Re: [ccp4bb] decrease of background with distance?

[Jim Pflugrath]

The source for the X-ray background are points along the air path
post-collimator including the sample with loop and cryoprotecdant (or
capillary and mother liquor).  So the 1/r^2 falloff is noticable going from
100 mm to 200 mm.  The same counts in a 2x2 pixel area is now seen in a 4x4
pixel area.

The source for Bragg reflections at a synchtrotron is upstream a couple
dozen meters.  The divergence is not large as well, so the spread in the
spots (for a source ~30 meters upstream) goes from 1/(30.1 * 30.1)^2 to
1/(30.2 * 30.2)^2 which is really not that noticable.

In contrast, a modern home lab system with Osmic multilayer optics, the
source is much closer to the sample and the divergence is larger.  For folks
with home lab systems, you can see your spots getting larger as you increase
your distance.  The total background under the (sum of all) pixels of the
Bragg reflections does not change that much with changing distance.  It's
just that everything (spot & background) are spread out over more pixels.
Thus moving the detector backwards will not do the same thing that it does
for you at a beamline.  Folks with such home lab systems can do the
experiment to see this for themselves.

So when your synchtrotron buddies talk about all this, be sure to buy them
beers to help with their thinking. :)

Jim

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of James
Holton
Sent: Tuesday, November 24, 2009 1:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] decrease of background with distance?

Spots don't fall off with the inverse square law.  It is a very easy
experiment to do.  Just take exposures at several distances and scale the
data together, noting the correction for air absorption. 

A good reference for the underlying theory is Chapter 6 of M. M. 
Woolfson's book (1997).  But briefly: Diffuse scattering falls off with the
inverse square law as one would naturally expect from anything that spreads
out in all directions.  Spots, however, are "reflections" and their source
point is the source of the incident beam, which is usually far away or at
worst comparable to the sample-to-detector distance.

For those who do not like my "long posts" I now direct you to the "delete"
button which should have come with your email client.  Everyone else, read
on.


...

It is an interesting point, however, that simultaneously increasing the
detector distance and increasing the photon energy to keep the resolution at
the edge of the detector constant has absolutely NO effect on the
photons/spot from Bragg vs diffuse scattering.  It took me a while to become
convinced of this, and I still owe Colin Nave a few beers as a result.
Nevertheless, the experimental evidence was given by Gonzalez, Denny & Nave
in 1994.

HTH

-James Holton
MAD Scientist

Richard Gillilan wrote:
> It seems to be widely known and observed that diffuse background 
> scattering decreases more rapidly with increasing detector-to-sample 
> distance than Bragg reflections. For example, Jim Pflugrath, in his
> 1999 paper (Acta Cryst 1999 D55 1718-1725) says "Since the X-ray 
> background falls off as the square of the distance, the expectation is 
> that a larger crystal-to-detector distance is better for reduction of 
> the x-ray background. ..."
>
> Does anyone know of a more rigorous discussion of why background 
> scatter fades while Bragg reflections remain collimated with distance?
>
>
> Richard Gillilan
> MacCHESS

[ccp4bb] Methylation of macromolecular complexes

[Jan Rash]

Hi all,
 I have a question regarding the methylation of macromolecular complexes. Is
there any report where a macromolecular complex has been successfully
methylated and later on crystallized. Is methylation a method of choice to
chemically modify macromolecular complexes where the entropical
contributions by lysines might be a cause for not obtaining crystals, or is
there any other better ways?

thanks,
Jan Rash

Re: [ccp4bb] decrease of background with distance?

[Colin Nave]

 Jim's point is well taken and can perhaps be generalised to say one is
limited either by the crystal or by the set up for recording the
diffraction. To optimise things it is useful to know where the limits
are.

With some tightly focused synchrotron beams you can also see your spots
getting larger as you increase the detector distance because the focused
beam can have a high divergence. By having a low divergence, one can
gain on spot to background ratio by moving the detector further away. To
fully exploit this, a larger detector (more angular resolution elements)
is needed. The gain of having a lower divergence beam and larger
detector will continue until dominated by the divergence introduced by
the crystal (James Holton has probably covered this somewhere in his
emails - I only read the bit about the beer!).

