Crank not working in CCP4 interface 7.
#CCP4I VERSION CCP4Interface 7.0.003
#CCP4I SCRIPT LOG bp3
#CCP4I DATE 15 Feb 2016 12:39:08
#CCP4I USER satyshur
#CCP4I PROJECT ACT-Tip
#CCP4I JOB_ID 1
#CCP4I SCRATCH /home/satyshur/tmp
#CCP4I HOSTNAME xingxtal
#CCP4I PID 2857
<hr>
crank->Error:crank::crank binary and crank XML version do not match
***************************************************************************
* Information from CCP4Interface script
***************************************************************************
The program run with command: /home/xray/ccp4/ccp4-7.0/ccp4-7.0/bin/tclsh /home/xray/ccp4/ccp4-7.0/ccp4-7.0/bin/crank /home/satyshur/tmp/ACT-Tip_1_1_com.tmp
has failed with error message
crank::crank binary and crank XML version do not match
while executing
"error $message"
(procedure "crank_error" line 8)
invoked from within
"crank_error "crank::crank binary and crank XML version do not match""
invoked from within
"if { [info exists XMLParse([join "crank parameters version" __])] } {
if { $version != $XMLParse([join "crank parameters version" __]) } {
cr..."
(file "/home/xray/ccp4/ccp4-7.0/ccp4-7.0/bin/crank" line 71)
***************************************************************************
#CCP4I TERMINATION STATUS 0 "crank::crank binary and crank XML version do not match while executing "error $message" (procedure "crank_error" line 8) invoked from within "crank_error "crank::crank binary and crank XML version do not match"" invoked from within "if { [info exists XMLParse([join "crank parameters version" __])] } { if { $version != $XMLParse([join "crank parameters version" __]) } { cr..." (file "/home/xray/ccp4/ccp4-7.0/ccp4-7.0/bin/crank" line 71)"
#CCP4I TERMINATION TIME 15 Feb 2016 12:39:08
#CCP4I TERMINATION OUTPUT_FILES /home/satyshur/Tiprl/ACT-Tip/1_crank
#CCP4I MESSAGE Task failed
Kenneth A. Satyshur, M.S., Ph.D.
Senior Scientist
University of Wisconsin-Madison
Madison, Wisconsin, 53706
608-215-5207
________________________________________
From: CCP4 bulletin board <[log in to unmask]> on behalf of CCP4BB automatic digest system <[log in to unmask]>
Sent: Sunday, February 14, 2016 6:00 PM
To: [log in to unmask]
Subject: CCP4BB Digest - 13 Feb 2016 to 14 Feb 2016 (#2016-45)
There are 11 messages totaling 1672 lines in this issue.
Topics of the day:
1. post-doc position in KAUST
2. Weird screen artefacts in Coot after "upgrading"
3. Protein:ligand ratio for co-crystallization (4)
4. Another reason... (4)
5. MBP fusion
----------------------------------------------------------------------
Date: Sun, 14 Feb 2016 03:03:39 +0100
From: Stefan Arold <[log in to unmask]>
Subject: post-doc position in KAUST
Dear All,
A post-doctoral position is currently available in my group for highly
motivated individuals who have obtained their PhD less than 5 years ago.
And who don't like rain.
With best regards
Stefan
A post-doc position in structural biology is currently available at KAUST
*Project:* Structure-function of key proteins in eukaryotic signalling and
adhesion (see for example *Cheung et al. eLIFE 2015*
<http://dx.doi.org/10.7554/eLife.06866> or *Brami-Cherrier et al. EMBO J
2014* <http://emboj.embopress.org/content/33/4/356.long>).
*Methods:* Hybrid approaches in structural biology, including X-ray
crystallography, SAXS, NMR, cryo-EM, computational methods, bioinformatics,
molecular biophysics and biochemistry.
*Localisation:* StruBE group at the King Abdullah University of Science and
Technology (KAUST
<https://bese.kaust.edu.sa/our-people/Pages/stefan-arold-faculty-lab.aspx>).
