



How can I tell if this crystal is protein or not? Do you think its worth
trying to see how it diffracts?



Also, does Izit affect diffraction/ protein structures at all? Could I
use a crystal with Izit in a diffraction experiment and ultimately to
get the structure?



Best

Careina


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Re: [ccp4bb] crystal dehydration

[Hargreaves, David]

Judith,
If you can, try monitoring the diffraction during the dehydration experiment. 
There are plenty of ways of doing this. Perhaps the simplest is to shoot your 
crystals in-situ after swapping the precipitant for a more concentrated one. 
The is also the Free Mounting System.

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
Discovery Sciences, Structure  Biophysics
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Judith A 
Ronau
Sent: 15 January 2013 14:47
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystal dehydration

Greetings!

I have recently been attempting crystal dehydration experiments to improve 
diffraction following the procedures from the ERSF in which crystals are 
exposed to increased concentrations of precipitant.  I would like to know if 
anyone knew of any alternative methods for dehydration of protein crystals. 
Thanks!

Best,
Judith

Re: [ccp4bb] To cryo or not to cryo...

[Hargreaves, David]

Hi Yuri,
I've had a very similar experience last week which ended badly. I opted to cryo 
my new crystals using my favourite cryo. Diffraction showed at least the 
crystals were protein but not much more. A classic example of cryo killing 
crystals. I mounted the remaining tiny fragment from the drop in a room 
temperature mount and got 15 images before it died. From this I got a cell and  
lattice even though it only diffracted to 6Ang. If had mounted a larger piece 
of crystal first off I might of been able to get a dataset with much shorter 
exposures. I would encourage either an in-situ test you have the equipment or 
use a capillary in whichever way suits. There are methods that allow recovery 
of the crystal after the test. At least this way you can (a) feel satisfied 
when you crush it up afterwards for seed because it didn't diffract at all or 
(b) successfully cryo your nicely diffracting crystal.
Good Luck!


David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yuri 
Pompeu
Sent: 01 December 2012 01:23
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] To cryo or not to cryo...

Dear community,
I have what seems to be a pretty decent single crystal that grew from a screen 
set up 2 weeks ago.
I am trying to reproduce it but so far I have not succeeded. I am however 
afraid the crystal that did form will start to deteriorate. So this brings me 
to dilemma, I feel like I should try and mount this crystal and shoot it. But 
since I only have 1 sample, I do not want to mess this up...  I am inclined to 
try cryo conditions, but I am afraid the addition of a cryo such as glycerol 
could destroy the little guy.
The crystal formed in 30% PEG 4000, 0.1M NaCitrate pH5.6 and 0.2M NH4AcO, I 
wonder if this is a cryo condition already?
Any suggestions would be appreciated.

best,

Re: [ccp4bb] Problems in crystallization

[Hargreaves, David]

I've seen this effect too. In the case I saw the drops were growing (and 
spreading) due to a reverse diffusion gradient. Optimised conditions used 
salting in method i.e. crystallising as the [pptnt] decreases during 
equilibration due to dilution as water is Tx from well to drop. Rare but not 
unknown.

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
Please consider the environment before printing this e-mail



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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eva 
Bligt-Lindén
Sent: 07 November 2012 12:07
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems in crystallization

Dear ccp4 users,

I have a problem in the crystallization of my target protein. Whenever
I set up a vapour diffusion experiment, either hanging or sitting
drops, the drops spread out. The surface tension is completely lost in
80-90% of the droplets. Have any one experienced something similar?
What could be the reason for this strange behaviour? I have tried
three different commercial screens with 96 condition each and there is
no difference between the screens. There is no difference between
manual or robotic setups either. The protein buffer is 40 mM Tris, 2
mM MgCl2 buffer, pH 7.4. The buffer controls are all ok.

Kind regards,
Eva



Eva Bligt-Lindén (M.Sc.)
PhD student
Structural Bioinformatics Laboratory

Department of Biosciences,
Åbo Akademi University
BioCity, Tykistökatu 6A
FI-20520 Turku
Finland

Re: [ccp4bb] Question about weird diffraction map

[Hargreaves, David]

Hi Zhao,



I would try a crystal that has not been treated with any dye. Small
molecules, particularly drug like compounds, crystallise out in soaking
experiments and can give diffraction similar to yours. My worry is that
you have non-diffracting protein crystals coated in some small molecule
crystalsthe question then is whether the addition of the dye has
destroyed your diffraction. Try in-situ or a capillary mount to be more
sure. A crush test might also tell you someting.