One could however go further as the divergence due to the crystal will
not be smooth but will contain some structure describing the crystal
disorder (e.g. domain structure). By matching the beam divergence and
detector angular resolution to this structure one should be able to
record this and get a still better ratio of spot intensity to
background.

The limit is to illuminate the crystal with a coherent beam and record
the scattering with an angular resolution appropriate to the whole
crystal. One would then reconstruct the crystal from the oversampled
scattering and average the unit cell contents in real space. It would
allow one to properly handle disorder and "background" from the crystal.

Most people would say that this last suggestion is way in the future and
perhaps not worth doing. However, it is being investigated by those
interested in looking at sub micron (nano) crystals with coherent x-ray
sources.

  Colin
  

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Jim Pflugrath
Sent: 24 November 2009 15:25
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] decrease of background with distance?

The source for the X-ray background are points along the air path
post-collimator including the sample with loop and cryoprotecdant (or
capillary and mother liquor).  So the 1/r^2 falloff is noticable going
from 100 mm to 200 mm.  The same counts in a 2x2 pixel area is now seen
in a 4x4 pixel area.

The source for Bragg reflections at a synchtrotron is upstream a couple
dozen meters.  The divergence is not large as well, so the spread in the
spots (for a source ~30 meters upstream) goes from 1/(30.1 * 30.1)^2 to
1/(30.2 * 30.2)^2 which is really not that noticable.

In contrast, a modern home lab system with Osmic multilayer optics, the
source is much closer to the sample and the divergence is larger.  For
folks with home lab systems, you can see your spots getting larger as
you increase your distance.  The total background under the (sum of all)
pixels of the Bragg reflections does not change that much with changing
distance.  It's just that everything (spot & background) are spread out
over more pixels.
Thus moving the detector backwards will not do the same thing that it
does for you at a beamline.  Folks with such home lab systems can do the
experiment to see this for themselves.

So when your synchtrotron buddies talk about all this, be sure to buy
them beers to help with their thinking. :)

Jim

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
James Holton
Sent: Tuesday, November 24, 2009 1:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] decrease of background with distance?

Spots don't fall off with the inverse square law.  It is a very easy
experiment to do.  Just take exposures at several distances and scale
the data together, noting the correction for air absorption. 

A good reference for the underlying theory is Chapter 6 of M. M. 
Woolfson's book (1997).  But briefly: Diffuse scattering falls off with
the inverse square law as one would naturally expect from anything that
spreads out in all directions.  Spots, however, are "reflections" and
their source point is the source of the incident beam, which is usually
far away or at worst comparable to the sample-to-detector distance.

For those who do not like my "long posts" I now direct you to the
"delete"
button which should have come with your email client.  Everyone else,
read on.


...

It is an interesting point, however, that simultaneously increasing the
detector distance and increasing the photon energy to keep the
resolution at the edge of the detector constant has absolutely NO effect
on the photons/spot from Bragg vs diffuse scattering.  It took me a
while to become convinced of this, and I still owe Colin Nave a few
beers as a result.
Nevertheless, the experimental evidence was given by Gonzalez, Denny &
Nave in 1994.

HTH

-James Holton
MAD Scientist

Richard Gillilan wrote:
> It seems to be widely known and observed that diffuse background 
> scattering decreases more rapidly with increasing detector-to-sample 
> distance than

Re: [ccp4bb] Methylation of macromolecular complexes

[Tim]

Jan Rash wrote:
> Hi all,
>  I have a question regarding the methylation of macromolecular
> complexes. Is there any report where a macromolecular complex has been
> successfully methylated and later on crystallized. Is methylation a
> method of choice to chemically modify macromolecular complexes where
> the entropical contributions by lysines might be a cause for not
> obtaining crystals, or is there any other better ways?
>
> thanks,
> Jan Rash
You could also try to use the surface entropy reduction server
(http://nihserver.mbi.ucla.edu/SER/) for identification of sites that
are most suitable for mutation designed to enhance crystallizability.
Methylation is probably more invasive.

