Re: [ccp4bb] P21221 to P21212 conversion

[Shya Biswas]

Hi,
i just repeated the MR using the reindexed dataset (hkl to lhk) and it gave
me the right solution.
thanks,
Shya

On Mon, May 7, 2012 at 5:39 PM, Edward A. Berry  wrote:

> Now given that the MR soluiton was obtained in the (preparing to duck)
> "nonstandard" setting, what is the transform to apply to that solution
> in pdbset to get the solution in the "standard" setting? Or is it easier
> to just repeat the MR?
> eab
>
> Shya Biswas wrote:
>
>> Hi Matt,
>> It worked really well in HKL 2000 reindex option, sorry about the
>> confusion before, I wanted hkl to lhk. as you pointed out the second one
>> gave me what I wanted.
>> thanks,
>> Shya
>>
>> On Mon, May 7, 2012 at 4:47 PM, Matthew Franklin > > wrote:
>>
>>On 5/7/12 4:09 PM, Shya Biswas wrote:
>>
>>Hi all,
>>I was wondering if anyone knows how to convert the P21221 to
>>P21212 spacegroup in HKL2000. I scaled the data set in P21212 in
>>HKL 2000 but I got a correct MR solution in P21221 spacegroup. I
>>have a script file that runs with scalepack but was wondering if
>>there is an easier way to do it with HKL2000 gui mode.
>>thanks,
>>Shya
>>
>>Hi Shya -
>>
>>Under the Scale tab of HKL2000, you'll see a button near the bottom
>>labeled "Reindex".  Clicking this brings up a dialog box with the
>>reindexing conventions appropriate to your spacegroup.  For P
>>orthorhombic, there are only two choices: hkl -> klh, or hkl -> lhk.
>>  If you have P21221, and want to go to P21212, you want hkl ->lhk.
>>
>>HOWEVER, there seems to be a bug in this particular reindexing (or
>>maybe the options are written confusingly).  You want to choose the
>>"wrong" option (number 2 in the list presented), as the two options
>>seem to be reversed.  You'll know that you got it right by
>>inspection of the scaling log file - look at the systematic absence
>>table at the bottom.  Also check that the unit cell axes were
>>permuted in the correct way - you want your old b axis to be your
>>new c axis.
>>
>>Once you select the correct reindexing (make sure you apply it to
>>all datasets being scaled at the same time), you click "Reindex" in
>>the popup dialog, and this triggers another round of scaling with
>>the reindexing included.  This reindexing is "sticky" - you don't
>>need to select it again for subsequent rounds of scaling.
>>
>>Hope that helps - feel free to contact me if you want more explanation.
>>
>>- Matt
>>
>>
>>--
>>Matthew Franklin, Ph. D.
>>Senior Scientist
>>New York Structural Biology Center
>>89 Convent Avenue, New York, NY 10027
>>(212) 939-0660 ext. 9374 
>>
>>
>>
>>

Re: [ccp4bb] P21221 to P21212 conversion

[Edward A. Berry]

Now given that the MR soluiton was obtained in the (preparing to duck)
"nonstandard" setting, what is the transform to apply to that solution
in pdbset to get the solution in the "standard" setting? Or is it easier
to just repeat the MR?
eab

Shya Biswas wrote:

Hi Matt,
It worked really well in HKL 2000 reindex option, sorry about the
confusion before, I wanted hkl to lhk. as you pointed out the second one
gave me what I wanted.
thanks,
Shya

On Mon, May 7, 2012 at 4:47 PM, Matthew Franklin mailto:mfrank...@nysbc.org>> wrote:

On 5/7/12 4:09 PM, Shya Biswas wrote:

Hi all,
I was wondering if anyone knows how to convert the P21221 to
P21212 spacegroup in HKL2000. I scaled the data set in P21212 in
HKL 2000 but I got a correct MR solution in P21221 spacegroup. I
have a script file that runs with scalepack but was wondering if
there is an easier way to do it with HKL2000 gui mode.
thanks,
Shya

Hi Shya -

Under the Scale tab of HKL2000, you'll see a button near the bottom
labeled "Reindex".  Clicking this brings up a dialog box with the
reindexing conventions appropriate to your spacegroup.  For P
orthorhombic, there are only two choices: hkl -> klh, or hkl -> lhk.
  If you have P21221, and want to go to P21212, you want hkl ->lhk.

HOWEVER, there seems to be a bug in this particular reindexing (or
maybe the options are written confusingly).  You want to choose the
"wrong" option (number 2 in the list presented), as the two options
seem to be reversed.  You'll know that you got it right by
inspection of the scaling log file - look at the systematic absence
table at the bottom.  Also check that the unit cell axes were
permuted in the correct way - you want your old b axis to be your
new c axis.

