Re: [ccp4bb] How to help crystal grow bigger

[ChenTiantian]

>"vary the precipitant/protein ratio"
Agree with Enrico.
Reducing the concentration of your protein sample to make it form less
nuclei, then it might grow bigger.

Best R,

tiantian

On Mon, Sep 5, 2011 at 7:48 PM, Enrico Stura  wrote:

> dear SnowDeer,
>
> The first step to see if bigger crystals can be grown is to use bigger
> protein drops and vary
> the precipitant/protein ratio at the beginning of the vapour diffusion
> experiment.
> You can work out for yourself why this
> should give you all the informations that you need in subsequent
> experiments.
>
> Enrico.
>
>
>
> On Mon, 05 Sep 2011 10:06:22 +0200, SnowDeer  wrote:
>
>  Dear All:
>>
>> Recently I am working on a protein which can already grow nice
>> pyramid-like
>> crystals after the condition was optimized, while the crystals are too
>> small
>> to be picked up. The crystals grew quite fast and densely, so I tried to
>> put
>> 100ul paraffin oil inside the 600ul reservoir solution or put the plate
>> under 16 degree to slow down the evaporation, while the crystals were
>> still
>> the same. I also tried macro or micro seeding with or without the paraffin
>> oil. Macroseeding would give a larger crystal (not very nice) with many
>> small crystals in the drop even I washed the seeds carefully. For
>> microseeding, the same small crystals grew.
>>
>> I don't have many experiences in crystallography, so I have no idea how to
>> make it grow bigger...
>>
>> Any suggestion is most welcome.
>>
>> Thanks.
>> SnowDeer
>>
>
>
> --
> Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
> Room 19, Bat.152,   Tel: 33 (0)1 69 08 9449Lab
> LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
> http://www-dsv.cea.fr/en/**institutes/institute-of-**
> biology-and-technology-saclay-**ibitec-s/unites-de-recherche/**
> department-of-molecular-**engineering-of-proteins-**
> simopro/molecular-toxinology-**and-biotechnology-laboratory-**
> ltmb/crystallogenesis-e.-stura
> http://www.chem.gla.ac.uk/**protein/mirror/stura/index2.**html
> e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
>



-- 
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park,
Shanghai, 201203

Re: [ccp4bb] Trying to "digest" PISA results

[Huang, Jing]

Ideally, the more nonspecific the protease, the better. (That would be a 
separate protein engineering project, or chemical catalyst project..) For now, 
we found that Pronase works well enough. Cheers, Jing

--
Jing Huang, Ph.D.
Associate Professor
UCLA
Department of Molecular & Medical Pharmacology
310-825-4329
jinghuang at mednet dot ucla dot edu
http://labs.pharmacology.ucla.edu/huanglab/


From: Jacob Keller [j-kell...@fsm.northwestern.edu]
Sent: Monday, September 05, 2011 3:44 PM
To: Huang, Jing
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Trying to "digest" PISA results

Good ol' trypsin--any reason why not?

JPK

On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing  wrote:
> Excellent point indeed. We always include (at least one, preferably multiple) 
> control proteins that are proteolysed equally across to make sure that the 
> observed "target stabilization" is not due to fortuitous protease inhibition.
>
> What protease did you use for your nucleotide binding case? The protease 
> sometimes matters. For example, thermolysin mainly only digests proteins that 
> are unfolded, whereas pronase, which is a mixture of various proteases, can 
> digest both folded and unfolded proteins.
>
> Best,
> Jing
>
> --
> Jing Huang, Ph.D.
> Associate Professor
> UCLA
> Department of Molecular & Medical Pharmacology
> 310-825-4329
> jinghuang at mednet dot ucla dot edu
> http://labs.pharmacology.ucla.edu/huanglab/
>
> 
> From: Jacob Keller [j-kell...@fsm.northwestern.edu]
> Sent: Monday, September 05, 2011 12:18 PM
> To: Huang, Jing
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>
> I did a similar assay years ago, but since the results were negative,
> never published anything--it was seeing whether nucleotides bound to
> my protein of interest by time courses of proteolysis +/- nucleotide.
> One tricky part of the assay, however, is to be sure that the compound
> of interest doesn't inhibit the protease--did you address that? I
> guess you would have to have some control proteins for that...
>
> Jacob
>
>
> g
>>
>> --
>> Jing Huang, Ph.D.
>> Associate Professor
>> UCLA
>> Department of Molecular & Medical Pharmacology
>> 310-825-4329
>> jinghuang at mednet dot ucla dot edu
>> http://labs.pharmacology.ucla.edu/huanglab/
>>
>> 
>> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller 
>> [j-kell...@fsm.northwestern.edu]
>> Sent: Monday, September 05, 2011 10:10 AM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>>
>> NMR...take that!
>>
>> JPK
>>
>> 2011/9/5 Andreas Förster :
>>> AUC !
>>>
>>>
>>> Andreas
>>>
>>>
>>>
>>> On 05/09/2011 6:00, Jacob Keller wrote:

 mea culpa! How about FRET?

 JPK

 On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:
>
> Hi Jacob,
> you forgot cross-linking to stabilize a weak complex and verify that it
> exists.
> Jürgen
> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:
>
> Well, I guess I have always been curious what is the gold standard
> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
> polydisperse sample with weak oligomerization, or SPR a very weak
> binding constant? Do we then revert to a functional assay? Or what if
> the functional assay does not show anything, but the binding constant
> is really strong? Or vice versa, the binding is completely
> undetectable, but the functional assay shows something?
>
> JPK
>
>>>
>>>
>>> --
>>>Andreas Förster, Research Associate
>>>Paul Freemont & Xiaodong Zhang Labs
>>> Department of Biochemistry, Imperial College London
>>>http://www.msf.bio.ic.ac.uk
>>>
>>
>>
>>
>> --
>> ***
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> cel: 773.608.9185
>> email: j-kell...@northwestern.edu
>> ***
>>
>> IMPORTANT WARNING:  This email (and any attachments) is only intended for 
>> the use of the person or entity to which it is addressed, and may contain 
>> information that is privileged and confidential.  You, the recipient, are 
>> obligated to maintain it in a safe, secure and confidential manner.  
>> Unauthorized redisclosure or failure to maintain confidentiality may subject 
>> you to federal and state penalties. If you are not the intended recipient, 
>> please immediately notify us by return email, and delete this message from 
>> your computer.
>>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
>
> IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
> u

Re: [ccp4bb] Trying to "digest" PISA results

[Jacob Keller]

Good ol' trypsin--any reason why not?