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
gengxiang zhao
Sent: 23 July 2012 21:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Question about weird diffraction map



Dear CCP4BB,

I have a question about my current diffraction map for one of crystals
which can be found in the attachment. Basically, the crystals were dyed
to initially testify that it belongs to protein. But from this
diffraction, even in the low diffract angle, no diffraction spots
theremeaning-this is not a protein crystal?

Any experienced idea/questions welcomed to discuss here.

Zhao,


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Re: [ccp4bb] The effect of His-tag location on crystallization

[Hargreaves, David]

In trying to reproduce a very nice public structure a cloning mis-hap put a 
-GS- prior to the C-term 6His tag. The resultant crystals had a 500Ang C 
dimension and 16 molecules in the au. Even a single amino acid could make all 
the difference

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of weliu
Sent: 27 June 2012 02:07
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] The effect of His-tag location on crystallization

Dear all,

We crystallized a protein and found that crystal quality greatly depended on 
the location of His-tag. When a His-tag was added at the C-terminus, only 
crystalline precipitate or spherical quasi crystals were grown. However, when 
the His-tag was moved to the N-terminus, single crystals were grown under a 
number of conditions, and the best one diffracted to 1.7 angstrom after 
optimization. I was wondering if there were published reports describing 
similar cases.

Thank you in advance

Wei Liu

Re: [ccp4bb] Crystallization with antibody

[Hargreaves, David]

There are plenty of reports (some even successful) where small concentrations 
of proteases have been used to enable crystallisation (with soluble proteins). 
Having put the effort into getting your material in the first place it's a 
small extra step to try. The professional way is to run a time coarse 
proteolysis and analyse the protein fragment you get or you can just add a bit 
of you favourite protease to the drop. I guess if your complex is stable enough 
it might survive this treatment.

There are companies that will raise specific antibodies, scFvs or other 
immunoglobulin (and possibly non-immunoglobulin) like molecules to your 
components or complex using technologies like phage/ribosome display. From 
experience this option could be very time consuming and expensive requiring 
much iteration. Generating entities with sufficient affinity that bind a 
desirable epitope and producing them on a crystallisation scale is not easy.

You could screen more conditions but there is a law of diminishing returns 
(redundancy in crystallisation conditions)

Try the simple things first...

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
Please consider the environment before printing this e-mail



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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa 
Hsu
Sent: 28 June 2012 13:26
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallization with antibody

Dear crystallographers

Trying to crystallize a membrane protein complex of 100 kDa with a soluble 
protein of 20 kDa which is interact with the membrane protein. So far, no 
co-crystals in  200 conditions. Some conditions gave crystals but mass spec of 
crystals show only either one protein present. I am thinking of antibody but 
don't know where to start. Can I use the anti-His tag antibody?

Thank you.

Re: [ccp4bb] effect of Izit dye within the crystal structure

[Hargreaves, David]

I've collected data from crystal soaked with brightly coloured ligands
which partition into the solvent channels. Although the crystal becomes
highly coloured (mopping up most of the colour from the drop) the
structures remained resolutely empty. I am intrigued by this observation
and wonder how mutating the residues that constitute the solvent
channels would affect soakbility and other crystal properties.



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
adam andres
Sent: 11 November 2011 04:34
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] effect of Izit dye within the crystal structure







Hi crystallographers



Has anyone actually collected data on a crystal that
has been treated with Izit dye? If so did you
see any structural effects or know any structure that present this dye?



Cheers



Adam Campos




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Re: [ccp4bb] Protein preps become a jelly

[Hargreaves, David]

Hi Aidong,

I've seen this in protein samples that have been snap frozen then thawed on 
ice. Thawing at room temperature (or in the hand) stopped this state forming. 
Could temperature be the issue? Maybe try 10C or leave the samples on the bench 
for a while and see if the state is reversible?

