[ccp4bb] co-crystallization

[yangliuqing]

Hello,everyone,
I have a question for cocrystallization, is there some relationship between Km 
value and substrate concentration when making cocrystallization? How can I know 
the substrate is enough for binding?
Thank you very much!
liuqing
_
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Re: [ccp4bb] co-crystallization

[Juergen Bosch]

The Km changes with your reservoir, so predictions are limited. In  
general if you have a low Km this is favourable but not a given that  
your ligand will be found in the electron density map. As a starting  
point try a molar ratio of >3 of the ligand to your protein and you  
can go as high as you want (assuming it's soluble and does not cost  
you an arm and a leg). 50 fold excess has been used in some cases. And  
remember for your cryo-step to include your ligand in sufficient  
amounts !


Jürgen

On 1 Dec 2008, at 05:31, yangliuqing wrote:


Hello,everyone,
I have a question for cocrystallization, is there some relationship  
between Km value and substrate concentration when making  
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing

八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！


-
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone:   +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch

Re: [ccp4bb] co-crystallization

[mesters]

Yes!, there is:

the fraction of occupied protein with substance can be calculated: S / 
(S + Km) with S being the concentration of the compound.


So, if S = Km, half of the sites are occupied (it follows from 
Michaelis-Menten theory).


In order to saturate the enzyme for 90,90909 % with the compound:

1) S = 10 x Kd (concentration of S at least 10 times the Kd)
and
2) S > P (total concentration of S must be larger than total 
concentration of protein or "binding sites")


Depending on the solubility of the compound, this is not always 
possible. In such a case, you need to use DMSO and/or add solid compound 
to the protein solution and leave it for quite some time for the 
compound to finally bind to the protein.


- J. -


yangliuqing wrote:

Hello,everyone,
I have a question for cocrystallization, is there some relationship 
between Km value and substrate concentration when making 
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 




--
Dr. Jeroen R. Mesters
Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
Institut für Biochemie, Universität zu Lübeck
Zentrum für Medizinische Struktur- und Zellbiologie
Ratzeburger Allee 160, D-23538 Lübeck
Tel: +49-451-5004065, Fax: +49-451-5004068
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.selfish-brain.org
Http://www.opticryst.org
--
If you can look into the seeds of time and say
which grain will grow and which will not - speak then to me  (Macbeth)
--

Re: [ccp4bb] co-crystallization

[Engin Ozkan]

As said, your Km is different in mother liquor than in your reaction 
conditions, but even that is not the end of it: You ligand/substrate might be 
inducing the slightest of all conformations in your protein that interferes 
with crystallization, or might block crystal contacts.  Then, you can either 
(1) get no crystals any longer, or (2) get no substrate in your crystals, 
depending on whichever wins energetically, substrate binding or crystal 
formation.  Therefore, being orders of magnitude above physiological Km/Kd is 
still not a guarantee for getting crystals of a complex. So, dwelling 
obsessively on molar ratios and scratching your head becomes irrelevant after a 
while, one should just do the experiment with a good molar ratio as suggested 
in this thread, and accept the verdict of the gods of crystallization.

Interestingly enough, your substrate-protein complex might crystallize more 
efficiently, or crystallize in different conditions, so not all is negative.

Engin

P.S. You are talking about Km and a substrate, but I hope you have a substrate 
analog, inactive enzyme or you are trying to capture the product complex, 
otherwise you might be in for a surprise when you solve the structure.

- Original Message -
From: "Juergen Bosch" <[EMAIL PROTECTED]>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, December 1, 2008 6:35:24 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] co-crystallization

The Km changes with your reservoir, so predictions are limited. In general if 
you have a low Km this is favourable but not a given that your ligand will be 
found in the electron density map. As a starting point try a molar ratio of >3 
of the ligand to your protein and you can go as high as you want (assuming it's 
soluble and does not cost you an arm and a leg). 50 fold excess has been used 
in some cases. And remember for your cryo-step to include your ligand in 
sufficient amounts ! 