-- 
Tim Schulte
Ruhr-Universität Bochum
LS Biophysik 
AG Proteinkristallographie
Universitätsstr. 150
44780 Bochum
Germany
Tel. +49-234-32-25754

Re: [ccp4bb] Methylation of macromolecular complexes

[Jan Rash]

Hi all,
What I really want to know is whether macromolecular complexes ( more than
200kDa) can tolerate methylation?
thanks,
 Jan  Rash

2009/11/24 Hüsnü Topal 

>  Hi Jan,
>
>
>
> Maybe this link
> http://www.jenabioscience.com/cms/en/1/catalog/529_jbs_methylation_kit.htmlor 
> these references helps…
>
>
>
> References
> [1] Kim *et al. *(2008) Large-scale evaluation of protein reductive
> methylation for improving protein crystallization. *Nature Methods* *5*,
> 853.
>
> [2] Fogg *et al*. (2006) Application of the use of high-throughput
> technologies to the determination of protein structures of bacterial and
> viral pathogens. *Acta Cryst*. D*62*:1196.
>
> [3] Walter *et al*. (2006) Lysine methylation as a routine rescue strategy
> for protein crystallization. *Structure* *14*:1617.
>
> I’m not sure if this answers your question. Good luck.
>
>
>
> best regards,
>
> Hüsnü  Topal
>
> Ruhr-Universität Bochum
> Institut für Physiologische Chemie, MA 2/141
> Abteilung für Biochemie supramolekularer Systeme
> Universitätsstr. 150
>
> 44801 Bochum, Germany
>
> phone: +49 (0) 234 32 24934
>
>  Internet: http://www.ruhr-uni-bochum.de/physiolchem/steegborn/
>
> Diese E-Mail enthält vertrauliche und/oder rechtlich geschützte
> Informationen. Wenn Sie nicht der richtige Adressat sind oder diese E-Mail
> irrtümlich erhalten haben, informieren Sie bitte sofort den Absender und
> vernichten Sie diese Mail. Das unerlaubte Kopieren sowie die unbefugte
> Weitergabe dieser Mail ist nicht gestattet.
>
> This e-mail may contain confidential and/or privileged information. If you
> are not the intended recipient (or have received this e-mail in error)
> please notify the sender immediately and destroy this e-mail. Any
> unauthorized copying, disclosure or distribution of the material in this
> e-mail is strictly forbidden.
>
> J *Go green* Bitte denken Sie an die Umwelt und den Klimaschutz und
> drucken diese E-Mail nur aus, wenn unbedingt notwendig! Please consider
> the environment before printing this email!
>
>
>   --
>
> *Von:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *Im Auftrag von
> *Jan Rash
> *Gesendet:* Dienstag, 24. November 2009 18:28
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [ccp4bb] Methylation of macromolecular complexes
>
>
>
> Hi all,
>  I have a question regarding the methylation of macromolecular complexes.
> Is there any report where a macromolecular complex has been successfully
> methylated and later on crystallized. Is methylation a method of choice to
> chemically modify macromolecular complexes where the entropical
> contributions by lysines might be a cause for not obtaining crystals, or is
> there any other better ways?
>
> thanks,
> Jan Rash
>
<>

[ccp4bb] AW: [ccp4bb] Methylation of macromolecular complexes

[Hüsnü Topal]

Hi Jan,

 

Maybe this link

http://www.jenabioscience.com/cms/en/1/catalog/529_jbs_methylation_kit.html
or these references helps…

 

References 
[1] Kim et al. (2008) Large-scale evaluation of protein reductive
methylation for improving protein crystallization. Nature Methods 5, 853. 

[2] Fogg et al. (2006) Application of the use of high-throughput
technologies to the determination of protein structures of bacterial and
viral pathogens. Acta Cryst. D62:1196. 

[3] Walter et al. (2006) Lysine methylation as a routine rescue strategy for
protein crystallization. Structure 14:1617.

I’m not sure if this answers your question. Good luck.