JPK

On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing  wrote:
> Excellent point indeed. We always include (at least one, preferably multiple) 
> control proteins that are proteolysed equally across to make sure that the 
> observed "target stabilization" is not due to fortuitous protease inhibition.
>
> What protease did you use for your nucleotide binding case? The protease 
> sometimes matters. For example, thermolysin mainly only digests proteins that 
> are unfolded, whereas pronase, which is a mixture of various proteases, can 
> digest both folded and unfolded proteins.
>
> Best,
> Jing
>
> --
> Jing Huang, Ph.D.
> Associate Professor
> UCLA
> Department of Molecular & Medical Pharmacology
> 310-825-4329
> jinghuang at mednet dot ucla dot edu
> http://labs.pharmacology.ucla.edu/huanglab/
>
> 
> From: Jacob Keller [j-kell...@fsm.northwestern.edu]
> Sent: Monday, September 05, 2011 12:18 PM
> To: Huang, Jing
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>
> I did a similar assay years ago, but since the results were negative,
> never published anything--it was seeing whether nucleotides bound to
> my protein of interest by time courses of proteolysis +/- nucleotide.
> One tricky part of the assay, however, is to be sure that the compound
> of interest doesn't inhibit the protease--did you address that? I
> guess you would have to have some control proteins for that...
>
> Jacob
>
>
> g
>>
>> --
>> Jing Huang, Ph.D.
>> Associate Professor
>> UCLA
>> Department of Molecular & Medical Pharmacology
>> 310-825-4329
>> jinghuang at mednet dot ucla dot edu
>> http://labs.pharmacology.ucla.edu/huanglab/
>>
>> 
>> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller 
>> [j-kell...@fsm.northwestern.edu]
>> Sent: Monday, September 05, 2011 10:10 AM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>>
>> NMR...take that!
>>
>> JPK
>>
>> 2011/9/5 Andreas Förster :
>>> AUC !
>>>
>>>
>>> Andreas
>>>
>>>
>>>
>>> On 05/09/2011 6:00, Jacob Keller wrote:

 mea culpa! How about FRET?

 JPK

 On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:
>
> Hi Jacob,
> you forgot cross-linking to stabilize a weak complex and verify that it
> exists.
> Jürgen
> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:
>
> Well, I guess I have always been curious what is the gold standard
> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
> polydisperse sample with weak oligomerization, or SPR a very weak
> binding constant? Do we then revert to a functional assay? Or what if
> the functional assay does not show anything, but the binding constant
> is really strong? Or vice versa, the binding is completely
> undetectable, but the functional assay shows something?
>
> JPK
>
>>>
>>>
>>> --
>>>        Andreas Förster, Research Associate
>>>        Paul Freemont & Xiaodong Zhang Labs
>>> Department of Biochemistry, Imperial College London
>>>            http://www.msf.bio.ic.ac.uk
>>>
>>
>>
>>
>> --
>> ***
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> cel: 773.608.9185
>> email: j-kell...@northwestern.edu
>> ***
>>
>> IMPORTANT WARNING:  This email (and any attachments) is only intended for 
>> the use of the person or entity to which it is addressed, and may contain 
>> information that is privileged and confidential.  You, the recipient, are 
>> obligated to maintain it in a safe, secure and confidential manner.  
>> Unauthorized redisclosure or failure to maintain confidentiality may subject 
>> you to federal and state penalties. If you are not the intended recipient, 
>> please immediately notify us by return email, and delete this message from 
>> your computer.
>>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
>
> IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
> use of the person or entity to which it is addressed, and may contain 
> information that is privileged and confidential.  You, the recipient, are 
> obligated to maintain it in a safe, secure and confidential manner.  
> Unauthorized redisclosure or failure to maintain confidentiality may subject 
> you to federal and state penalties. If you are not the intended recipient, 
> please immediately notify us by return email, and delete this message from 
> your computer.
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email

Re: [ccp4bb] Trying to "digest" PISA results

[Huang, Jing]

Excellent point indeed. We always include (at least one, preferably multiple) 
control proteins that are proteolysed equally across to make sure that the 
observed "target stabilization" is not due to fortuitous protease inhibition.

What protease did you use for your nucleotide binding case? The protease 
sometimes matters. For example, thermolysin mainly only digests proteins that 
are unfolded, whereas pronase, which is a mixture of various proteases, can 
digest both folded and unfolded proteins.

Best,
Jing

--
Jing Huang, Ph.D.
Associate Professor
UCLA
Department of Molecular & Medical Pharmacology
310-825-4329
jinghuang at mednet dot ucla dot edu
http://labs.pharmacology.ucla.edu/huanglab/


From: Jacob Keller [j-kell...@fsm.northwestern.edu]
Sent: Monday, September 05, 2011 12:18 PM
To: Huang, Jing
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Trying to "digest" PISA results

I did a similar assay years ago, but since the results were negative,
never published anything--it was seeing whether nucleotides bound to
my protein of interest by time courses of proteolysis +/- nucleotide.
One tricky part of the assay, however, is to be sure that the compound
of interest doesn't inhibit the protease--did you address that? I
guess you would have to have some control proteins for that...

Jacob


g
>
> --
> Jing Huang, Ph.D.
> Associate Professor
> UCLA
> Department of Molecular & Medical Pharmacology
> 310-825-4329
> jinghuang at mednet dot ucla dot edu
> http://labs.pharmacology.ucla.edu/huanglab/
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller 
> [j-kell...@fsm.northwestern.edu]
> Sent: Monday, September 05, 2011 10:10 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>
> NMR...take that!
>
> JPK
>
> 2011/9/5 Andreas Förster :
>> AUC !
>>
>>
>> Andreas
>>
>>
>>
>> On 05/09/2011 6:00, Jacob Keller wrote:
>>>
>>> mea culpa! How about FRET?
>>>
>>> JPK
>>>
>>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:

 Hi Jacob,
 you forgot cross-linking to stabilize a weak complex and verify that it
 exists.
 Jürgen
 On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:

 Well, I guess I have always been curious what is the gold standard
 here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
 polydisperse sample with weak oligomerization, or SPR a very weak
 binding constant? Do we then revert to a functional assay? Or what if
 the functional assay does not show anything, but the binding constant
 is really strong? Or vice versa, the binding is completely
 undetectable, but the functional assay shows something?

 JPK

>>
>>
>> --
>>Andreas Förster, Research Associate
>>Paul Freemont & Xiaodong Zhang Labs
>> Department of Biochemistry, Imperial College London
>>http://www.msf.bio.ic.ac.uk
>>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
>
> IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
> use of the person or entity to which it is addressed, and may contain 
> information that is privileged and confidential.  You, the recipient, are 
> obligated to maintain it in a safe, secure and confidential manner.  
> Unauthorized redisclosure or failure to maintain confidentiality may subject 
> you to federal and state penalties. If you are not the intended recipient, 
> please immediately notify us by return email, and delete this message from 
> your computer.
>



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

IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
use of the person or entity to which it is addressed, and may contain 
information that is privileged and confidential.  You, the recipient, are 
obligated to maintain it in a safe, secure and confidential manner.  
Unauthorized redisclosure or failure to maintain confidentiality may subject 
you to federal and state penalties. If you are not the intended recipient, 
please immediately notify us by return email, and delete this message from your 
computer.