KAUST, founded in 2009, is located at the Red Sea shores in Saudi Arabia,
and currently ranks nr. 1 in citations per faculty (QS World University
Rankings 2015/2016). We have outstanding research facilities, a community
with more than 100 nationalities, pristine coral reefs and about two hours
of rain per year.
*Remuneration:* Salary 50 – 60 kUSD per year (tax free), commensurate with
experience. Free housing.
*Contact:* [log in to unmask]
*Stefan T Arold, PhD*
*Associate Professor*
*Division of Biological and Environmental Sciences and Engineering*
*Computational Bioscience Research Center (CBRC)*
*King Abdullah University of Science and Technology (KAUST)*
*Thuwal 23955-6900*
*Kingdom of Saudi Arabia*
------------------------------
Date: Sun, 14 Feb 2016 11:27:54 +0000
From: Jon Sayers <[log in to unmask]>
Subject: Re: Weird screen artefacts in Coot after "upgrading"
Dear All,
Thanks for the numerous suggestions: Here is what worked for me:
After suggestions that it may be a hardware issue I checked Applediscussions and found an article graphics problems on MacBook Pros. They have two graphics cards.
(see the full discussion on https://discussions.apple.com/thread/6619352?start=0&tstart=0)
However, I installed gfxCardStatus from https://gfx.io <https://gfx.io/> . It allows you to switch between the two graphics cards. I can run Coot etc quite happily now from one of the GFX cards but not the other so I guess it is a hardware issue.
BW Jon
> On 12 Feb 2016, at 17:22, Jon Sayers <[log in to unmask]> wrote:
>
> Has anyone seen this type of artefact (and better yet do you know of a fix)? It looks like some sort of scene from an Indiana Jones movie.
> It started on my MacBookPro running Yosemite when I installed the latest CCP4 a few weeks back, and persists when I updated the Mac OS to El Capitan. It works fine on my iMac which is running same system/ccp4 versions. Any suggestion gratefully received.
>
>
> <weird_coot.png>
> Best wishes,
> Jon
>
> Prof. Jon R Sayers FRSB
> Florey Institute,
> Dept. of Infection, Immunity and Cardiovascular Disease
> University of Sheffield Medical School
> Beech Hill Rd
> Sheffield S10 2RX
> United Kingdom,
>
> Tel 0114 271 3027
> Email [log in to unmask] <mailto:[log in to unmask]>
> www.shef.ac.uk/medicine/infectionandimmunity/staffprofiles/sayers.html <http://www.shef.ac.uk/medicine/infectionandimmunity/staffprofiles/sayers.html>
>
>
>
>
>
>
>
>
>
>
>
Best wishes,
Jon
Prof. Jon R Sayers FRSB
Florey Institute,
Dept. of Infection, Immunity and Cardiovascular Disease
University of Sheffield Medical School
Beech Hill Rd
Sheffield S10 2RX
United Kingdom,
Tel 0114 271 3027
Email [log in to unmask] <mailto:[log in to unmask]>
www.shef.ac.uk/medicine/infectionandimmunity/staffprofiles/sayers.html
------------------------------
Date: Sun, 14 Feb 2016 20:02:12 +0530
From: Kavyashree Manjunath <[log in to unmask]>
Subject: Re: Protein:ligand ratio for co-crystallization
Dear George,
Ok. MST does not give the percentage binding. The value
I mentioned is from the graph, might not be accurate.
The calculation mentioned does not include the concentration
of protein. According to this, so irrespective of the protein
concentration, the [L] required for a kd of 50microM would be 950microM.
But, this would be incorrect if the protein concentration itself is
950microM or more.
If a biophysical technique detects a binding at a ratio
Of protein:ligand=1:100 @ [P] --> 20nM, then the same technique
might not detect the binding if the ratio is less than
1:100 at [P] --> 20microM. Please correct me if I am wrong.
Accordingly, I thought the same ratio is needed for co-crystallization.
What is the minimum occupancy of the ligand, that can be
detected in the electron density?