Jürgen 



On 1 Dec 2008, at 05:31, yangliuqing wrote: 



Hello,everyone, 
I have a question for cocrystallization, is there some relationship between Km 
value and substrate concentration when making cocrystallization? How can I know 
the substrate is enough for binding? 
Thank you very much! 
liuqing 


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 






- 
Jürgen Bosch 
University of Washington 
Dept. of Biochemistry, K-426 
1705 NE Pacific Street 
Seattle, WA 98195 
Box 357742 
Phone: +1-206-616-4510 
FAX: +1-206-685-7002 
Web: http://faculty.washington.edu/jbosch 

[ccp4bb] CCP4 Study Weekend, SR User Group Meeting programme

[Flaig, R (Ralf)]

Dear colleagues,

like in previous years there will be the SR User Group Meeting
organised as a satellite meeting of the main CCP4 Study Weekend.
Details about the meeting programme can be found here:

http://www.cse.scitech.ac.uk/events/CCP4_2009/user_group.html

The meeting will take place on Saturday 3rd January 2009
from 15:00h to 17:30h.

We hope that you will find the programme interesting and that
you can make it to the meeting.


Katherine McAuley + Ralf Flaig

Beamline Scientists, Macromolecular Crystallography
Diamond Light Source, Harwell Science and Innovation Campus
Chilton, Didcot OX11 0DE, United Kingdom
http://www.diamond.ac.uk
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Re: [ccp4bb] co-crystallization

[Edward A. Berry]

mesters wrote:

Yes!, there is:

the fraction of occupied protein with substance can be calculated: S / 
(S + Km) with S being the concentration of the compound.


So, if S = Km, half of the sites are occupied (it follows from 
Michaelis-Menten theory).


But- one warning (perhaps obvious but I think worth pointing out):
S here is the _free_ concentration. The total concentration you have
to add must include the bound ligand also. To achieve 50% saturation
you need to add 50% of the protein concentration plus 1 x Km.

In enzyme assays, where the enzyme may be picomolar, the amount
bound is often insignificant. But in crystallization experiments
the protein is often hundreds of micromolar, and Ki's < 1 uM,
the Ki becomes irrelevant- just add stoichiometric ligand and
a little extra.
Ed



In order to saturate the enzyme for 90,90909 % with the compound:

1) S = 10 x Kd (concentration of S at least 10 times the Kd)
and
2) S > P (total concentration of S must be larger than total 
concentration of protein or "binding sites")


Depending on the solubility of the compound, this is not always 
possible. In such a case, you need to use DMSO and/or add solid compound 
to the protein solution and leave it for quite some time for the 
compound to finally bind to the protein.


- J. -


yangliuqing wrote:

Hello,everyone,
I have a question for cocrystallization, is there some relationship 
between Km value and substrate concentration when making 
cocrystallization? How can I know the substrate is enough for binding?

Thank you very much!
liuqing


八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 

Re: [ccp4bb] Program to fill unitcell randomly

[Edward A. Berry]

Ethan A Merritt wrote:

On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:

Dear all,
does anybody know a program to
fill an unit cell a,b,c randomly by an arbitrary number
of spheres (atoms)?


First you would need to define "random".
 Uniform density throughout the lattice?
 Uniform distribution of neighbor-neighbor distances?
 Uniform fractional coodinates?
 Must the placement conform to space group symmetry?


Although I am sure it was not intended, this might suggest
to some that uniform is equivalent to random-
actually they are the opposite: a random distribution would
have large areas with nothing and other places where two or
three spheres are almost on top of each other.
A uniform distribution is, well, uniform.

Most programming languages have a function to generate a random
number evenly distributed between 0 and 1. Decide how many atoms
you want, get three random numbers for each atom, and those are
your fractional coordinates of your random spheres. Coordconv will
convert to orthogonal angstroms given your cell parameters.

Applesoft came with a BASIC program to do this in two dimensions-
start with a white screen and paint random pixels black for as
long as you let it run. Some black patches developed early on,
and some pixels remained white for hours, not because the random
number generator was faulty, but because that is the nature of
randomness.

Re: [ccp4bb] Program to fill unitcell randomly

[Ethan Merritt]

On Monday 01 December 2008 10:28:34 Edward A. Berry wrote:
> Ethan A Merritt wrote:
> > On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:
> >> Dear all,
> >> does anybody know a program to
> >> fill an unit cell a,b,c randomly by an arbitrary number
> >> of spheres (atoms)?
> > 
> > First you would need to define "random".
> >  Uniform density throughout the lattice?
> >  Uniform distribution of neighbor-neighbor distances?
> >  Uniform fractional coodinates?
> >  Must the placement conform to space group symmetry?
> > 
> Although I am sure it was not intended, this might suggest
> to some that uniform is equivalent to random-
> actually they are the opposite: a random distribution would
> have large areas with nothing and other places where two or
> three spheres are almost on top of each other.
> A uniform distribution is, well, uniform.