Re: [ccp4bb] Trying to "digest" PISA results

[Jacob Keller]

I did a similar assay years ago, but since the results were negative,
never published anything--it was seeing whether nucleotides bound to
my protein of interest by time courses of proteolysis +/- nucleotide.
One tricky part of the assay, however, is to be sure that the compound
of interest doesn't inhibit the protease--did you address that? I
guess you would have to have some control proteins for that...

Jacob


g
>
> --
> Jing Huang, Ph.D.
> Associate Professor
> UCLA
> Department of Molecular & Medical Pharmacology
> 310-825-4329
> jinghuang at mednet dot ucla dot edu
> http://labs.pharmacology.ucla.edu/huanglab/
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller 
> [j-kell...@fsm.northwestern.edu]
> Sent: Monday, September 05, 2011 10:10 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Trying to "digest" PISA results
>
> NMR...take that!
>
> JPK
>
> 2011/9/5 Andreas Förster :
>> AUC !
>>
>>
>> Andreas
>>
>>
>>
>> On 05/09/2011 6:00, Jacob Keller wrote:
>>>
>>> mea culpa! How about FRET?
>>>
>>> JPK
>>>
>>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:

 Hi Jacob,
 you forgot cross-linking to stabilize a weak complex and verify that it
 exists.
 Jürgen
 On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:

 Well, I guess I have always been curious what is the gold standard
 here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
 polydisperse sample with weak oligomerization, or SPR a very weak
 binding constant? Do we then revert to a functional assay? Or what if
 the functional assay does not show anything, but the binding constant
 is really strong? Or vice versa, the binding is completely
 undetectable, but the functional assay shows something?

 JPK

>>
>>
>> --
>>        Andreas Förster, Research Associate
>>        Paul Freemont & Xiaodong Zhang Labs
>> Department of Biochemistry, Imperial College London
>>            http://www.msf.bio.ic.ac.uk
>>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
>
> IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
> use of the person or entity to which it is addressed, and may contain 
> information that is privileged and confidential.  You, the recipient, are 
> obligated to maintain it in a safe, secure and confidential manner.  
> Unauthorized redisclosure or failure to maintain confidentiality may subject 
> you to federal and state penalties. If you are not the intended recipient, 
> please immediately notify us by return email, and delete this message from 
> your computer.
>



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

Re: [ccp4bb] Trying to "digest" PISA results

[Huang, Jing]

Jacob,

You may also try DARTS (drug affinity responsive target stability). In this 
method, small molecule (or protein) binding leads to specific protection of its 
target protein from protease digestion. It is completely label free, and 
appears to be able to detect very low affinity interactions (high microM for 
small molecules). We'd be happy to send detailed protocols if you are 
interested.

http://www.pnas.org/content/106/51/21984.full

Best,
Jing

--
Jing Huang, Ph.D.
Associate Professor
UCLA
Department of Molecular & Medical Pharmacology
310-825-4329
jinghuang at mednet dot ucla dot edu
http://labs.pharmacology.ucla.edu/huanglab/


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller 
[j-kell...@fsm.northwestern.edu]
Sent: Monday, September 05, 2011 10:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Trying to "digest" PISA results

NMR...take that!

JPK

2011/9/5 Andreas Förster :
> AUC !
>
>
> Andreas
>
>
>
> On 05/09/2011 6:00, Jacob Keller wrote:
>>
>> mea culpa! How about FRET?
>>
>> JPK
>>
>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:
>>>
>>> Hi Jacob,
>>> you forgot cross-linking to stabilize a weak complex and verify that it
>>> exists.
>>> Jürgen
>>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:
>>>
>>> Well, I guess I have always been curious what is the gold standard
>>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
>>> polydisperse sample with weak oligomerization, or SPR a very weak
>>> binding constant? Do we then revert to a functional assay? Or what if
>>> the functional assay does not show anything, but the binding constant
>>> is really strong? Or vice versa, the binding is completely
>>> undetectable, but the functional assay shows something?
>>>
>>> JPK
>>>
>
>
> --
>Andreas Förster, Research Associate
>Paul Freemont & Xiaodong Zhang Labs
> Department of Biochemistry, Imperial College London
>http://www.msf.bio.ic.ac.uk
>



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

IMPORTANT WARNING:  This email (and any attachments) is only intended for the 
use of the person or entity to which it is addressed, and may contain 
information that is privileged and confidential.  You, the recipient, are 
obligated to maintain it in a safe, secure and confidential manner.  
Unauthorized redisclosure or failure to maintain confidentiality may subject 
you to federal and state penalties. If you are not the intended recipient, 
please immediately notify us by return email, and delete this message from your 
computer.

Re: [ccp4bb] Trying to "digest" PISA results

[Bosch, Juergen]

Free flow electrophoresis would be another option, by the way anybody on the 
East Coast who has one of those instruments ? I'd be interested to get an email 
directly.

Thanks,

Jürgen

On Sep 5, 2011, at 1:00 PM, Jacob Keller wrote:

mea culpa! How about FRET?

JPK

On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen 
mailto:jubo...@jhsph.edu>> wrote:
Hi Jacob,
you forgot cross-linking to stabilize a weak complex and verify that it
exists.
Jürgen
On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:

Well, I guess I have always been curious what is the gold standard
here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
polydisperse sample with weak oligomerization, or SPR a very weak
binding constant? Do we then revert to a functional assay? Or what if
the functional assay does not show anything, but the binding constant
is really strong? Or vice versa, the binding is completely
undetectable, but the functional assay shows something?

JPK

On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans 
mailto:p...@mrc-lmb.cam.ac.uk>> wrote:

I get confused by these figures. As I understand it the "interface area"
given in Pisa is half the loss of accessible area on forming the complex: is
that right?

We're working on a complex with interface area ~500A^2, where the complex is
stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa
estimate of DelG -2.3. Does that sound sensible?

Phil

On 5 Sep 2011, at 10:33, Eugene Krissinel wrote:

720 is not an impressive size for a stable interface, but it may do
depending on molecule size and exact chemistry of the interface (h-bonds,
salt bridges, disulphides, charges etc etc). Everything is subject to
chemical environment and concentration, as usual. For these entries, PISA
gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol
estimated (guessed) accuracy of PISA, this may or may not be a stable thing.
And yes, it has about 70-80% chances to be simply an artefact of crystal
packing, according to some sort of derivations that I did in 2nd PISA paper
in J.Comp.Chem. in January last year.

Having said all this, PISA is not an oracle and does not pretend to be
correct in 100% of instances.

Eugene.