Thank you
Regards
Kavya
> Dear Kavya,
> Assuming
> Kd=50 microM with protein concertation = 20nM for 95% bound:
> Kd=(P)(L)/(PL) ---> 50 microM=(5%)(L)/(95%) ---> (50 microM) (95)/(5)=
> (L) --->(50 microM) (19)= (L) --->(950 microM) = (L)
> (P)=protein free (unbound) concentration (L) =ligand free (unbound)
> concentration (PL)= protein-ligand complex concentration
>
> So in order to get 95% bound (using your numbers), you need around 950
> microM ligand (Sebastian conclusion is correct), which is far from 250
> microM.
>
> Now let assume that Kd =50 microM is correct (from the above it looks
> unlikely. In order to get the complex in co-crystallization experiment
> with
> a protein concentration of 100 microM, for 90% bound (90% occupancy)
> you need a ligand concentration 540 microM in the drop (around 10fold
> above the KD as Sebastian pointed out) or protein:ligand 1:5.4 (molar
> ratio).
>
> George
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Falk,
> Sebastian
> Sent: Thursday, February 11, 2016 10:15 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
>
> Dear Kavya,
>
> with a KD of 50 micro-molar, and a highest ligand concentration of 250
> micro-molar, you re just 5 times above the KD. I cannot image that you
> will
> have 95% of complex then. Usually you will have 90% binding when 10fold
> above the KD.
>
> With a KD of 50 micro-molar it is going to be tricky to obtain crystals,
> but
> not impossible. Even complexes with Kd> 80-90 micro-molar have
> crystallised
> and the structure determined.
>
> Best,
> Sebastian
>
>
>
>> On 11 Feb 2016, at 08:10, Kavyashree Manjunath <[log in to unmask]>
> wrote:
>>
>> Dear George,
>>
>> For one of the ligand the kd was 50 micromole, the protein
>> concentration used in MST was 20nM and at 250micromolar ligand
>> concentration, it was ~95% bound.
>>
>> Thank you
>> Regards
>> Kavya
>>
>>
>>> Almost means 88%? 93% ? 95%? or 99%?
>>>
>>> That why it is much easier when Kd is known because we do not need
>>> all the above detail information just one number (Kd).
>>> Give me also the protein concentration of protein during bioassay.
>>>
>>> G.
>>>
>>> -----Original Message-----
>>> From: [log in to unmask] [mailto:[log in to unmask]]
>>> Sent: Wednesday, February 10, 2016 1:17 PM
>>> To: George Kontopidis
>>> Cc: [log in to unmask]
>>> Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
>>>
>>> Dear George,
>>>
>>> Sorry for the late reply. 1:500 the MST curve almost reaches the
>>> saturation.
>>>
>>> Thank you
>>> Regards
>>> Kavya
>>>
>>>> Dear Kavya,
>>>>
>>>> The biophysical techniques like MST shows a binding of 20%, 40% or
>>>> 99% (protein:ligand)?
>>>> when molar ratio of protein:ligand is greater than 1:500 we need
>>>> this number to give you an answer
>>>>
>>>> All depend of the Ka or Kd (1/Ka). That why you have obtained ligand
>>>> bound structure even with molar ratio of protein:ligand down to 1:10.
>>>>
>>>>
>>>> Regards,
>>>>
>>>> George
>>>>
>>>> -----Original Message-----
>>>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
>>>> Of Kavyashree Manjunath
>>>> Sent: Tuesday, February 09, 2016 7:30 AM
>>>> To: [log in to unmask]
>>>> Subject: [ccp4bb] Protein:ligand ratio for co-crystallization
>>>>
>>>> Dear users,
>>>>
>>>> I would like to know the minimum protein:ligand ratio that will
>>>> yield a cocrystal, provided there are some binding values from
>>>> biophysical techniques.
>>>>
>>>> Suppose, biophysical techniques like MST shows a binding when molar
>>>> ratio of protein:ligand is greater than 1:500, is it necessary to
>>>> have this ratio while co-crystallizing?
>>>>
>>>> I have previous experiences in which I had obtained ligand bound
>>>> structure when I used 1:10 to 1:30 (molar ratio). But, it is not
>>>> working
>>> now.
>>>>
>>>> Thank you
>>>> Regards
>>>> Kavya
>>>>
>>>>
>>>> --
>>>> This message has been scanned for viruses and dangerous content by
>>>> MailScanner, and is believed to be clean.