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
Please consider the environment before printing this e-mail


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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of aidong
Sent: 30 August 2011 16:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein preps become a jelly

Dear Buddies,

Sorry for bothering you with an off-ccp4 question.  We recently are
experiencing a very strange phenomena.  A couple of protein preps with
reasonably high concentration (10-20mg/ml) become a jelly after
storages for overnight or a couple of days at 4C.  All of them have
been purified by gel filtration.  Some of these proteins behave like
this from very first preps but some of them had been very kind to us
previously.  We have googled extensively in CCP4BB and www but it
appears this only happens to us.  It would be highly appreciated that
you could exchange their experiences or provide your suggestions.

Aidong Han, Ph.D

Department of Biomedical Sciences
School of Life Sciences
Xiamen University
Xiamen, Fujian 361005
China
Phone: 0592-218-8172 (O)
   0592-218-8173 (L)
Web: http://life.xmu.edu.cn/adhanlab/

Re: [ccp4bb] Aging PEGs

[Hargreaves, David]

I went through  the peg solid stocks in our cupboard and noticed some of them 
smelled acrid. I probably won’t use them. Not sure whether they’re chemically 
decomposing. Maybe fossilising?



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von 
Delft
Sent: 24 August 2011 22:21
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Aging PEGs



And now,   does anybody know of systematic data indicating how consistently 
all this matters?
phx

On 24/08/2011 21:45, Prince, D Bryan wrote:

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan


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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent: Wednesday, August 24, 2011 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all old vs new PEG issues
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Methods for dehydrating crystals

[Hargreaves, David]

Hi Andrea,

If you haven't already done so it might be worth trying a room temperature 
mount (capillary or in-situ) to get a feeling for how well the crystals 
diffract to start with.

Dave

David Hargreaves
Associate Principal Scientist
_
AstraZeneca
DECS, CPSS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF
Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693
David.Hargreaves @astrazeneca.com
 
Please consider the environment before printing this e-mail


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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrea L 
Edwards
Sent: 26 August 2011 21:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Methods for dehydrating crystals

Hi all,

What are the most successful methods you know of for dehydrating a crystal 
prior to freezing it? I am trying to push the resolution of my crystals.

Thanks,
Andrea

Re: [ccp4bb] Bypassing phase separation for nice crystals.

[Hargreaves, David]

Hi Timur,



I've had a crystal which sounds similar to yours. When I came to freeze
it noticed that it was not exactly solid more amorphous like treacle.
Laughingly I went ahead and screened it anyway and was shocked to see
diffraction to around 15Ang. I was lucky enough to have other crystals
in the same screen but if that had been the only hit maybe some
techniques like re-annealing, dehydrating, shifting pH  osmotic shock
might help. Any diffraction (order!) is better than no diffraction.

Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of F.
Timur Senguen
Sent: 18 July 2011 14:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Bypassing phase separation for nice crystals.



Hi everyone,



I have been issues with a particular protein. I have been close for a
while, but yet so far.



Rather than going from a clear drop to crystal, my protein first
undergoes phase separation (large oily drops) in which one phase
contains most, if not all, of the protein. This phase separation occurs
within a day of preparing the drop. A day after phase separation the
oily phase is now a large disordered crystalline mass which does not
diffract very well. I have tried changing buffer concentrations,
precipitant amounts, ionic strengths and pH and in all cases this
phenomenon is observed. I even screened protein concentrations to see if
reducing protein concentration would prevent the phase separation.



Is there any way to bypass this phase separation, which I think prevents
me from obtaining nice crystals. Should I try detergents, chaotropes, or
other additives?



Thanks in advance.



Timur

--
F. Timur Senguen, Ph.D.

Postdoctoral Research Fellow

Boston Biomedical Research Institute

64 Grove Street,
Watertown,
MA 02472 USA




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Re: [ccp4bb] generate large symmetry model

[Hargreaves, David]

Thank you to all for the help. There’s obviously more than one way to skin a 
cat!  In the end pdbcur did the job for me. Great!



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
martyn.w...@stfc.ac.uk
Sent: 30 June 2011 15:08
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] generate large symmetry model



As well as pdbset, you can use pdbcur. A combination of keywords genunit, symop 
and symcommit.

I’d probably use genunit (applicable to whatever spacegroup you have), followed 
by a second call using symop/symcommit to apply unit cell translations.