On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:

Like Jan, I find it very useful to sort out the clear cut cases. Otherwise
it is easy to get things wrong..

But isnt a buried surface area of 720 rather small for a stable interface?
 If there is other confirming evidence like 2 diff space groups then you
feel more secure!!

On 09/01/2011 02:27 PM, Yuri Pompeu wrote:

This is regarding Ethan´s point, particularly:

2) the protein has crystallized as a monomer even though it

[sometimes] exists in solution as a dimer.  The interface

seen in the crystal is not the "real" dimer interface and

thus the PISA score is correct.

I see the same exact interface in a crystal of a close homologue that
belongs to a different space group (hexagonal vs tetragonal system)




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

..
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://web.mac.com/bosch_lab/








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

..
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://web.mac.com/bosch_lab/

Re: [ccp4bb] Trying to "digest" PISA results

[Jacob Keller]

NMR...take that!

JPK

2011/9/5 Andreas Förster :
> AUC !
>
>
> Andreas
>
>
>
> On 05/09/2011 6:00, Jacob Keller wrote:
>>
>> mea culpa! How about FRET?
>>
>> JPK
>>
>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:
>>>
>>> Hi Jacob,
>>> you forgot cross-linking to stabilize a weak complex and verify that it
>>> exists.
>>> Jürgen
>>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:
>>>
>>> Well, I guess I have always been curious what is the gold standard
>>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
>>> polydisperse sample with weak oligomerization, or SPR a very weak
>>> binding constant? Do we then revert to a functional assay? Or what if
>>> the functional assay does not show anything, but the binding constant
>>> is really strong? Or vice versa, the binding is completely
>>> undetectable, but the functional assay shows something?
>>>
>>> JPK
>>>
>
>
> --
>        Andreas Förster, Research Associate
>        Paul Freemont & Xiaodong Zhang Labs
> Department of Biochemistry, Imperial College London
>            http://www.msf.bio.ic.ac.uk
>



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

Re: [ccp4bb] Trying to "digest" PISA results

[Andreas Förster]

AUC !


Andreas



On 05/09/2011 6:00, Jacob Keller wrote:

mea culpa! How about FRET?

JPK

On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:

Hi Jacob,
you forgot cross-linking to stabilize a weak complex and verify that it
exists.
Jürgen
On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:

Well, I guess I have always been curious what is the gold standard
here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
polydisperse sample with weak oligomerization, or SPR a very weak
binding constant? Do we then revert to a functional assay? Or what if
the functional assay does not show anything, but the binding constant
is really strong? Or vice versa, the binding is completely
undetectable, but the functional assay shows something?

JPK




--
Andreas Förster, Research Associate
Paul Freemont & Xiaodong Zhang Labs
Department of Biochemistry, Imperial College London
http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] Trying to "digest" PISA results

[Jacob Keller]

mea culpa! How about FRET?

JPK

On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen  wrote:
> Hi Jacob,
> you forgot cross-linking to stabilize a weak complex and verify that it
> exists.
> Jürgen
> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:
>
> Well, I guess I have always been curious what is the gold standard
> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
> polydisperse sample with weak oligomerization, or SPR a very weak
> binding constant? Do we then revert to a functional assay? Or what if
> the functional assay does not show anything, but the binding constant
> is really strong? Or vice versa, the binding is completely
> undetectable, but the functional assay shows something?
>
> JPK
>
> On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans  wrote:
>
> I get confused by these figures. As I understand it the "interface area"
> given in Pisa is half the loss of accessible area on forming the complex: is
> that right?
>
> We're working on a complex with interface area ~500A^2, where the complex is
> stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa
> estimate of DelG -2.3. Does that sound sensible?
>
> Phil
>
> On 5 Sep 2011, at 10:33, Eugene Krissinel wrote:
>
> 720 is not an impressive size for a stable interface, but it may do
> depending on molecule size and exact chemistry of the interface (h-bonds,
> salt bridges, disulphides, charges etc etc). Everything is subject to
> chemical environment and concentration, as usual. For these entries, PISA
> gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol
> estimated (guessed) accuracy of PISA, this may or may not be a stable thing.
> And yes, it has about 70-80% chances to be simply an artefact of crystal
> packing, according to some sort of derivations that I did in 2nd PISA paper
> in J.Comp.Chem. in January last year.
>
> Having said all this, PISA is not an oracle and does not pretend to be
> correct in 100% of instances.
>
> Eugene.
>
>
> On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:
>
> Like Jan, I find it very useful to sort out the clear cut cases. Otherwise
> it is easy to get things wrong..
>
> But isnt a buried surface area of 720 rather small for a stable interface?
>  If there is other confirming evidence like 2 diff space groups then you
> feel more secure!!
>
> On 09/01/2011 02:27 PM, Yuri Pompeu wrote:
>
> This is regarding Ethan´s point, particularly:
>
> 2) the protein has crystallized as a monomer even though it
>
> [sometimes] exists in solution as a dimer.  The interface
>
> seen in the crystal is not the "real" dimer interface and
>
> thus the PISA score is correct.
>
> I see the same exact interface in a crystal of a close homologue that
> belongs to a different space group (hexagonal vs tetragonal system)
>
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: j-kell...@northwestern.edu
> ***
>
> ..
> 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://web.mac.com/bosch_lab/
>
>
>
>
>



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

Re: [ccp4bb] Trying to "digest" PISA results

[Bosch, Juergen]

Hi Jacob,

you forgot cross-linking to stabilize a weak complex and verify that it exists.

Jürgen

On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote:

Well, I guess I have always been curious what is the gold standard
here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
polydisperse sample with weak oligomerization, or SPR a very weak
binding constant? Do we then revert to a functional assay? Or what if
the functional assay does not show anything, but the binding constant
is really strong? Or vice versa, the binding is completely
undetectable, but the functional assay shows something?

JPK

On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans 
mailto:p...@mrc-lmb.cam.ac.uk>> wrote:
I get confused by these figures. As I understand it the "interface area" given 
in Pisa is half the loss of accessible area on forming the complex: is that 
right?

We're working on a complex with interface area ~500A^2, where the complex is 
stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa 
estimate of DelG -2.3. Does that sound sensible?

Phil

On 5 Sep 2011, at 10:33, Eugene Krissinel wrote:

720 is not an impressive size for a stable interface, but it may do depending 
on molecule size and exact chemistry of the interface (h-bonds, salt bridges, 
disulphides, charges etc etc). Everything is subject to chemical environment 
and concentration, as usual. For these entries, PISA gives dissociation free 
energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy 
of PISA, this may or may not be a stable thing. And yes, it has about 70-80% 
chances to be simply an artefact of crystal packing, according to some sort of 
derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year.

Having said all this, PISA is not an oracle and does not pretend to be correct 
in 100% of instances.

Eugene.