 

best regards,

Hüsnü  Topal

Ruhr-Universität Bochum
Institut für Physiologische Chemie, MA 2/141
Abteilung für Biochemie supramolekularer Systeme
Universitätsstr. 150

44801 Bochum, Germany

phone: +49 (0) 234 32 24934



Internet:  
http://www.ruhr-uni-bochum.de/physiolchem/steegborn/

Diese E-Mail enthält vertrauliche und/oder rechtlich geschützte
Informationen. Wenn Sie nicht der richtige Adressat sind oder diese E-Mail
irrtümlich erhalten haben, informieren Sie bitte sofort den Absender und
vernichten Sie diese Mail. Das unerlaubte Kopieren sowie die unbefugte
Weitergabe dieser Mail ist nicht gestattet.

This e-mail may contain confidential and/or privileged information. If you
are not the intended recipient (or have received this e-mail in error)
please notify the sender immediately and destroy this e-mail. Any
unauthorized copying, disclosure or distribution of the material in this
e-mail is strictly forbidden.

J Go green Bitte denken Sie an die Umwelt und den Klimaschutz und drucken
diese E-Mail nur aus, wenn unbedingt notwendig! Please consider the
environment before printing this email!

 

  _  

Von: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] Im Auftrag von Jan
Rash
Gesendet: Dienstag, 24. November 2009 18:28
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Methylation of macromolecular complexes

 

Hi all,
 I have a question regarding the methylation of macromolecular complexes. Is
there any report where a macromolecular complex has been successfully
methylated and later on crystallized. Is methylation a method of choice to
chemically modify macromolecular complexes where the entropical
contributions by lysines might be a cause for not obtaining crystals, or is
there any other better ways?

thanks,
Jan Rash

<>

[ccp4bb] Age Concern edition of the Computing Commission and Teaching Commission newsletter

[Ralf W. Grosse-Kunstleve]

Posted on behalf of Lachlan Cranswick:

The Computing Commission and Teaching Commission has produced a joint
November 2009 newsletter on the theme of "Age Concern", which deals with
issue concerning "the slow march towards retirement of the major
generation of crystallographic programmers", as further explained by
David Watkin within the newsletter.

 
http://www.iucr.org/resources/commissions/crystallographic-computing/newsletters/10

or via

  
http://www.iucr.org/resources/commissions/crystallographic-teaching/newsletters/3

---

This issue's articles include :

- the latest background and information from the UK based EPSRC funded
Age Concern Project.

- history and science within the Caltech based CRYRM single crystal
suite; DIMS - Direct methods In Multidimensional Space software; Crunch
1.5 Direct Methods Software; the Glasgow based GX single crystal suite;
the Italian based CAOS structure refinement software; Ton Spek's Platon;
and a list of programs archived at Armel Le Bail's Crystallography
Source Code Museum.

- experience in conversion of large crystallographic Fortran-77 to C++;
as well as the place of the Quick and Dirty Crystallographic Computer
Program

- Historical group photographs from some of the Crystallographic
Computing and Teaching School of the 1970's.

- Archived manuals and source code from the above mentioned software.

- Teaching material of Isabella L. Karle, as she presented at the 1978
Erice School on Direct Methods for Solving Crystal Structures

- A reprint of the The Analytical Theory of Point Systems (1923) by J.
D. Bernal (1901-1971)

Further Computing Commission newsletters are intended on theme of Age
Concern : capturing the history and the crystallographic science behind
past and present Small Molecule, Powder Diffraction, Protein and other
crystallographic software. Submissions on this topic are welcome and
encouraged.

  -  Lachlan Cranswick  ( lachlan.cransw...@nrc.gc.ca )

Re: [ccp4bb] Methylation of macromolecular complexes

[Li Chan]

 Hi all,

 

I have a small complex, one component is 13 kDa with structure available and 
the other is 7 kDa, which could not be able to grew crystals after lots of 
efforts. It grew crystals after methylation and diffracted to 2.3 A, however, I 
could not be able to solve the structure using the structure of the big 
component as a template for molecular replacement, and heavy atoms soaking was 
not successful. I plan to do selenomethionine expression next. Does the 
methylation change protein structures a lot? otherwise, why does molecular 
replacement not work?  I would much appreciate any idea and suggestions how to 
solve the structure using the data and the template avaliable.