I fear you are muddying the waters rather than clarifying.
What you refer to as "random distribution" is better described
as random sampling from a uniform distribution.  

> Most programming languages have a function to generate a random
> number evenly distributed between 0 and 1. 

My point was that simple random sampling is not correct in the
context of crystallographic symmetry.  If you use this procedure to 
"fill the unit cell", as originally requested, you will violate
the crystal symmetry.  If you use it to fill the asymmetric unit,
then the distribution that describes placement within the full
unit cell is no longer the same distribution as you sampled from,
since it is now perturbed by the additional placements generated
by crystallographic symmetric rather than by random sampling.
That may be acceptable, or it may not, depending on the
intended application.

> Decide how many atoms 
> you want, get three random numbers for each atom, and those are
> your fractional coordinates of your random spheres. Coordconv will
> convert to orthogonal angstroms given your cell parameters.

That was the "uniform fractional coordinates" case that I listed.
It is unlikely to be the correct choice (although as always it depends
on the question). This problem is that since it is based on fractional
coordinates rather than the true cartesian coordinates, the resulting
density of atomic centers will be strongly anisotropic. The density
along each axis will be inversely proportional to the cell edge.
You would do better to define a cartesian coordinate grid that fills
the region of interest, and then assign an atom to each grid point with
probability 1/N. This produces artifacts of its own, of course, since
the distribution of interatomic distances is now discrete rather than
continuous.

The question "what is random?" is very deep, and the answer
depends strongly on the intended application.

-- 
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

[ccp4bb] Diffraction problems

[garima singh]

Hi All,
I am working on a 80 KDa single amino acid mutant of a protein expressed in
E.Coli. The protein is very pure and I get very nice looking crystals in
different conditions. I used different cryo protectants and tried room temp
testing of diffraction for these crystals. I don't get any diffraction at
all in most of them. A few were salt, so I was sure that there is no other
instrumental problem. If anyone has faced the same problem or someone can
give some insight about it other than the papers that suggest post
crystallization treatments , something that has worked for them, I would be
grateful

Thanks

Garima

[ccp4bb] Quikchange cloning: Insert length

[Raji Edayathumangalam]

Hi Folks,

Sorry for the non-xtallo posting.

I am curious to hear what is the longest insert anyone has cloned  
using a modification of the Quikchange cloning strategy. Basically,  
ligation-independent cloning by strapping on homologous regions of  
the vector onto the primers which also generate the initial PCR  
product. I plan to proceed with my insert which is ~ 2kb and am  
curious to get some feedback if you have successfully cloned inserts  
> 1.5kb using the above strategy.


Many thanks.
Raji

[ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

[Fischmann, Thierry]

Dear fellow crystallographers,

 

This is a question which is not CCP4-related.

 

Is anybody aware of a protein which is known to be a dimer in solution
(say by SEC), and yet crystallizes as a monomer? Wouldn't the high
concentration in the crystallization drop further favor dimerization?

 

In other words, if a protein crystallizes as a monomer, can I conclude
that it does not form biologically relevant dimers in solution?

 

Thank you in advance for your replies.

 

Thierry

 

*
This message and any attachments are solely for the
intended recipient. If you are not the intended recipient,
disclosure, copying, use or distribution of the information 
included in this message is prohibited -- Please 
immediately and permanently delete.

Re: [ccp4bb] Program to fill unitcell randomly

[Edward A. Berry]

Thanks, Ethan,
For your third point- I realized (after sending) that the distribution
would be stretched along the long axis- but actually I'm having
a hard time coming to grips with that conceptually- if there
are n atoms in the cell, they will necessarily be distributed
more sparsely in projection along the long cell axis than the
short axes, and you can't add more atoms along the long axis
to increase it's density without increasing density along the other two.

As for the rest, I think it is semantics or a question how precisely
we want to say something. Yes, what I was describing was a randomly
chosen sample from a uniform probability distribution, but it is this
sample that the OP is requesting- so I would rephrase your question:
does he want _a random sampling from_ a uniform probability distribution
throughout the lattice, or ...
Ed

Ethan Merritt wrote:

On Monday 01 December 2008 10:28:34 Edward A. Berry wrote:

Ethan A Merritt wrote:

On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:

Dear all,
does anybody know a program to
fill an unit cell a,b,c randomly by an arbitrary number
of spheres (atoms)?