On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:

Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it 
is easy to get things wrong..

But isnt a buried surface area of 720 rather small for a stable interface?  If 
there is other confirming evidence like 2 diff space groups then you feel more 
secure!!

On 09/01/2011 02:27 PM, Yuri Pompeu wrote:
This is regarding Ethan´s point, particularly:
2) the protein has crystallized as a monomer even though it
[sometimes] exists in solution as a dimer.  The interface
seen in the crystal is not the "real" dimer interface and
thus the PISA score is correct.
I see the same exact interface in a crystal of a close homologue that belongs 
to a different space group (hexagonal vs tetragonal system)




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

..
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://web.mac.com/bosch_lab/

Re: [ccp4bb] Trying to "digest" PISA results

[Jacob Keller]

Well, I guess I have always been curious what is the gold standard
here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a
polydisperse sample with weak oligomerization, or SPR a very weak
binding constant? Do we then revert to a functional assay? Or what if
the functional assay does not show anything, but the binding constant
is really strong? Or vice versa, the binding is completely
undetectable, but the functional assay shows something?

JPK

On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans  wrote:
> I get confused by these figures. As I understand it the "interface area" 
> given in Pisa is half the loss of accessible area on forming the complex: is 
> that right?
>
> We're working on a complex with interface area ~500A^2, where the complex is 
> stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa 
> estimate of DelG -2.3. Does that sound sensible?
>
> Phil
>
> On 5 Sep 2011, at 10:33, Eugene Krissinel wrote:
>
>> 720 is not an impressive size for a stable interface, but it may do 
>> depending on molecule size and exact chemistry of the interface (h-bonds, 
>> salt bridges, disulphides, charges etc etc). Everything is subject to 
>> chemical environment and concentration, as usual. For these entries, PISA 
>> gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol 
>> estimated (guessed) accuracy of PISA, this may or may not be a stable thing. 
>> And yes, it has about 70-80% chances to be simply an artefact of crystal 
>> packing, according to some sort of derivations that I did in 2nd PISA paper 
>> in J.Comp.Chem. in January last year.
>>
>> Having said all this, PISA is not an oracle and does not pretend to be 
>> correct in 100% of instances.
>>
>> Eugene.
>>
>>
>> On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:
>>
>>> Like Jan, I find it very useful to sort out the clear cut cases. Otherwise 
>>> it is easy to get things wrong..
>>>
>>> But isnt a buried surface area of 720 rather small for a stable interface?  
>>> If there is other confirming evidence like 2 diff space groups then you 
>>> feel more secure!!
>>>
>>> On 09/01/2011 02:27 PM, Yuri Pompeu wrote:
 This is regarding Ethan´s point, particularly:
> 2) the protein has crystallized as a monomer even though it
> [sometimes] exists in solution as a dimer.  The interface
> seen in the crystal is not the "real" dimer interface and
> thus the PISA score is correct.
 I see the same exact interface in a crystal of a close homologue that 
 belongs to a different space group (hexagonal vs tetragonal system)
>



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

[ccp4bb] Group Leader position in Structural Biology

[Danny Huang]

The Beatson Institute for Cancer Research is committed to carry out world-class 
research into the biology of cancer and to help develop better treatments to 
improve the quality of life for cancer patients. We are core funded by Cancer 
Research UK and provide an outstanding environment for our scientists.
We are now recruiting a Group Leader with an excellent track record and 
expertise in X-ray crystallography to develop and head a programme in 
Structural Biology. We invite applications from successful and motivated 
scientists who would like to develop an independent research programme in this 
area and join our well-established structural biology department. We offer 
either a tenure track and tenured position, depending on previous experience, 
and extremely generous support that includes core funding for staff and 
consumables as well as access to leading-edge facilities and research support 
(including state-of-the art X-ray crystallography, DNA sequencing, IT and 
proteomics).

Further information on the Beatson's research activities, infrastructure and 
facilities is available on our website www.beatson.gla.ac.uk
Applications, including a one to two page statement of research interests and 
goals, CV, list of publications and the names of three referees, should be sent 
to Professor Karen Vousden, Director, the Beatson Institute for Cancer 
Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, Scotland 
(email: k.vous...@beatson.gla.ac.uk).
Applications should be received before 31st October 2011.

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, CP&SS

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



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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Confidentiality Notice: This message is private and may contain confidential, 
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Disclaimer: Email messages may be subject to delays, interception, non-delivery 
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is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by 
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Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
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Conduct and Policies.
-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] 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, CP&SS
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


--
AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
Confidentiality Notice: This message is private and may contain confidential, 
proprietary and legally privileged information. If you have received this 
message in error, please notify us and remove it from your system and note that 
you must not copy, distribute or take any action in reliance on it. Any 
unauthorised use or disclosure of the contents of this message is not permitted 
and may be unlawful.
Disclaimer: Email messages may be subject to delays, interception, non-delivery 
and unauthorised alterations. Therefore, information expressed in this message 
is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by 
an authorised representative independent of this message. No contractual 
relationship is created by this message by any person unless specifically 
indicated by agreement in writing other than email.
Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
for the purposes of the prevention and detection of crime, ensuring the 
security of our computer systems and checking Compliance with our Code of 
Conduct and Policies.
-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/

[ccp4bb] Workshop on Dynamic X-ray Scattering in Structural Biology 2011

[Vukica Srajer]

Second Announcement – Registration is now open at

http://cars9.uchicago.edu/biocars/pages/DXSworkshop11/index.shtml

Workshop on Dynamic X-ray Scattering in Structural Biology 2011
November 2 - 4, 2011
Hosted by BioCARS, The University of Chicago
Held at Advanced Photon Source, Argonne National Laboratory

Organized by Zhong Ren,r...@uchicago.edu, 630-252-0498
Administrative contact: Katie Tietz,ti...@cars.uchicago.edu, 630-252-0450

This workshop will provide hands-on training in designing and conducting 
time-resolved experiments principally in macromolecular crystallography, 
but also in fiber diffraction and solution scattering from biological 
samples. BioCARS and BioCAT users will learn about the exciting 
capabilities in dynamic scattering at beamlines 14-ID and 18-ID of the APS.


The workshop will feature three Lecture Sessions. The first will focus 
on the experimental techniques of time-resolved crystallography using 
either Laue or monochromatic diffraction. The second concerns a notable 
challenge of dynamic crystallography lying in data analysis and 
structure refinement. The third will showcase recent examples of 
structural dynamics that go beyond classical time-resolved 
crystallography. Four hands-on sessions (Crystallization, Experiment, 
Software and Case Study) are designed to offer an overview of various 
experimental approaches to capturing dynamic structures. In the 
Crystallization Session, we will demonstrate novel crystallization 
techniques for room temperature diffraction, often a key requirement for 
dynamic crystallography. The Software Session will provide training on 
data analysis and structure refinement of time-dependent structural 
changes. In the Experiment Session, attendees are invited to bring their 
own crystal samples and to conduct preliminary testing of their 
diffraction quality, often the first step in developing time-resolved 
projects. After the participants have familiarized themselves with a 
variety of theoretical and practical aspects of time-resolved 
experiments, we encourage attendees to present their specific cases in 
structural biology and prospective projects in the Case Study Session. 
Through one-on-one discussions with BioCARS and BioCAT staff or in a 
group discussion, this session will provide an opportunity to identify 
promising experimental approaches, potential difficulties and feasible 
solutions to the participants’ specific problems.