 

Many thanks for your help.

 

Peter


Date: Tue, 24 Nov 2009 18:27:49 +0100
From: jan...@googlemail.com
Subject: [ccp4bb] Methylation of macromolecular complexes
To: CCP4BB@JISCMAIL.AC.UK

Hi all,
 I have a question regarding the methylation of macromolecular complexes. Is 
there any report where a macromolecular complex has been successfully 
methylated and later on crystallized. Is methylation a method of choice to 
chemically modify macromolecular complexes where the entropical contributions 
by lysines might be a cause for not obtaining crystals, or is there any other 
better ways?

thanks,
Jan Rash
  
_
Keep your friends updated―even when you’re not signed in.
http://www.microsoft.com/middleeast/windows/windowslive/see-it-in-action/social-network-basics.aspx?ocid=PID23461::T:WLMTAGL:ON:WL:en-xm:SI_SB_5:092010

Re: [ccp4bb] Methylation of macromolecular complexes

[Eric Larson]

Hi Peter,

How do you know you have crystallized the complex?  Perhaps MR is not working 
because your crystals only contain the small protein.

good luck,

Eric



 Hi all,
 
I have a small complex, one component is 13 kDa with structure available and
the other is 7 kDa, which could not be able to grew crystals after lots of
efforts. It grew crystals after methylation and diffracted to 2.3 A,
however, I could not be able to solve the structure using the structure of
the big component as a template for molecular replacement, and heavy atoms
soaking was not successful. I plan to do selenomethionine expression
next. Does the methylation change protein structures a lot? otherwise, why
does molecular replacement not work?  I would much appreciate any idea and
suggestions how to solve the structure using the data and the template
avaliable.
 
Many thanks for your help.
 
Peter


Date: Tue, 24 Nov 2009 18:27:49 +0100
From: jan...@googlemail.com
Subject: [ccp4bb] Methylation of macromolecular complexes
To: CCP4BB@JISCMAIL.AC.UK

Hi all,
 I have a question regarding the methylation of macromolecular complexes. Is
there any report where a macromolecular complex has been successfully
methylated and later on crystallized. Is methylation a method of choice to
chemically modify macromolecular complexes where the entropical
contributions by lysines might be a cause for not obtaining crystals, or is
there any other better ways?

thanks,
Jan Rash


Keep your friends updated— even when you’re not signed in.

Re: [ccp4bb] Methylation of macromolecular complexes

[Sean Seaver]

>I have a small complex, one component is 13 kDa with structure available
and the other is 7 kDa, which could not be able to grew crystals after lots
of efforts. It grew crystals after methylation and diffracted to 2.3 A,
however, I could not be able to solve the structure using the structure of
the big component as a template for molecular replacement, and heavy atoms
soaking was not successful. I plan to do selenomethionine expression next.
Does the methylation change protein structures a lot? otherwise, why does
molecular replacement not work?  I would much appreciate any idea and
suggestions how to solve the structure using the data and the template
avaliable.

---
A couple of questions that I would consider in regards to the molecular
replacement not working:

Is the binding between the proteins 1:1?  
Does the Matthews coefficient reflect this?

MR maybe difficult if you have a number of 7 kDa proteins binding to a 13
kDa protein.

Hope that Helps,

Sean

[ccp4bb] Help request: Failed MR using the same molecule as model

[Chao Quan]

Dear CCP4 community:

I am a beginner to crystallography and therefore my apologies if this question 
is too 
simple.

Basically we obtained several crystal forms of the same molecule, which is a 
hetero-
trimer containing protein A(18kD), protein B(16kD) and a RNA segment(40nt or 
about 
15kD).