>>>>
>>>
>>>
>>>
>>>
>
>
------------------------------
Date: Sun, 14 Feb 2016 16:27:31 +0100
From: Anastassis Perrakis <[log in to unmask]>
Subject: Re: Protein:ligand ratio for co-crystallization
Ok, lets start from the basics. ;-)
Y= x/(kD+x)
… maximal binding=1 for simplicity
Y is the fraction bound
x is the ligand cocnentration
and kD is your KD
so …
x= (Y * KD) / (1 - Y)
… you can plot that and stick it in your desk really, for a few K_D
… and to make a long story short, the numbers are more or less exactly as Sebastian says ...
However, life is more complicated.
When a protein interacts with a ligand and the reaction has reached equilibrium (which in most cases happens in the milliseconds to several seconds scale) , although we call that a “steady state” this is till a dynamic interaction. Molecules of the protein and the ligand associate and disassociate all the time, but the system is in equilibrium.
P+L <—> PL
This equilibrium can then be described by the association rate, or K_on, that depends on the absolute concentration of the reactants and is expressed as events per molarity per time M^(-1)sec^(-1)
The disassociation or K_off, does not depend on the concentration of the reactants, but is only a function of time, e.g. how many complexes disassociate per second, and is expressed in sec^(-1)
The infamous disassociation constant we have all been talking of, K_D, is then the K_off/K_on, thus has Molarity as a unit, and it tells you the concentration in which as many complexes are made as they associate as a function of time. In practice, when you measure association with one reactant in very low concentration and titrate the other one one, K_D is the point that you have 50% of “saturation”.
Now, back to your question: when you crystallise you change the system I just described, so Kon, Koff, and thus KD will change. So, you cannot just predict. However, some simple rules:
1. Better have more ligand than protein, as the protein needs to be “saturated", and you will never do that with less ligand than protein ...
1. You better be at least 3x or preferably 10x over the K_D with your ligand, to make sure most of your mixture is bound.
2. You better have a low K_off. If a K_off is a minute you have a good chance to capture that in a crystal, if its 10ms tough luck: transient complexes are hard to crystallise.
Lazy Sunday afternoon - sorry for the long essay and sorry for some oversimplifications I made and some seasoned biochemists might be chuckling about ;-)
A.
------------------------------
Date: Sun, 14 Feb 2016 19:12:40 +0100
From: Bernhard Rupp <[log in to unmask]>
Subject: Another reason...
...to avoid win10 if you want to use crystallographic applets:
In Windows 10, the Edge browser does not support plug-ins and therefore
will not run Java. Switch to a different browser (Firefox or Internet
Explorer 11) to run the Java plug-in. More info
<http://java.com/en/download/faq/win10_faq.xml>
...and if you switch to a different (mozilla) browser, defender starts to
slug it out at 100% disk -
at least on my now obsolete schlepptop...
Prepare for a gravitational wave when I ditch that thing into a black hole.
Best, BR
--
-----------------------------------------------------------------
Bernhard Rupp
001 (925) 209-7429
+43 (676) 571-0536
[log in to unmask]
[log in to unmask]
http://www.ruppweb.org/
-----------------------------------------------------------------
The hard part about playing chicken
is to know when to flinch
-----------------------------------------------------------------
------------------------------
Date: Sun, 14 Feb 2016 19:41:05 +0100
From: Victor Lamzin <[log in to unmask]>
Subject: Re: Protein:ligand ratio for co-crystallization
Hi,
The fraction of the ligand bound depends not only on the ligand
concentration and Kd, but also on the concentration of the protein.
Indeed, it is better to have more ligand and less protein and here the
size of the protein matters. The larger it is, the lower the protein's
molar concentration, and thus with the same concentration of the ligand
the higher its occupancy in the structure will be.
A rule-of-thumb that the concentration of the ligand should be 10 times
Kd is well applicable provided that the molar amount of ligand still
exceeds the molar amount of protein.
Say one co-crystallises the protein with MW of 30 kDa, Kd is 100 mkM.
One mixes a drop of protein at 10 mg/ml with the same size a drop of
ligand at its concentration 10 times higher than Kd, 1 mM. The occupancy
of the ligand in the structure will be around 85%.