Please note that due to time limitation, hands-on sessions will be able 
to accommodate up to 25 participants for the BioCARS experiment and 
software sessions and up to 10 participants for the BioCAT experiment 
session. Lecture sessions are open to all registered participants.


Please send questions and comments to Zhong Ren (r...@uchicago.edu) or 
Katie Tietz (ti...@cars.uchicago.edu).


~~~
Vukica Srajer Ph.D.
University of Chicago/CARS
9700 South Cass Ave, Bldg 434B
Argonne, IL 60439
tel: (630) 252-0455
fax: (630) 252-0443

Re: [ccp4bb] How to help crystal grow bigger

[Enrico Stura]

dear SnowDeer,

The first step to see if bigger crystals can be grown is to use bigger  
protein drops and vary
the precipitant/protein ratio at the beginning of the vapour diffusion  
experiment.

You can work out for yourself why this
should give you all the informations that you need in subsequent  
experiments.


Enrico.


On Mon, 05 Sep 2011 10:06:22 +0200, SnowDeer  wrote:


Dear All:

Recently I am working on a protein which can already grow nice  
pyramid-like
crystals after the condition was optimized, while the crystals are too  
small
to be picked up. The crystals grew quite fast and densely, so I tried to  
put

100ul paraffin oil inside the 600ul reservoir solution or put the plate
under 16 degree to slow down the evaporation, while the crystals were  
still
the same. I also tried macro or micro seeding with or without the  
paraffin

oil. Macroseeding would give a larger crystal (not very nice) with many
small crystals in the drop even I washed the seeds carefully. For
microseeding, the same small crystals grew.

I don't have many experiences in crystallography, so I have no idea how  
to

make it grow bigger...

Any suggestion is most welcome.

Thanks.
SnowDeer



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

Re: [ccp4bb] Trying to "digest" PISA results

[Phil Evans]

I get confused by these figures. As I understand it the "interface area" given 
in Pisa is half the loss of accessible area on forming the complex: is that 
right?

We're working on a complex with interface area ~500A^2, where the complex is 
stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa 
estimate of DelG -2.3. Does that sound sensible?

Phil

On 5 Sep 2011, at 10:33, Eugene Krissinel wrote:

> 720 is not an impressive size for a stable interface, but it may do depending 
> on molecule size and exact chemistry of the interface (h-bonds, salt bridges, 
> disulphides, charges etc etc). Everything is subject to chemical environment 
> and concentration, as usual. For these entries, PISA gives dissociation free 
> energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy 
> of PISA, this may or may not be a stable thing. And yes, it has about 70-80% 
> chances to be simply an artefact of crystal packing, according to some sort 
> of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last 
> year.
> 
> Having said all this, PISA is not an oracle and does not pretend to be 
> correct in 100% of instances.
> 
> Eugene.
> 
> 
> On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:
> 
>> Like Jan, I find it very useful to sort out the clear cut cases. Otherwise 
>> it is easy to get things wrong..
>> 
>> But isnt a buried surface area of 720 rather small for a stable interface?  
>> If there is other confirming evidence like 2 diff space groups then you feel 
>> more secure!!
>> 
>> On 09/01/2011 02:27 PM, Yuri Pompeu wrote:
>>> This is regarding Ethan´s point, particularly:
 2) the protein has crystallized as a monomer even though it
 [sometimes] exists in solution as a dimer.  The interface
 seen in the crystal is not the "real" dimer interface and
 thus the PISA score is correct.
>>> I see the same exact interface in a crystal of a close homologue that 
>>> belongs to a different space group (hexagonal vs tetragonal system)

[ccp4bb] MOLREP TFcntrst

[Sven Dahms]

Dear all,

Does anyone know the definition of the "TFcntrst" value in MOLREPv10.2.35 and 
the difference of "TFcntrst" to "Cntrst"?

an example from a MOLREP log-file:

  Time_elapsed: 0h  0m 18s Remained: 0h  0m 40s
 Sol_ RF TF   Tf/sig  TFcntrst  PFind  PFPFmin  wRfac Scor Scor_max Cntrst
 Sol___1__1   25.38  2.848   1.00  1.00   1.00  0.725  0.143  0.143   0.00
 Sol___2_12   30.78  2.136   1.00  1.00   1.00  0.736  0.120  0.143   0.00
 Sol___3_11   24.33  2.538   1.00  1.00   1.00  0.733  0.124  0.143   0.00
 Sol___4__1   29.08  8.246   1.00  1.00   1.00  0.665  0.266  0.266   0.00
 Sol___5__1   34.66  7.999   1.00  1.00   1.00  0.666  0.265  0.266   0.00
 Sol___6_14   27.33  2.200   1.00  1.00   1.00  0.724  0.135  0.266   0.00
...

If anyone could help me, I'd be very grateful. Thanks!

Sven
-- 
Empfehlen Sie GMX DSL Ihren Freunden und Bekannten und wir
belohnen Sie mit bis zu 50,- Euro! https://freundschaftswerbung.gmx.de

Re: [ccp4bb] Trying to "digest" PISA results

[Eugene Krissinel]

720 is not an impressive size for a stable interface, but it may do depending 
on molecule size and exact chemistry of the interface (h-bonds, salt bridges, 
disulphides, charges etc etc). Everything is subject to chemical environment 
and concentration, as usual. For these entries, PISA gives dissociation free 
energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy 
of PISA, this may or may not be a stable thing. And yes, it has about 70-80% 
chances to be simply an artefact of crystal packing, according to some sort of 
derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year.

Having said all this, PISA is not an oracle and does not pretend to be correct 
in 100% of instances.

Eugene.


On 5 Sep 2011, at 10:14, Eleanor Dodson wrote:

> Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it 
> is easy to get things wrong..
> 
> But isnt a buried surface area of 720 rather small for a stable interface?  
> If there is other confirming evidence like 2 diff space groups then you feel 
> more secure!!
> 
> On 09/01/2011 02:27 PM, Yuri Pompeu wrote:
>> This is regarding Ethan´s point, particularly:
>>   >2) the protein has crystallized as a monomer even though it
>>   >[sometimes] exists in solution as a dimer.  The interface
>>   >seen in the crystal is not the "real" dimer interface and
>>   >thus the PISA score is correct.
>> I see the same exact interface in a crystal of a close homologue that 
>> belongs to a different space group (hexagonal vs tetragonal system)

Re: [ccp4bb] question about sfall output

[Eleanor Dodson]

Hmm - Firstly: your final FC PHIC calculation are just wrong. I cant 
tell why without more details - is the SG set correctly, because those 
phases cannot reflect the SG symmetry.. Can you send the log file?


For the scaling against Fobs Qs. It looks to me as though you are using 