We have solved the structure of one crystal form(form_1); its information is as 
follows:
space group = P 42 22;
unit cell = 126.514  126.514   76.766  90.00  90.00  90.00
resolution = 2.7A;
Rwork = 0.25, Rfree = 0.265;
solvent content = 60%;
Number of molecules per Asymmetric Unit = 1;
Data redundancy = 5;
Data Completeness = 94%;

I am now trying to solve the structure of form_2 crystal using molecular 
replacement. So 
far the information I know about form_2 is as follows:
space group = I 422 or I 41 22;
unit cell = 180.096  180.096   152.530  90.00  90.00  90.00
(unit cell is about 4 times the size of form_1)
resolution = 3.3A; (which is low)
Number of molecules per Asymmetric Unit = 2 or 3;
Data redundancy = 4;
Data Completeness = 92%;
There is no twinning(as shown by Sfcheck);
As shown in"Analyse Data for MR", the first peak is 100 and second is 68.92; I 
am not 
sure if this indicates translation in a Asymmetric Unit;

The problem is, I can not get a good solution by MR using Phaser (both I422 and 
I4122 
are tried). When I searched for 3 molecules per Asymmetric Unit, Phaser did not 
give 
solutions at all. 

When I used 2/ASU instead, I was able to get some solutions, with typical 
statistics as 
follows:
RFZ=14, TFZ=35.9, PAK=0, LLG=1036; 
However, these solutions had high R values(like Rwork=0.59 and Rfree=0.58), 
which 
indicated that they are not solutions at all. Still, I tried refinement using 
refmac5, but R 
values did not go down even after 50 rounds; sometimes they even increased 
after 
refinement.

Besides, the RMS values bond length, bond angle and chiral center were all 0 as 
show by 
refmac5.

I tried limiting resolution range to 15-4A in Phaser, which did not help either.

Now I am completely stuck. Could anyone give me some advice? I know this 
situation is 
very strange, because I am using the SAME molecule for MR but can not can a 
solution.

Thanks a lot,

P.S. 
1) Both form_1 and form_2 crystals were grown using Selenomethionine-containing 
samples. There are 3 Sel_Met in protein A and 1 in protein B. 
2) A 10-aa internal segment of protein B is missing in the solved structure, 
which may 
indicate high flexibility.

Chao

Re: [ccp4bb] Methylation of macromolecular complexes

[mjvdwoerd]

 There is a reasonable experiment you can do: 

Take the unit cell of your (presumed) crystal complex and put the search model 
into the until cell in a way that seems reasonable (use coot, or your favorite 
program). Make structure factors (with CCP4 or your favorite program) and throw 
away the phases. Then rotate your MR search model randomly and put the search 
model and structure factors into the MR program. See if it finds a solution. 
You can repeat it with structure factor data to which noise has been added. You 
can use this method to optimize your MR search parameters.

Don't be surprised if you cannot find a MR solution at all (even if you know 
for sure it should exist in your artificial problem).

Once upon a time I tried to solve the structure of a small poly-peptide and 
with the method above I proved to myself that it was not possible to find a 
solution (with the programs that were available at that time, when Dinosaurs 
still roamed the Earth). I found that MR is very finicky when applied to small 
peptides. At least you will be able to determine what the optimal parameters 
are (resolution, search radius) and whether it can succeed at all. 

To pursue Se-Met is smart. Small peptide crystals resist heavy atom soaking. If 
your resolution is high enough, you can also try direct methods. On such a 
small peptide that should be easy (provided your crystals are well-behaved). 
Frequently the diffraction resolution of small peptide crystals is high enough 
that direct methods work very well.

Mark

 

 

-Original Message-
From: Sean Seaver 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tue, Nov 24, 2009 5:13 pm
Subject: Re: [ccp4bb] Methylation of macromolecular complexes


>I have a small complex, one component is 13 kDa with structure available
and the other is 7 kDa, which could not be able to grew crystals after lots
of efforts. It grew crystals after methylation and diffracted to 2.3 A,
however, I could not be able to solve the structure using the structure of
the big component as a template for molecular replacement, and heavy atoms
soaking was not successful. I plan to do selenomethionine expression next.
Does the methylation change protein structures a lot? otherwise, why does
molecular replacement not work?  I would much appreciate any idea and
suggestions how to solve the structure using the data and the template
avaliable.

---
A couple of questions that I would consider in regards to the molecular
replacement not working:

Is the binding between the proteins 1:1?  
Does the Matthews coefficient reflect this?

MR maybe difficult if you have a number of 7 kDa proteins binding to a 13
kDa protein.