Now imagine that for the same size protein its Kd is 10 times better, 10
mkM, and the ligand concentration is again 10 times higher than Kd, 100
mkM. The ligand occupancy will be about 30%.
A hypothetical but anecdotal case would be when one fuses this protein
to one molecule of GFP thus increasing MW of the complex to 57 kDa.
Assuming Kd staying at 10 mkM, the crystals are still growing under the
same conditions as above, and the binding site is unaffected - the same
ligand concentration of 100 mkM will provide bound ligand with somewhat
reasonable occupancy of 50% ;-)
The above thoughts apply to co-crystallisation. In case of soaking there
are additional factors which will affect the occupancy of the bound
ligand. The smaller the size of the crystals and the larger the volume
of the soaking drop - the more likely the efforts will be rewarded in a
nicely-looking ligand density.
Cheers,
Victor
On 14/02/2016 16:27, Anastassis Perrakis wrote:
> Ok, lets start from the basics. ;-)
>
> Y= x/(kD+x)
>
> … maximal binding=1 for simplicity
> Y is the fraction bound
> x is the ligand cocnentration
> and kD is your KD
>
> so …
>
> x= (Y * KD) / (1 - Y)
>
> … you can plot that and stick it in your desk really, for a few K_D
>
> … and to make a long story short, the numbers are more or less exactly as Sebastian says ...
>
> However, life is more complicated.
>
> When a protein interacts with a ligand and the reaction has reached equilibrium (which in most cases happens in the milliseconds to several seconds scale) , although we call that a “steady state” this is till a dynamic interaction. Molecules of the protein and the ligand associate and disassociate all the time, but the system is in equilibrium.
>
> P+L <—> PL
>
> This equilibrium can then be described by the association rate, or K_on, that depends on the absolute concentration of the reactants and is expressed as events per molarity per time M^(-1)sec^(-1)
>
> The disassociation or K_off, does not depend on the concentration of the reactants, but is only a function of time, e.g. how many complexes disassociate per second, and is expressed in sec^(-1)
>
> The infamous disassociation constant we have all been talking of, K_D, is then the K_off/K_on, thus has Molarity as a unit, and it tells you the concentration in which as many complexes are made as they associate as a function of time. In practice, when you measure association with one reactant in very low concentration and titrate the other one one, K_D is the point that you have 50% of “saturation”.
>
> Now, back to your question: when you crystallise you change the system I just described, so Kon, Koff, and thus KD will change. So, you cannot just predict. However, some simple rules:
>
> 1. Better have more ligand than protein, as the protein needs to be “saturated", and you will never do that with less ligand than protein ...
> 1. You better be at least 3x or preferably 10x over the K_D with your ligand, to make sure most of your mixture is bound.
> 2. You better have a low K_off. If a K_off is a minute you have a good chance to capture that in a crystal, if its 10ms tough luck: transient complexes are hard to crystallise.
>
> Lazy Sunday afternoon - sorry for the long essay and sorry for some oversimplifications I made and some seasoned biochemists might be chuckling about ;-)
>
> A.
------------------------------
Date: Sun, 14 Feb 2016 19:36:11 +0000
From: Jurgen Bosch <[log in to unmask]>
Subject: Re: Another reason...
Time to switch BR.
J?rgen
......................
J?rgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street<x-apple-data-detectors://4>, W8708
Baltimore, MD 21205<x-apple-data-detectors://5/0>
Office: +1-410-614-4742<tel:%2B1-410-614-4742>
Lab: +1-410-614-4894<tel:%2B1-410-614-4894>
Fax: +1-410-955-2926<tel:%2B1-410-955-2926>
http://lupo.jhsph.edu<http://lupo.jhsph.edu/>
On Feb 14, 2016, at 13:13, Bernhard Rupp <[log in to unmask]<mailto:[log in to unmask]>> wrote:
...to avoid win10 if you want to use crystallographic applets:
In Windows 10, the Edge browser does not support plug-ins and therefore will not run Java. Switch to a different browser (Firefox or Internet Explorer 11) to run the Java plug-in. More info<http://java.com/en/download/faq/win10_faq.xml>
...and if you switch to a different (mozilla) browser, defender starts to slug it out at 100% disk -
at least on my now obsolete schlepptop...