First you would need to define "random".
 Uniform density throughout the lattice?
 Uniform distribution of neighbor-neighbor distances?
 Uniform fractional coodinates?
 Must the placement conform to space group symmetry?


Although I am sure it was not intended, this might suggest
to some that uniform is equivalent to random-
actually they are the opposite: a random distribution would
have large areas with nothing and other places where two or
three spheres are almost on top of each other.
A uniform distribution is, well, uniform.


I fear you are muddying the waters rather than clarifying.
What you refer to as "random distribution" is better described
as random sampling from a uniform distribution.  


Most programming languages have a function to generate a random
number evenly distributed between 0 and 1. 


My point was that simple random sampling is not correct in the
context of crystallographic symmetry.  If you use this procedure to 
"fill the unit cell", as originally requested, you will violate

the crystal symmetry.  If you use it to fill the asymmetric unit,
then the distribution that describes placement within the full
unit cell is no longer the same distribution as you sampled from,
since it is now perturbed by the additional placements generated
by crystallographic symmetric rather than by random sampling.
That may be acceptable, or it may not, depending on the
intended application.

Decide how many atoms 
you want, get three random numbers for each atom, and those are

your fractional coordinates of your random spheres. Coordconv will
convert to orthogonal angstroms given your cell parameters.


That was the "uniform fractional coordinates" case that I listed.
It is unlikely to be the correct choice (although as always it depends
on the question). This problem is that since it is based on fractional
coordinates rather than the true cartesian coordinates, the resulting
density of atomic centers will be strongly anisotropic. The density
along each axis will be inversely proportional to the cell edge.
You would do better to define a cartesian coordinate grid that fills
the region of interest, and then assign an atom to each grid point with
probability 1/N. This produces artifacts of its own, of course, since
the distribution of interatomic distances is now discrete rather than
continuous.

The question "what is random?" is very deep, and the answer
depends strongly on the intended application.

Re: [ccp4bb] co-crystallization

[Phoebe Rice]

Just to be a pedantic pain - Km is not necessarily Kd.  I
think that assumption only holds if the chemical step
following substrate binding is rate-limiting.
   Phoebe


 Original message 
>Date: Mon, 1 Dec 2008 15:34:59 +0100
>From: mesters <[EMAIL PROTECTED]>  
>Subject: Re: [ccp4bb] co-crystallization  
>To: CCP4BB@JISCMAIL.AC.UK
>
>Yes!, there is:
>
>the fraction of occupied protein with substance can be
calculated: S / 
>(S + Km) with S being the concentration of the compound.
>
>So, if S = Km, half of the sites are occupied (it follows from 
>Michaelis-Menten theory).
>
>In order to saturate the enzyme for 90,90909 % with the compound:
>
>1) S = 10 x Kd (concentration of S at least 10 times the Kd)
>and
>2) S > P (total concentration of S must be larger than total 
>concentration of protein or "binding sites")
>
>Depending on the solubility of the compound, this is not always 
>possible. In such a case, you need to use DMSO and/or add
solid compound 
>to the protein solution and leave it for quite some time for the 
>compound to finally bind to the protein.
>
>- J. -
>
>
>yangliuqing wrote:
>> Hello,everyone,
>> I have a question for cocrystallization, is there some
relationship 
>> between Km value and substrate concentration when making 
>> cocrystallization? How can I know the substrate is enough
for binding?
>> Thank you very much!
>> liuqing
>>
>>

>> 八卦娱乐包打听，MSN资讯速递帮你忙！ 了解详细！ 
>> 
>
>
>-- 
>Dr. Jeroen R. Mesters
>Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
>Institut für Biochemie, Universität zu Lübeck
>Zentrum für Medizinische Struktur- und Zellbiologie
>Ratzeburger Allee 160, D-23538 Lübeck
>Tel: +49-451-5004065, Fax: +49-451-5004068
>Http://www.biochem.uni-luebeck.de
>Http://www.iobcr.org
>Http://www.selfish-brain.org
>Http://www.opticryst.org
>--
>If you can look into the seeds of time and say
>which grain will grow and which will not - speak then to me 
(Macbeth)
>--
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them 
  both in one book
Please do take a 
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

[Fischmann, Thierry]

Thank you Patrick for the reply, as well to another person who has
replied directly to me.