Hope that Helps,

Sean

 

[ccp4bb] Help request: Failed MR using the same molecule as model

[Chao Quan]

Dear CCP4 community:

Sorry if there is a duplicate post. I am a beginner to crystallography and 
therefore my 
apologies if this question is too simple.

Basically we obtained several crystal forms of the same molecule, which is a 
hetero-
trimer containing protein A(18kD), protein B(16kD) and a RNA segment(40nt or 
about 
15kD).

We have solved the structure of one crystal form(form_1); its information is as 
follows:
space group = P 42 22;
unit cell = 126.514  126.514   76.766  90.00  90.00  90.00
resolution = 2.7A;
Rwork = 0.25, Rfree = 0.265;
solvent content = 60%;
Number of molecules per Asymmetric Unit = 1;
Data redundancy = 5;
Data Completeness = 94%;

I am now trying to solve the structure of form_2 crystal using molecular 
replacement. So 
far the information I know about form_2 is as follows:
space group = I 422 or I 41 22;
unit cell = 180.096  180.096   152.530  90.00  90.00  90.00
(unit cell is about 4 times the size of form_1)
resolution = 3.3A; (which is low)
Number of molecules per Asymmetric Unit = 2 or 3;
Data redundancy = 4;
Data Completeness = 92%;
There is no twinning(as shown by Sfcheck);
As shown in "Analyse Data for MR", the first peak is 100 and second is 68.92; I 
am not 
sure if this indicates translation in a Asymmetric Unit;

The problem is, I can not get a good solution by MR using Phaser (both I422 and 
I4122 
are tried). When I searched for 3 molecules per Asymmetric Unit, Phaser did not 
give 
solutions at all. 

When I used 2/ASU instead, I was able to get some solutions, with typical 
statistics as 
follows:
RFZ=14, TFZ=35.9, PAK=0, LLG=1036; 
However, these solutions had high R values(like Rwork=0.59 and Rfree=0.58), 
which 
indicated that they are not solutions at all. Still, I tried refinement using 
refmac5, but R 
values did not go down even after 50 rounds; sometimes they even increased 
after 
refinement.

Besides, the RMS values bond length, bond angle and chiral center were all 0 as 
show by 
refmac5.

I tried limiting resolution range to 15-4A in Phaser, which did not help 
either. 

Now I am completely stuck. Could anyone give me some advice? I know this 
situation is 
very strange, because I am using the SAME molecule for MR but can not can a 
solution.

Thanks a lot,

P.S. 
1) Both form_1 and form_2 crystals were grown using Selenomethionine-containing 
samples. There are 3 Sel_Met in protein A and 1 in protein B. 
2) A 10-aa internal segment of protein B is missing in the solved structure, 
which may 
indicate high flexibility.

Chao

Re: [ccp4bb] Methylation of macromolecular complexes

[Artem Evdokimov]

Yes,

 

I’ve methylated a ~300 kDa complex once, just for kicks – and it still
functioned. MS was fairly good as well, showing that essentially all
accessible amines were altered. Notably I did not get the entire 300 kDa
complex on MS – it did not survive TFA/Acetonitrile and I saw components
instead.

 

There should not be a limit based on mass, as long as you’re careful to
maintain conditions that are both conducive to methylation and to the
survival of your complex. Methylaiton can affect complex stability – in
either direction.

 

Artem

 

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jan
Rash
Sent: Tuesday, November 24, 2009 11:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Methylation of macromolecular complexes

 

Hi all,
What I really want to know is whether macromolecular complexes ( more than
200kDa) can tolerate methylation? 
thanks,
 Jan  Rash

2009/11/24 Hüsnü Topal 

Hi Jan,

 

Maybe this link

http://www.jenabioscience.com/cms/en/1/catalog/529_jbs_methylation_kit.html
or these references helps…

 

References 
[1] Kim et al. (2008) Large-scale evaluation of protein reductive
methylation for improving protein crystallization. Nature Methods 5, 853. 

[2] Fogg et al. (2006) Application of the use of high-throughput
technologies to the determination of protein structures of bacterial and
viral pathogens. Acta Cryst. D62:1196. 