Prepare for a gravitational wave when I ditch that thing into a black hole.
Best, BR
--
-----------------------------------------------------------------
Bernhard Rupp
001 (925) 209-7429
+43 (676) 571-0536
[log in to unmask]<mailto:[log in to unmask]>
[log in to unmask]<mailto:[log in to unmask]>
http://www.ruppweb.org/
-----------------------------------------------------------------
The hard part about playing chicken
is to know when to flinch
-----------------------------------------------------------------
------------------------------
Date: Sun, 14 Feb 2016 21:43:52 +0100
From: Bernhard Rupp <[log in to unmask]>
Subject: Re: Another reason...
The shelx programs work perfectly well because they are solid Fortran code
that runs - since decades now - fine as console apps under windows. Even
my old cgi server apps in Fortran returned trivial html code that 1992
Mosaic could interpret and even Edge now understands. The issue I referred
to is with Java extensions with their almost postmodern update paranoia
and incompatibilities. Client side apps rely on code in interpreted
languages that may or may nor be supported/updated/etc by the dominant
OSes. Kudos to the ShelXle developers for divining the fact that the
mainstream W10 browser would not support Java, which quite a number of
other, very useful macromolecular crystallography web apps use.....
I am just perplexed by that insanity of Edge not supporting Java. Maybe
Ellison and Gates should shoot a round of golf together....
And, no, I am not going to by a Mac (my appreciation for industrial design
not withstanding) and installing X11 under various Linux flavor VMs was to
phrase it mildly an abortive task whose details need not be described.
Cheers, BR
On Sun, Feb 14, 2016 at 7:29 PM, George Sheldrick <
[log in to unmask]> wrote:
> Dear Bernhard,
>
> Most SHELX users happily use Windows, including Windows 10. They use
> several third party GUIs to help them, e.g. Christian Huebschle's shelXle,
> which however does not use Java (just qt4 and C++). So your warning does
> not apply to small molecule crystallographers.
>
> Best wishes, George
>
>
>
> On 02/14/2016 07:12 PM, Bernhard Rupp wrote:
>
> ...to avoid win10 if you want to use crystallographic applets:
>
> In Windows 10, the Edge browser does not support plug-ins and therefore
> will not run Java. Switch to a different browser (Firefox or Internet
> Explorer 11) to run the Java plug-in. More info
> <http://java.com/en/download/faq/win10_faq.xml>
>
> ...and if you switch to a different (mozilla) browser, defender starts to
> slug it out at 100% disk -
> at least on my now obsolete schlepptop...
>
> Prepare for a gravitational wave when I ditch that thing into a black hole.
>
> Best, BR
>
>
>
> --
> -----------------------------------------------------------------
> Bernhard Rupp
> 001 (925) 209-7429
> +43 (676) 571-0536
> [log in to unmask]
> [log in to unmask]
> http://www.ruppweb.org/
> -----------------------------------------------------------------
> The hard part about playing chicken
> is to know when to flinch
> -----------------------------------------------------------------
>
>
>
> --
> Prof. George M. Sheldrick FRS
> Dept. Structural Chemistry,
> University of Goettingen,
> Tammannstr. 4,
> D37077 Goettingen, Germany
> Tel. +49-551-39-33021 or -33068
> Fax. +49-551-39-22582
>
>
--
-----------------------------------------------------------------
Bernhard Rupp (Hofkristallrat a. D)
001 (925) 209-7429
+43 (676) 571-0536
[log in to unmask]
[log in to unmask]
http://www.ruppweb.org/
-----------------------------------------------------------------
The hard part about playing chicken
is to know when to flinch
-----------------------------------------------------------------
------------------------------
Date: Sun, 14 Feb 2016 22:49:27 +0200
From: George Kontopidis <[log in to unmask]>
Subject: Re: Protein:ligand ratio for co-crystallization
Dear Kavya,
the protein concentration should be included in the calculation.
In the first case was neglected because according to your statement was
20nM. So would not make difference to say 950 microM or the correct one
which is 950.02 microM
In second example I mentioned that protein concentration is 100 microM and
it was included in the calculations.