 

Please provide with examples if you know of any (say a reference or a
PDB id ), as it would allow for comparison between published results and
my own crystallization system.

 

To answer your points:

 

The crystallization conditions in the case at hand are as follow : the
precipitant is PEG, the amount of salt is relatively low (0.1 M buffer +
some NaCl etc.), and the pH is 8.0. The SEC experimental conditions are
not too far away from the crystallization conditions except, of course,
for the presence of PEG.

 

Thierry

 

 

From: Patrick Loll [mailto:[EMAIL PROTECTED] 
Sent: Monday, December 01, 2008 05:59 PM
To: Fischmann, Thierry
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution
crystallize as a monomer?

 

depends on the crystallization conditions (and the SEC conditions).
E.g., if the crystals grow in 4 M salt at pH 3, maybe that disrupts a
complex that forms under more physiological conditions

 

 

On 1 Dec 2008, at 5:47 PM, Fischmann, Thierry wrote:





Dear fellow crystallographers,

 

This is a question which is not CCP4-related.

 

Is anybody aware of a protein which is known to be a dimer in solution
(say by SEC), and yet crystallizes as a monomer? Wouldn't the high
concentration in the crystallization drop further favor dimerization?

 

In other words, if a protein crystallizes as a monomer, can I conclude
that it does not form biologically relevant dimers in solution?

 

Thank you in advance for your replies.

 

Thierry

 

*
This message and any attachments are solely for the
intended recipient. If you are not the intended recipient,
disclosure, copying, use or distribution of the information 
included in this message is prohibited -- Please 
immediately and permanently delete.

 


---

Patrick J. Loll, Ph. D.   

Professor of Biochemistry & Molecular Biology

Director, Biochemistry Graduate Program

Drexel University College of Medicine

Room 10-102 New College Building

245 N. 15th St., Mailstop 497

Philadelphia, PA  19102-1192  USA

 

(215) 762-7706

[EMAIL PROTECTED]

 

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disclosure, copying, use or distribution of the information 
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Re: [ccp4bb] Quikchange cloning: Insert length

[Michael Giffin]

Have you seen these papers:

M Geiser, R Cebe, D Drewello, and R Schmitz. Integration of pcr
fragments at any specific site within
cloning vectors without the use of restriction enzymes and dna ligase.
Biotechniques, 31(1):88–90, 2001.

W Wang and B A Malcolm. Two-stage pcr protocol allowing introduction
of multiple mutations,
deletions and insertions using quikchange site-directed mutagenesis.
Biotechniques, 26(4):680–682, 1999.


If i recall correctly, Geiser el al inserted a >1kb fragment with a
modified Quickchange method.


On Mon, Dec 1, 2008 at 12:54 PM, Raji Edayathumangalam
<[EMAIL PROTECTED]> wrote:
> Hi Folks,
>
> Sorry for the non-xtallo posting.
>
> I am curious to hear what is the longest insert anyone has cloned using a
> modification of the Quikchange cloning strategy. Basically,
> ligation-independent cloning by strapping on homologous regions of the
> vector onto the primers which also generate the initial PCR product. I plan
> to proceed with my insert which is ~ 2kb and am curious to get some feedback
> if you have successfully cloned inserts > 1.5kb using the above strategy.
>
> Many thanks.
> Raji
>

Michael Giffin
The Scripps Research Institute
Department of Molecular and Experimental Medicine
10550 North Torrey Pines Road, MEM-131
La Jolla, CA 92037
email:  [EMAIL PROTECTED]
lab:  858-784-7758

Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

[William Scott]

Yo Thierry:

The periplasmic domain of the aspartate receptor, in the absence of  
ligand, 1lih, is a dimer, but crystallizes as a monomer in the sense  
that there is one monomer per asymmetric unit.  There is a disulphide  
bond between two Cys36 that maintains it as a dimer (and indeed  
reduction of this bond inhibits crystallization).  Each of two ligand  
binding sites spans both monomers. So based on that, the biologically  
relevant form is definitely a dimer, so you can't conclude otherwise  
based on the fact that it crystallizes as one monomer per asymmetric  
unit.  Now if it were to crystallize in a space group lacking a  
crystallographic 2-fold coincident with the natural dimer axis, that  
might be a different story.