values of Fobs already scaled to match the model by some program.. The 
scaling algorithms in different programs are subtly different - there is 
no clear "correct" answer in to how to handle a dolvent continuum for 
instance - so I suspect the differences reflect that.
In sfall (and I think most programs) the model B factors are changed to 
give best agreement with the Wilson scale for the observed data, and a 
scale factor is applied to the Fobs to bring it to match the fcalc.


So I guess sfall has changed the overall B for the model slightly 
changing the higher resolution Fcs a bit..

Eleanor


On 09/03/2011 08:07 PM, Pawel wrote:

Dear all,

First of all just "hello" to everyone since I am new to this forum. Not
being a crystallographer, bear with me if I say something crazy.

I was recently tinkering with Sfall and noticed that the Fcalc output by
Sfall have different values depending on if one feeds Sfall with an
input mtz file containing Fobs ("mode sfcalc xyzin" vs. "mode sfcalc
xyzin hklin") and also depending on if one tells Sfall to scale the Fobs
or not ("noscale" on or off). This puzzles me as I would have thought
that since Fcalc are calculated from the atomic coordinates, they should
be affected by neither the Fobs fed to Sfall nor the scaling of the Fobs.

As an example, Sfall calculations on PDB:1AHO. In each case the Fcalc
column is the penultimate one. Here the difference in Fcalc between
scaling or not scaling the Fobs is not so pronounced, but it still
puzzles me that there would be any change at all. Also, in this case
when the Sfall is run without hklin, there is a substantial change in PHIC.

1. HKLIN was provided and scaling turned on.
LIST OF REFLECTIONS
===
H K L FP SIGFP FC PHIC
0 1 2 162.32 3.86 238.41 270.00
0 1 4 97.72 2.34 190.32 90.00
0 1 5 230.11 12.78 256.90 270.00
0 1 6 205.83 7.00 322.93 90.00
0 2 2 53.28 2.14 198.16 0.00
0 2 4 137.66 5.46 2.63 0.00
0 2 5 249.32 5.84 293.85 0.00
0 2 6 203.96 5.37 303.16 180.00

2. HKLIN was provided but scaling was turned off.
LIST OF REFLECTIONS
===
H K L FP SIGFP FC PHIC
0 1 2 158.53 3.77 238.28 270.00
0 1 4 95.44 2.29 189.92 90.00
0 1 5 224.74 12.48 256.07 270.00
0 1 6 201.02 6.84 321.43 90.00
0 2 2 52.04 2.09 198.01 0.00
0 2 4 134.45 5.33 2.63 0.00
0 2 5 243.50 5.70 292.83 0.00
0 2 6 199.20 5.24 301.69 180.00

3. HKLIN with Fobs was not provided (both FC and PHIC are quite different)
LIST OF REFLECTIONS
===
H K L FC PHIC
0 1 2 260.16 -108.18
0 1 4 122.43 -7.47
0 1 5 38.13 81.93
0 1 6 209.85 125.96
0 2 2 386.47 -78.65
0 2 4 145.47 -82.02
0 2 5 107.70 -15.80
0 2 6 101.94 138.75

If anyone could explain, I'd be very grateful. Thanks!

Pawel Janowski

Re: [ccp4bb] Low resolution structure determination advice

[Eleanor Dodson]

Are thw Se SAd and native data isomorphous - because if so you can just 
cad the data sets together - generate phases for the Se set, then use 
those as a starting set for the native data to phase extend. Parrot is 
the more up to date version of DM which does this in the GUI.


The AutoSHARP will use SOLOMON I believe to do this.

Eleanor

 On 09/01/2011 08:28 PM, Bosch, Juergen wrote:

How about phase extension using DM, sure you say you only have one mol per asu 
but it might still be worth trying various approaches of solvent 
flattening/flipping.
Don't know what you used to detect your sites and refine them, but it also 
might be worth sticking them into Sharp with your partial model and see if the 
phases improve.
You say you have 450/750 residues what makes you believe that you placed them 
correctly ?
Also do you have space from the crystal lattice packing for the additional 300 
residues ? In other words are you certain that what you have crystalized is the 
full length version of your protein ?
Adding side chains leading to worse Rfactors would suggest that you are most 
likely not in frame.
Since you have a partial backbone you could try finding a "homolog" via EBI SSM 
to serve as a better starting model, aligning your sequence to it and replacing the side 
chains accordingly.

Another suggestion keep changing programs don't stick to Refmac visit Phenix or 
the other way round, and as a last resort you could give GraphENT a try.

Jürgen

On Sep 1, 2011, at 3:00 PM, Pete Meyer wrote:

Hi,
Depending on how many zn sites you have, you may be able to do zn-mad
for your native crystals.  You don't mention if you've tried combining
your various sources of phase information; if not, it's worth looking into.

You may also want to look into various multi-crystal techniques
(averaging, phasing and/or merging) - I've had decent luck with
multi-crystal phasing off zn at low resolutions.

Good luck,
Pete

Basudeb Bhattacharyya wrote:
Dear all,

We're looking for some advice about how to proceed with a structure we're 
working on.  Our protein is 750 amino acids and naturally binds zinc.  We have 
a SeMet data set that goes down to 3.7 angstroms.  4 of 8 selenium sites are 
ordered and visible in addition to our zincs and we've modeled about 450 
residues of C-alpha backbone off a pure SAD density map
(we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best 
maps--clear density and visible secondary structures--we get are off Se SAD).  
We have one monomer per AU (and we have secondary structure coverage over our 
entire protein based on looking at conserved domains of our 
protein--unfortunately, MR is not working for this project).
R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 
and P31, which haven't worked).  We also have a native set down to 2.7 
angstroms.  We are able to place our working model into the native data set, 
but we are unable to further refine the structure in Refmac (density doesn’t 
improve and the stats creep up).  Addition of side chains only makes our stats 
worse.  The data sets are clean (no twinning, etc.).  While we understand that 
we may need more phasing information (i.e. our initial model may still be quite 
inaccurate given resolution and size of the protein among other things--we are 
trying to improve this), we're wondering if anyone might have some other 
suggestions or insights about how we can move forward given the data that we 
currently have. Thanks in advance for any advice.

Sincerely,

Basu

..
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://web.mac.com/bosch_lab/

Re: [ccp4bb] Trying to "digest" PISA results

[Eleanor Dodson]

Like Jan, I find it very useful to sort out the clear cut cases. 
Otherwise it is easy to get things wrong..


But isnt a buried surface area of 720 rather small for a stable 
interface?  If there is other confirming evidence like 2 diff space 
groups then you feel more secure!!