Regarding minimum occupancy of the ligand that can be detected in the
electron density?
It was able to detect 30% occupancy at around 2.1 A resolution. Now days
you may be able to go even lower.
Having the unliganded structure in other words 0% occupancy, it will help
you a lot to determine more accurately the occupancy.
George
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Kavyashree Manjunath
Sent: Sunday, February 14, 2016 4:32 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
Dear George,
Ok. MST does not give the percentage binding. The value I mentioned is from
the graph, might not be accurate.
The calculation mentioned does not include the concentration of protein.
According to this, so irrespective of the protein concentration, the [L]
required for a kd of 50microM would be 950microM.
But, this would be incorrect if the protein concentration itself is
950microM or more.
If a biophysical technique detects a binding at a ratio Of
protein:ligand=1:100 @ [P] --> 20nM, then the same technique might not
detect the binding if the ratio is less than
1:100 at [P] --> 20microM. Please correct me if I am wrong.
Accordingly, I thought the same ratio is needed for co-crystallization.
What is the minimum occupancy of the ligand, that can be detected in the
electron density?
Thank you
Regards
Kavya
> Dear Kavya,
> Assuming
> Kd=50 microM with protein concertation = 20nM for 95% bound:
> Kd=(P)(L)/(PL) ---> 50 microM=(5%)(L)/(95%) ---> (50 microM) (95)/(5)=
> (L) --->(50 microM) (19)= (L) --->(950 microM) = (L) (P)=protein free
> (unbound) concentration (L) =ligand free (unbound)
> concentration (PL)= protein-ligand complex concentration
>
> So in order to get 95% bound (using your numbers), you need around
> 950 microM ligand (Sebastian conclusion is correct), which is far
> from 250 microM.
>
> Now let assume that Kd =50 microM is correct (from the above it looks
> unlikely. In order to get the complex in co-crystallization
> experiment with a protein concentration of 100 microM, for 90% bound
> (90% occupancy) you need a ligand concentration 540 microM in the
> drop (around 10fold above the KD as Sebastian pointed out) or
> protein:ligand 1:5.4 (molar ratio).
>
> George
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Falk, Sebastian
> Sent: Thursday, February 11, 2016 10:15 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
>
> Dear Kavya,
>
> with a KD of 50 micro-molar, and a highest ligand concentration of 250
> micro-molar, you re just 5 times above the KD. I cannot image that you
> will have 95% of complex then. Usually you will have 90% binding when
> 10fold above the KD.
>
> With a KD of 50 micro-molar it is going to be tricky to obtain
> crystals, but not impossible. Even complexes with Kd> 80-90
> micro-molar have crystallised and the structure determined.
>
> Best,
> Sebastian
>
>
>
>> On 11 Feb 2016, at 08:10, Kavyashree Manjunath
>> <[log in to unmask]>
> wrote:
>>
>> Dear George,
>>
>> For one of the ligand the kd was 50 micromole, the protein
>> concentration used in MST was 20nM and at 250micromolar ligand
>> concentration, it was ~95% bound.
>>
>> Thank you
>> Regards
>> Kavya
>>
>>
>>> Almost means 88%? 93% ? 95%? or 99%?
>>>
>>> That why it is much easier when Kd is known because we do not need
>>> all the above detail information just one number (Kd).
>>> Give me also the protein concentration of protein during bioassay.
>>>
>>> G.
>>>
>>> -----Original Message-----
>>> From: [log in to unmask] [mailto:[log in to unmask]]
>>> Sent: Wednesday, February 10, 2016 1:17 PM
>>> To: George Kontopidis
>>> Cc: [log in to unmask]
>>> Subject: Re: [ccp4bb] Protein:ligand ratio for co-crystallization
>>>
>>> Dear George,
>>>
>>> Sorry for the late reply. 1:500 the MST curve almost reaches the
>>> saturation.
>>>
>>> Thank you
>>> Regards
>>> Kavya
>>>
>>>> Dear Kavya,
>>>>
>>>> The biophysical techniques like MST shows a binding of 20%, 40% or
>>>> 99% (protein:ligand)?