When you add aspartate, it crystallizes as a dimer with only one of  
two potential binding sites occupied by the ligand.


Bill



William G. Scott

Contact info:
http://chemistry.ucsc.edu/~wgscott/

On Dec 1, 2008, at 2:47 PM, Fischmann, Thierry wrote:


Dear fellow crystallographers,



This is a question which is not CCP4-related.



Is anybody aware of a protein which is known to be a dimer in solution
(say by SEC), and yet crystallizes as a monomer? Wouldn't the high
concentration in the crystallization drop further favor dimerization?



In other words, if a protein crystallizes as a monomer, can I conclude
that it does not form biologically relevant dimers in solution?



Thank you in advance for your replies.



Thierry



*
This message and any attachments are solely for the
intended recipient. If you are not the intended recipient,
disclosure, copying, use or distribution of the information
included in this message is prohibited -- Please
immediately and permanently delete.

Re: [ccp4bb] Quikchange cloning: Insert length

[Dima Klenchin]

I am curious to hear what is the longest insert anyone has cloned
using a modification of the Quikchange cloning strategy. Basically,
ligation-independent cloning by strapping on homologous regions of
the vector onto the primers which also generate the initial PCR
product. I plan to proceed with my insert which is ~ 2kb and am
curious to get some feedback if you have successfully cloned inserts
> 1.5kb using the above strategy.


Here, the maximum we've tried was 3 kbp and it worked just as well as with 
the the smaller fragments. Most of the Quickchange cloning we did involved 
1500-300 bp and no obvious differences in success rate vs size come to mind.


Dima

Re: [ccp4bb] Quikchange cloning: Insert length

[Artem Evdokimov]

It helps to remember that PCR does have an upper limit of total
double-stranded DNA content (regardless of its molarity!) after which it
does not work any more (due to the competition of the polymerase for
non-specific dsDNA versus primer-substrate pairs).

Therefore the theoretical limits on this form of cloning are imposed by the
molarity of two DNA fragments, the fidelity/processivity of your PCR enzyme,
and the quality of the reaction mix. In practice it helps to also consider
the frequency of PCR errors as well as certain other factors. 

I've done this sort of cloning with ~4 KB inserts, however the use of such
long inserts required optimization of conditions and was not 100% successful
(unlike the relatively simple insertion of 0.5-1.5 KB).

With long inserts it is *critical* to have highly purified and highly
homogenous starter DNA as well as (individually selectable) a correct
template/insert ratio.

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Raji
Edayathumangalam
Sent: Monday, December 01, 2008 3:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Quikchange cloning: Insert length

Hi Folks,

Sorry for the non-xtallo posting.

I am curious to hear what is the longest insert anyone has cloned  
using a modification of the Quikchange cloning strategy. Basically,  
ligation-independent cloning by strapping on homologous regions of  
the vector onto the primers which also generate the initial PCR  
product. I plan to proceed with my insert which is ~ 2kb and am  
curious to get some feedback if you have successfully cloned inserts  
 > 1.5kb using the above strategy.

Many thanks.
Raji

Re: [ccp4bb] Program to fill unitcell randomly

[Ethan Merritt]

On Monday 01 December 2008 15:07:56 Edward A. Berry wrote:
> Thanks, Ethan,
> For your third point- I realized (after sending) that the distribution
> would be stretched along the long axis- but actually I'm having
> a hard time coming to grips with that conceptually- if there
> are n atoms in the cell, they will necessarily be distributed
> more sparsely in projection along the long cell axis than the
> short axes, and you can't add more atoms along the long axis
> to increase it's density without increasing density along the other two.

Heh. A new "Monty Hall" problem to demonstrate how probability
distributions mess with our minds.

You are of course correct that you cannot increase the number of
atoms in a fixed cell without increasing the density in all directions
through the crystal. And I phrased my original description badly,
if I made it sound like somehow this was possible.
I should not have used the word "density"; perhaps
"mean distance between particles along the path" is a better wording.

The main point I was trying to make is that if you ask for a 
"random distribution in 3-space" without mentioning crystals,
you are probably expecting certain properties. In particular,
unless otherwise stated, the criteria for "random" would
normally include isotropy. Or at least that's what I would assume.  
A distribution that had significantly different properties in
different directions would not be considered "random" in this
context.