[3] Walter et al. (2006) Lysine methylation as a routine rescue strategy for
protein crystallization. Structure 14:1617.

I’m not sure if this answers your question. Good luck.

 

best regards,

Hüsnü  Topal

Ruhr-Universität Bochum
Institut für Physiologische Chemie, MA 2/141
Abteilung für Biochemie supramolekularer Systeme
Universitätsstr. 150

44801 Bochum, Germany

phone: +49 (0) 234 32 24934

Internet:  
http://www.ruhr-uni-bochum.de/physiolchem/steegborn/

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  _  

Von: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] Im Auftrag von Jan
Rash
Gesendet: Dienstag, 24. November 2009 18:28
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Methylation of macromolecular complexes

 

Hi all,
 I have a question regarding the methylation of macromolecular complexes. Is
there any report where a macromolecular complex has been successfully
methylated and later on crystallized. Is methylation a method of choice to
chemically modify macromolecular complexes where the entropical
contributions by lysines might be a cause for not obtaining crystals, or is
there any other better ways?

thanks,
Jan Rash

 

<>

Re: [ccp4bb] Help request: Failed MR using the same molecule as model

[Frederic VELLIEUX]

Hi there,

2 things:

Are you sure that the relative orientations of your components are the same in 
the different crystal forms? If you haven't done it already, it would be worth 
while trying to search for the individual components as well (could be 
difficult because your components are not so large), or do something similar to 
"Patterson correlation refinement" that was introduced in X-PLOR many years ago 
(and which probably sits in CNS too). The idea there is to adjust the 
orientation of the components, before carrying out the translation search. Last 
time I have used this was at the time of X-PLOR so that I would not know how to 
do it in CNS.

Also, even though Phaser is a very good program, it might be worth it to run 
the molecular replacement search using AMoRe too, that will give a very 
comprehensive search (doing the translation search over many many rotation 
function peaks) rapidly. I personally prefer Jorge Navaza's stand alone version 
of AMoRe (perhaps because Jorge sits in the office next to mine so that my 
opinions are biased).


> I am a beginner to crystallography and therefore my apologies if this 
> question is too 
> simple.


No question's too simple for ccp4bb. Macromolecular crystallography is not as 
simple as some people think. Or would want others to think.


Ciao,

Fred.

[ccp4bb] MSD PISA

[Koustav Maity]

Hi All,
I am using MSD PISA web server to analyze the protein ligand interface in my
newly solved structure. The ligand in the structure is a new inhibitor
(generated using PRODRG), so there is no entry for the inhibitor in the PDB.
Therefore when I am uploading my pdb file to PISA server it is not
recognizing the ligand molecule. Probably PISA reads the ligand identifier
and matches with the database to process it as a ligand. Is there any way I
can specify my inhibitor library file ? or is there some other way to
resolve this issue?
Koustav

Re: [ccp4bb] MSD PISA

[Matthew Chu]

Hi Koustav,

I had the same question before and here is the answer from Eugene Krissinel,
the developer of PISA:

"Yes PISA has a database of compounds, which contains certain interaction
parameters. If a compound is not found in the database, then PISA does not
know how it interacts with other molecules and for this the compound is
ignored. The only way to repair this is to process your compound, which
cannot be done until it is deposited to our database. But even then, I am
afraid, nothing can be done because I am no longer with the EBI and full
knowledge of PISA was not digested by people left behind. Sorry for the
crude truth."

Good luck,
Matt

2009/11/25 Koustav Maity 

> Hi All,
> I am using MSD PISA web server to analyze the protein ligand interface in
> my newly solved structure. The ligand in the structure is a new inhibitor
> (generated using PRODRG), so there is no entry for the inhibitor in the PDB.
> Therefore when I am uploading my pdb file to PISA server it is not
> recognizing the ligand molecule. Probably PISA reads the ligand identifier
> and matches with the database to process it as a ligand. Is there any way I
> can specify my inhibitor library file ? or is there some other way to
> resolve this issue?
> Koustav
>



-- 

Matthew L.H. Chu, PhD
The University of Manchester, UK