>>>> when molar ratio of protein:ligand is greater than 1:500 we need
>>>> this number to give you an answer
>>>>
>>>> All depend of the Ka or Kd (1/Ka). That why you have obtained
>>>> ligand bound structure even with molar ratio of protein:ligand down to
1:10.
>>>>
>>>>
>>>> Regards,
>>>>
>>>> George
>>>>
>>>> -----Original Message-----
>>>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
>>>> Of Kavyashree Manjunath
>>>> Sent: Tuesday, February 09, 2016 7:30 AM
>>>> To: [log in to unmask]
>>>> Subject: [ccp4bb] Protein:ligand ratio for co-crystallization
>>>>
>>>> Dear users,
>>>>
>>>> I would like to know the minimum protein:ligand ratio that will
>>>> yield a cocrystal, provided there are some binding values from
>>>> biophysical techniques.
>>>>
>>>> Suppose, biophysical techniques like MST shows a binding when molar
>>>> ratio of protein:ligand is greater than 1:500, is it necessary to
>>>> have this ratio while co-crystallizing?
>>>>
>>>> I have previous experiences in which I had obtained ligand bound
>>>> structure when I used 1:10 to 1:30 (molar ratio). But, it is not
>>>> working
>>> now.
>>>>
>>>> Thank you
>>>> Regards
>>>> Kavya
>>>>
>>>>
>>>> --
>>>> This message has been scanned for viruses and dangerous content by
>>>> MailScanner, and is believed to be clean.
>>>>
>>>
>>>
>>>
>>>
>
>
------------------------------
Date: Sun, 14 Feb 2016 21:27:15 +0000
From: Ian Tickle <[log in to unmask]>
Subject: Re: Another reason...
On 14 February 2016 at 20:43, Bernhard Rupp <[log in to unmask]>
wrote:
>
> I am just perplexed by that insanity of Edge not supporting Java. Maybe
> Ellison and Gates should shoot a round of golf together....
>
Hi Bernhard
I'm afraid MS are only following the crowd on this: Ellison & Gates,
together with the CEOs of Google & Mozilla have already decided that Java
plug-ins have no future - and no doubt Apple will soon follow suit (if they
haven't done so already)! Google Chrome no longer supports NPAPI plug-ins
(which includes Java):
https://developer.chrome.com/extensions/npapi
https://support.google.com/chrome/answer/6213033 .
Mozilla plans to withdraw support from Firefox by the end of this year:
https://blog.mozilla.org/futurereleases/2015/10/08/npapi-plugins-in-firefo
<https://blog.mozilla.org/futurereleases/2015/10/08/npapi-plugins-in-firefox>
x
Quote: "Oracle recommends
<http://docs.oracle.com/javase/8/docs/technotes/guides/deploy/applet_dev_guide.html#JSDPG1032>
that sites currently using Java applets consider switching to plugin-free
solutions such as Java Web Start
<https://blogs.oracle.com/java-platform-group/entry/java_web_start_in_or>".
You can of course keep using IE11, but how many website developers are
going to continue using Java if only pre-W10 Windows installation can make
use of it?
There are much better alternatives such as WebGL which don't suffer from
the security issues that have plagued Java over the years (OK maybe they
just have different security issues ...). So the writing is on the wall to
website developers: time to switch!
Cheers
-- Ian
>
>
------------------------------
Date: Sun, 14 Feb 2016 23:17:31 +0000
From: "Fernandez, Elias J" <[log in to unmask]>
Subject: MBP fusion
Dear Colleagues,
We are attempting to express our target protein fused to MBP in BL21(DE3) and BL21(RIPL). Unfortunately, we get a mixture of MBP-target protein and MBP only, which severely depletes our overall yield of MBP-target protein. We've tried enriched media, such as 2xYT & TB and also optimal high-density induction in minimal media at temperatures ranging from 20-37deg and IPTG from 0.05-0.5microM.
Any suggestions to address this competitively high yield of MBP-only protein?
Cheers,
Elias
------------------------------
End of CCP4BB Digest - 13 Feb 2016 to 14 Feb 2016 (#2016-45)
************************************************************