But a crystal lattice is the antithesis of "random" in this 
sense, because it imposes by definition a requirement for an
exact direction-dependent repeat spacing determined by the lattice.

You cannot simultaneously satisfy a requirement for uniform
isotropic distribution in 3-space and a requirement for
crystalline symmetry, except in the degenerate case of density = 0.


> As for the rest, I think it is semantics or a question how precisely
> we want to say something. Yes, what I was describing was a randomly
> chosen sample from a uniform probability distribution, but it is this
> sample that the OP is requesting- so I would rephrase your question:
> does he want _a random sampling from_ a uniform probability distribution
> throughout the lattice, or ...
> Ed
> 
> Ethan Merritt wrote:
> > On Monday 01 December 2008 10:28:34 Edward A. Berry wrote:
> >> Ethan A Merritt wrote:
> >>> On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:
>  Dear all,
>  does anybody know a program to
>  fill an unit cell a,b,c randomly by an arbitrary number
>  of spheres (atoms)?
> >>> First you would need to define "random".
> >>>  Uniform density throughout the lattice?
> >>>  Uniform distribution of neighbor-neighbor distances?
> >>>  Uniform fractional coodinates?
> >>>  Must the placement conform to space group symmetry?
> >>>
> >> Although I am sure it was not intended, this might suggest
> >> to some that uniform is equivalent to random-
> >> actually they are the opposite: a random distribution would
> >> have large areas with nothing and other places where two or
> >> three spheres are almost on top of each other.
> >> A uniform distribution is, well, uniform.
> > 
> > I fear you are muddying the waters rather than clarifying.
> > What you refer to as "random distribution" is better described
> > as random sampling from a uniform distribution.  
> > 
> >> Most programming languages have a function to generate a random
> >> number evenly distributed between 0 and 1. 
> > 
> > My point was that simple random sampling is not correct in the
> > context of crystallographic symmetry.  If you use this procedure to 
> > "fill the unit cell", as originally requested, you will violate
> > the crystal symmetry.  If you use it to fill the asymmetric unit,
> > then the distribution that describes placement within the full
> > unit cell is no longer the same distribution as you sampled from,
> > since it is now perturbed by the additional placements generated
> > by crystallographic symmetric rather than by random sampling.
> > That may be acceptable, or it may not, depending on the
> > intended application.
> > 
> >> Decide how many atoms 
> >> you want, get three random numbers for each atom, and those are
> >> your fractional coordinates of your random spheres. Coordconv will
> >> convert to orthogonal angstroms given your cell parameters.
> > 
> > That was the "uniform fractional coordinates" case that I listed.
> > It is unlikely to be the correct choice (although as always it depends
> > on the question). This problem is that since it is based on fractional
> > coordinates rather than the true cartesian coordinates, the resulting
> > density of atomic centers will be strongly anisotropic. The density
> > along each axis will be inversely proportional to the cell edge.
> > You would do better to define a cartesian coordinate grid that fills
> > the region of interest, and then assign an atom to each grid point with
> > probability 1/N. This produces artifacts of it

Re: [ccp4bb] Program to fill unitcell randomly

[Daniel Anderson]

(I don't remember the motivation for the original question.)

Shake-and-Bake used to generate random atoms in an asymmetric unit, and 
the program kept the atoms spaced by at least a bond length. Since
PDB entry 2erl, I am not up to date on Shake-and-Bake's current set of 
tricks.


The crystal for 2erl was so densely packed that random atoms spaced by 
1.5A produced very good starting phase sets. (but I still don't know 
what's the motivation underlying the current discussion.)


Did that help?,
Dan



On Mon, 1 Dec 2008, Ethan Merritt wrote:


On Monday 01 December 2008 10:28:34 Edward A. Berry wrote:

Ethan A Merritt wrote:

On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:

Dear all,
does anybody know a program to
fill an unit cell a,b,c randomly by an arbitrary number
of spheres (atoms)?


First you would need to define "random".
 Uniform density throughout the lattice?
 Uniform distribution of neighbor-neighbor distances?
 Uniform fractional coodinates?
 Must the placement conform to space group symmetry?


Although I am sure it was not intended, this might suggest
to some that uniform is equivalent to random-
actually they are the opposite: a random distribution would
have large areas with nothing and other places where two or
three spheres are almost on top of each other.
A uniform distribution is, well, uniform.