On 09/01/2011 02:27 PM, Yuri Pompeu wrote:

This is regarding Ethan´s point, particularly:
   >2) the protein has crystallized as a monomer even though it
   >[sometimes] exists in solution as a dimer.  The interface
   >seen in the crystal is not the "real" dimer interface and
   >thus the PISA score is correct.
I see the same exact interface in a crystal of a close homologue that belongs 
to a different space group (hexagonal vs tetragonal system)

Re: [ccp4bb] Temperature Factor statistics

[Eleanor Dodson]

On 08/31/2011 01:32 AM, Yuri Pompeu wrote:

Quick newbie question,
After i get my output file from baverage containing the average b-factor and 
rms by residues,
How can I calculate and display the average (and or mean) B-factors?
Is there a way of calculating it by protein, ligands and solvent separately?
thank you



If these are in different chains - Yes.
Look at the plots..
Eleanor

Re: [ccp4bb] twinning in hexagonal system

[Eleanor Dodson]

Dear John,

In a hexagonal crystal class the possible point groups are
P3 (only a 3-fold axis)
P321(3-fold plus 2-fold relating hkl and kh-l)
P312 (3-fold and 2-fold relating (hkl and -k,-h,-l)
P6 ( 6 fold axis only)
P622 (6-fold plus 2-fold relating hkl and k,h,-l)

pointless will tell you which of these symmetry operators is well 
observed, and suggest a pooint group.


truncate gives graphs of indicators of possible twinning.
The safest indicators are:
the moments of  - if the moment of *4 is < 2.0 twinning is likely. 
However if you have non-crystallographic translation this can be misleading.

The L test done with the "safe" well-measured  data
The cumulative intensity test  with the "safe" well-measured  data.
If these are satisfactory in your assignment of point group to P622 then 
that is probably the correct one.


If they are not then you need to choose which symmetry element to drop.
ctruncate will suggest which is the most likely twin operator, and  the 
point group must be changed to remove that  sym op (or sym ops)


The H test is not very reliable. it is only useful when you have the 
point group right..It looks at the agreement between symmetry pairs so 
obviously if you really have point group P6 but you assign the point 
group as P3, it will tell you that the symmetry pair h,k,l and -h,-k,l 
agree very well and give an H score of ~ 0.5.




On 08/30/2011 08:45 PM, Garib N Murshudov wrote:

Hello

Space groups with point groups 622 and 432 merohedral twinning is not possible 
(they are the highest groups possible for proteins).
If you could merge in 622 it means that Rmerge was very small. It is very 
likely that point group is either 622 or 6 with very strong rotational symmetry 
that is perpendicular to 6 fold axis. In these cases H test (sfcheck based on 
this) and N(z) test (truncate is based on this) will overestimate (H) or 
underestimate (N(z)) if twin is present. At the moment better test for these 
cases seems to be L-test (that is in ctruncate I think).

Solving structure using molecular replacement in the presence of twinning does 
not seem to be a problem (unless data are very noisy and/or model is too far).
Pointless gives very good idea about "true" space group.
I would solve in P6_{x} space groups and then run zanuda (from ysbl server) to 
correct space group.

I hope it helps

regards
Garib


On 30 Aug 2011, at 20:31, john peter wrote:


Hello All,

This is regarding twinning in a data set.

I collected a native data set  to resolution, 1.8 A.  I used XDS suite
to process and scale the data set. It scaled well in P622 and I found
systematic absence (l=6n present).

Hence thought the space group may be P6122/P6522. SFCHECK  did not
show any twinning and also it did not detect pseudo-translation.  Twin
test in http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin
Detector: Padilla-Yeates Algorithm ) showed  perfect twinning.

Scaled in P61/65 and  SFCHECK reported  twinning fraction 0.272&  no
pseudo-translation.
http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin Detector:
Padilla-Yeates Algorithm ) showed  perfect twinning.
Another server from ucla  http://nihserver.mbi.ucla.edu/Twinning/
showed partial twinning  with twin fraction 0.23 as follows.

2 along a, b, a*, b*

No. Twin Law Related Reflections = 18033 (pairs)
No. Twin Law Pairs Considered = 9016

  = 0.266149
  = 0.095303

Twin Fraction = 0.233249 +/- 0.000602

(SHELXL Commands: TWIN 1 0 0 -1 -1 0 0 0 -1 and BASF 0.233249)


In P61/65, I got the following matthews-coeffs

mol/asym  Matthews Coeff  %solvent   P(1.73) P(tot)
_
  19.7587.39  .00  .00
  24.8874.79  .00  .01
  33.2562.18  .06  .14
  4 2.4449.57  .57  .63
  5 1.9536.97  .36  .21
  6 1.6324.36  .00  .00
  7 1.3911.75  .00  .00
_


May I ask what could be the real twin fraction and what is the
likelihood of solving the structure by molecular replacement by models
with 25 % sequence identity and 30 % sequence similarity.

Thank you so much for reading this mail during your  busy hours and
all suggestions, comments would be gratefully welcome&   appreciated.

thank you ccp4 mailing list.

John


Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
Web http://www.mrc-lmb.cam.ac.uk

[ccp4bb] How to help crystal grow bigger

[SnowDeer]

Dear All:

Recently I am working on a protein which can already grow nice pyramid-like
crystals after the condition was optimized, while the crystals are too small
to be picked up. The crystals grew quite fast and densely, so I tried to put
100ul paraffin oil inside the 600ul reservoir solution or put the plate
under 16 degree to slow down the evaporation, while the crystals were still
the same. I also tried macro or micro seeding with or without the paraffin
oil. Macroseeding would give a larger crystal (not very nice) with many
small crystals in the drop even I washed the seeds carefully. For
microseeding, the same small crystals grew.

I don't have many experiences in crystallography, so I have no idea how to
make it grow bigger...

Any suggestion is most welcome.

Thanks.
SnowDeer

Re: [ccp4bb] Saving distances drawn in Coot 0.6.1

[Paul Emsley]

On 04/09/11 19:58, Romain Talon wrote:

I would like to know if there is any way to save dotted lines distances
drawn in Coot in order to recover the information then opening a new
session, even if the program was closed before ?


If you mean the dotted lines from environment distances, then those 
should be saved in the state file (and recovered after restarting).  If 
that doesn't happen then that's a bug (you might like to try with the 
latest stable release, it works for me).


If you mean the dotted lines from Calculate Distances, then that is not 
saved in the state file. (It can be, of course, with a bit of effort.. 
should it be?)


Paul.