Cheers

Martyn



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Hargreaves, David
Sent: 30 June 2011 13:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] generate large symmetry model



Does anyone have a rigorous method (or script) for generating an extended 
lattice e.g 3x3x3 unit cells from any pdb file?

Any help gratefully received,



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail





AstraZeneca UK Limited is a company incorporated in England and Wales with 
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[ccp4bb] generate large symmetry model

[Hargreaves, David]

Does anyone have a rigorous method (or script) for generating an
extended lattice e.g 3x3x3 unit cells from any pdb file?

Any help gratefully received,



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

DECS, CPSS

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com



Please consider the environment before printing this e-mail




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AstraZeneca UK Limited is a company incorporated in England and Wales with 
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London, W2 6BD.
Confidentiality Notice: This message is private and may contain confidential, 
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Re: [ccp4bb] Preparation of seed-stocks without seed-beads

[Hargreaves, David]

Take two siliconised glass cover slips. Place 2-5ul of mother liquor on one and 
transfer a single crystal to this solution. Place the other cover slip on top 
and press down. The two optically flat surfaces crush the crystal (best 
observed using a microscope). Slide the two cover slips apart and use 25-50ul 
of mother liquor to hoover up the carnage. This is your seed stock. Use neat or 
up to x10,000 dilution. Freezing this stock can increase its potency. Try more 
ore less grinding for bigger or smaller seed fragments.

David



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-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk]on Behalf Of Ed
Pozharski
Sent: 14 September 2009 17:47
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Preparation of seed-stocks without seed-beads


You can use the glass cell disruption pestle

http://www.vwrsp.com/catjpg/mp0/mp0440.jpg

to grind them up.  They cost money, of course, and if you plan to buy
something, why not go for seed bead kit?  But if you want something very
cheap, then you'll find this amazing discovery interesting:

Outer diameter of the bottom of a 0.5 mL eppendorf tube is the same
as the inner diameter of a 1.5 mL eppendorf tube.

This is known as eppendorf mortar-pestle phenomenon :)

On Sat, 2009-09-12 at 13:52 +0530, james09 pruza wrote:
 Dear CCP4bbers,
 
 Can anyone suggests how to make seed-stocks if one is not having
 seed-beads... Is there any other methods to crush the crystals for the
 same purpose. What if it is simple vortexed. Off-course there wold be
 all sorts of sizes, the intact crystals as well. 
 Please suggest.
 
 Thanks a lot more for previous help.
 James.
-- 

[ccp4bb] Temporary Postdoctoral Research Scientist at AstraZeneca, Alderley Park

[Hargreaves, David]

Dear CCP4bb,
I like to take this opportunity to draw your attention to the following role 
(below).
Please follow the link to the AstraZeneca web site (job search DECS156) for 
more details and application forms. 
(please don't apply via this email!)

David Hargreaves

Postdoctoral Research Scientist - Structural Biology
Competitive salary + excellent benefits * Alderley Park, Cheshire

In this two-year fixed-term role, you'll work on an exciting and innovative 
cross-disciplinary project. 

About the role 
A key member of an international team, you'll provide cutting-edge scientific 
expertise to AstraZeneca's disease research groups. It's a fantastic 
opportunity to play a leading role in the development of a set of molecular 
tools that has the potential to widen the range of drug targets available for 
structure-aided drug design. You should be available to start work at the 
beginning of January 2010.

About you 
First and foremost, we're looking for a proven postdoctoral scientist with a 
relevant PhD - this could be in Biochemistry, Structural Biology or something 
similar. On top of that, it's vital that you have a strong record of practical 
laboratory work. This should include hands on experience of protein 
purification for structural studies and a sound understanding of biophysical 
techniques.

About us 
As one of the world's leading pharmaceutical companies, AstraZeneca is focused 
on providing innovative, effective medicines that make a real difference in key 
areas of healthcare. Join us and you'll be part of our Discovery Enabling 
Capabilities and Sciences department - a global team dedicated to providing 
cutting-edge scientific expertise to disease research teams. 

To find out more and to apply, please visit www.careers.astrazeneca.co.uk 
http://www.careers.astrazeneca.co.uk and search for job reference DECS156.

Closing date: 20th September 2009. 




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