I fear you are muddying the waters rather than clarifying.
What you refer to as "random distribution" is better described
as random sampling from a uniform distribution.


Most programming languages have a function to generate a random
number evenly distributed between 0 and 1.


My point was that simple random sampling is not correct in the
context of crystallographic symmetry.  If you use this procedure to
"fill the unit cell", as originally requested, you will violate
the crystal symmetry.  If you use it to fill the asymmetric unit,
then the distribution that describes placement within the full
unit cell is no longer the same distribution as you sampled from,
since it is now perturbed by the additional placements generated
by crystallographic symmetric rather than by random sampling.
That may be acceptable, or it may not, depending on the
intended application.


Decide how many atoms
you want, get three random numbers for each atom, and those are
your fractional coordinates of your random spheres. Coordconv will
convert to orthogonal angstroms given your cell parameters.


That was the "uniform fractional coordinates" case that I listed.
It is unlikely to be the correct choice (although as always it depends
on the question). This problem is that since it is based on fractional
coordinates rather than the true cartesian coordinates, the resulting
density of atomic centers will be strongly anisotropic. The density
along each axis will be inversely proportional to the cell edge.
You would do better to define a cartesian coordinate grid that fills
the region of interest, and then assign an atom to each grid point with
probability 1/N. This produces artifacts of its own, of course, since
the distribution of interatomic distances is now discrete rather than
continuous.

The question "what is random?" is very deep, and the answer
depends strongly on the intended application.




--
***
Daniel Anderson, Ph.D.
Email: [EMAIL PROTECTED]
Phone: 310-206-3642 Fax: 310-206-3914

Howard Hughes Medical Institute at
University of California Los Angeles

Lab: Paul Boyer Hall Room 219
For US Postal Service and 2-dimensional, use:
 Box 951662 MRL5-748
 Los Angeles, CA 90095-1662
For UPS, FedEx, DHL, or 2.5-3-dimensional, use:
 Boyer 219
 611 Charles Young Drive East
 Los Angeles, CA 90095-1570
 USA

[ccp4bb] Offtopic: FAD enzymatic assay

[michael nelson]

 was working to set up an FAD enzymatic assay. I wished to be able to use 450nM 
to continuously monitor the progress of the reaction. The substrate I used is 
the natural substrate of the enzyme and the protein is recombinant protein and 
I assume it's active since I do see changes in TLC plate. But no signal was 
observed at all using a spectrometer. Does anyone here have any suggestions on 
how to correctly carry out FAD enzymatic assay? 

Also I heard from one postdoc here that FADH2 is quickly reoxidized by O2. Is 
that right? Should I do the assay under anaerobic condition? If so, how? 

Your help is highly appreciated!

Thanks!

Best regards,

Mike




  


  

Re: [ccp4bb] Offtopic: FAD enzymatic assay

[conancao]

Mike:
 
  The trick may be doing the assay under anaerobic condition, especially if 
the FAD cofactor is sensitive to oxygen. 
  You need an anaerobic train and tanometers for the experiment. 
  Good refs: Hille, R. Biochemistry 1991 Sep 3;30(35):8522-9. Electron 
transfer within xanthine oxidase: a solvent kinetic isotope effect study. 
 Hunt, J., Massey, V.  J. Biol. Chem. 1994 Jul 
22;269(29):18904-14 Studies of the reductive half-reaction of milk xanthine 
dehydrogenase. 
 
Cheers,
Hongnan Cao
UC Riverside



Date: Mon, 1 Dec 2008 18:07:57 -0800From: [EMAIL PROTECTED]: [ccp4bb] Offtopic: 
FAD enzymatic assayTo: CCP4BB@JISCMAIL.AC.UK









was working to set up an FAD enzymatic assay. I wished to be able to use 450nM 
to continuously monitor the progress of the reaction. The substrate I used is 
the natural substrate of the enzyme and the protein is recombinant protein and 
I assume it's active since I do see changes in TLC plate. But no signal was 
observed at all using a spectrometer. Does anyone here have any suggestions on 
how to correctly carry out FAD enzymatic assay? Also I heard from one postdoc 
here that FADH2 is quickly reoxidized by O2. Is that right? Should I do the 
assay under anaerobic condition? If so, how? Your help is highly 
appreciated!Thanks!Best regards,Mike
_
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