Once you select the correct reindexing (make sure you apply it to
all datasets being scaled at the same time), you click "Reindex" in
the popup dialog, and this triggers another round of scaling with
the reindexing included.  This reindexing is "sticky" - you don't
need to select it again for subsequent rounds of scaling.

Hope that helps - feel free to contact me if you want more explanation.

- Matt


--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374 

Re: [ccp4bb] P21221 to P21212 conversion

[Shya Biswas]

Hi Matt,
It worked really well in HKL 2000 reindex option, sorry about the confusion
before, I wanted hkl to lhk. as you pointed out the second one gave me what
I wanted.
thanks,
Shya

On Mon, May 7, 2012 at 4:47 PM, Matthew Franklin wrote:

> On 5/7/12 4:09 PM, Shya Biswas wrote:
>
>> Hi all,
>> I was wondering if anyone knows how to convert the P21221 to P21212
>> spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
>> got a correct MR solution in P21221 spacegroup. I have a script file that
>> runs with scalepack but was wondering if there is an easier way to do it
>> with HKL2000 gui mode.
>> thanks,
>> Shya
>>
> Hi Shya -
>
> Under the Scale tab of HKL2000, you'll see a button near the bottom
> labeled "Reindex".  Clicking this brings up a dialog box with the
> reindexing conventions appropriate to your spacegroup.  For P orthorhombic,
> there are only two choices: hkl -> klh, or hkl -> lhk.  If you have P21221,
> and want to go to P21212, you want hkl ->lhk.
>
> HOWEVER, there seems to be a bug in this particular reindexing (or maybe
> the options are written confusingly).  You want to choose the "wrong"
> option (number 2 in the list presented), as the two options seem to be
> reversed.  You'll know that you got it right by inspection of the scaling
> log file - look at the systematic absence table at the bottom.  Also check
> that the unit cell axes were permuted in the correct way - you want your
> old b axis to be your new c axis.
>
> Once you select the correct reindexing (make sure you apply it to all
> datasets being scaled at the same time), you click "Reindex" in the popup
> dialog, and this triggers another round of scaling with the reindexing
> included.  This reindexing is "sticky" - you don't need to select it again
> for subsequent rounds of scaling.
>
> Hope that helps - feel free to contact me if you want more explanation.
>
> - Matt
>
>
> --
> Matthew Franklin, Ph. D.
> Senior Scientist
> New York Structural Biology Center
> 89 Convent Avenue, New York, NY 10027
> (212) 939-0660 ext. 9374
>
>
>

Re: [ccp4bb] P21221 to P21212 conversion

[Ethan Merritt]

On Monday, May 07, 2012 02:00:43 pm Phil Jeffrey wrote:
> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt
>  > Scaling is done in a point group, not a space group.
> 
> My quibble with this statement is that the output reflection data from 
> Scalepack differs depending on what space group you tell it, since 
> systematic absences along h00, 0k0 and 00l in P2x2x2x are not written 
> out.  The number of reflections affected is quite small, of course.

The statement is correct, but the scalepack behavior is IMHO a bad thing.
Therefore I always tell it to scale in the pointgroup (P222 in this case)
and I correct the space group later.

Ethan


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

Re: [ccp4bb] P21221 to P21212 conversion

[Jacob Keller]

Why do you like P21212 better than P22121?

JPK

On Mon, May 7, 2012 at 4:00 PM, Phil Jeffrey  wrote:

> The program that does the indexing in HKL is Denzo.  Denzo doesn't care
> about the space group.  It cares about the point group (cf. Ethan's point)
> and the cell dimensions, because it integrates the data without regard to
> the symmetry expressed in the intensities - however it does take notice of
> the restrictions placed on cell dimensions by point groups.  Denzo
> therefore picks primitive orthorhombic cells in a
> Scalepack scales the integrated data but does not reindex the data if you
> tell it the space group is P22121.  Therefore unit cell choice in HKL is by
> default driven by cell edge size.  Scalepack has the ability to reindex the
> data, for those of us that like to work in P21212 rather than P22121.
>
>
> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt
> > Scaling is done in a point group, not a space group.
>
> My quibble with this statement is that the output reflection data from
> Scalepack differs depending on what space group you tell it, since
> systematic absences along h00, 0k0 and 00l in P2x2x2x are not written out.
>  The number of reflections affected is quite small, of course.
>
>
> Phil Jeffrey
> Princeton
>
>
>
>
>
> On 5/7/12 4:48 PM, Jacob Keller wrote:
>
>> Is it true that HKL adopts the naming convention of putting the screw
>> axes first and then naming a> the cell a> or would it automatically be p21212?
>>
>> JPK
>>
>> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt > > wrote:
>>
>>On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
>> > Hi all,
>> > I was wondering if anyone knows how to convert the P21221 to P21212
>> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL
>>2000 but I
>> > got a correct MR solution in P21221 spacegroup.
>>
>>Shya:
>>
>>Scaling is done in a point group, not a space group.
>>
>>The point group P222 contains both space groups P2(1)22(1) and
>>P2(1)2(1)2,
>>so your original scaling is correct in either case.
>>
>>It is not clear from your query which of two things happened:
>>
>>1) The MR solution kept the same a, b, and c axis assignments but made
>> a
>>different call on whether each axis did or did not correspond to a
>>2(1) screw.
>>In this case you don't need to do anything to your files. Just make
>> sure
>>that you keep the new space group as you go forward into refinement.
>>
>>2) The MR solution kept the orginal screw-axis identifications but
>>permuted the axes to the standard setting (non-screw axis is
>>labelled "c").
>>In this case you will need to construct a file containing the permuted
>>indices. For example, the reflection originally labeled (h=1 k=2
>>l=3) is now
>>(h=3 k=1 l=2). There are several programs that can help you do this,
>>including the HKL2000 GUI. But you do not need to go back into HKL
>>if you don't want to. You could, for example, use the ccp4i GUI to
>>select
>>-> Reflection Data Utilities
>>-> Reindex Reflections
>>Define Transformation Matrix by entering reflection transformation
>>h=l k=h l=k
>>
>>
>>Ethan
>>
>>
>> > I have a script file that
>> > runs with scalepack but was wondering if there is an easier way
>>to do it
>> > with HKL2000 gui mode.
>> > thanks,
>> > Shya
>> >
>>
>>--
>>Ethan A Merritt
>>Biomolecular Structure Center, K-428 Health Sciences Bldg
>>University of Washington, Seattle 98195-7742
>>
>>
>>
>>
>> --
>> *
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> email: j-kell...@northwestern.edu 
>> > >
>> *
>>
>
>


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

Re: [ccp4bb] P21221 to P21212 conversion

[Felix Frolow]

If one make a proper transformation :-/ and supply a correct space group, 
absent reflections will be printed in the end of scale.log
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On May 8, 2012, at 24:00 , Phil Jeffrey wrote:

> The program that does the indexing in HKL is Denzo.  Denzo doesn't care about 
> the space group.  It cares about the point group (cf. Ethan's point) and the 
> cell dimensions, because it integrates the data without regard to the 
> symmetry expressed in the intensities - however it does take notice of the 
> restrictions placed on cell dimensions by point groups.  Denzo therefore 
> picks primitive orthorhombic cells in a 
> Scalepack scales the integrated data but does not reindex the data if you 
> tell it the space group is P22121.  Therefore unit cell choice in HKL is by 
> default driven by cell edge size.  Scalepack has the ability to reindex the 
> data, for those of us that like to work in P21212 rather than P22121.
> 
> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt
> > Scaling is done in a point group, not a space group.
> 
> My quibble with this statement is that the output reflection data from 
> Scalepack differs depending on what space group you tell it, since systematic 
> absences along h00, 0k0 and 00l in P2x2x2x are not written out.  The number 
> of reflections affected is quite small, of course.
> 
> 
> Phil Jeffrey
> Princeton
> 
> 
> 
> 
> On 5/7/12 4:48 PM, Jacob Keller wrote:
>> Is it true that HKL adopts the naming convention of putting the screw
>> axes first and then naming a> the cell a> or would it automatically be p21212?
>> 
>> JPK
>> 
>> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt > > wrote:
>> 
>>On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
>> > Hi all,
>> > I was wondering if anyone knows how to convert the P21221 to P21212
>> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL
>>2000 but I
>> > got a correct MR solution in P21221 spacegroup.
>> 
>>Shya:
>> 
>>Scaling is done in a point group, not a space group.
>> 
>>The point group P222 contains both space groups P2(1)22(1) and
>>P2(1)2(1)2,
>>so your original scaling is correct in either case.
>> 
>>It is not clear from your query which of two things happened:
>> 
>>1) The MR solution kept the same a, b, and c axis assignments but made a
>>different call on whether each axis did or did not correspond to a
>>2(1) screw.
>>In this case you don't need to do anything to your files. Just make sure
>>that you keep the new space group as you go forward into refinement.
>> 
>>2) The MR solution kept the orginal screw-axis identifications but
>>permuted the axes to the standard setting (non-screw axis is
>>labelled "c").
>>In this case you will need to construct a file containing the permuted
>>indices. For example, the reflection originally labeled (h=1 k=2
>>l=3) is now
>>(h=3 k=1 l=2). There are several programs that can help you do this,
>>including the HKL2000 GUI. But you do not need to go back into HKL
>>if you don't want to. You could, for example, use the ccp4i GUI to
>>select
>>-> Reflection Data Utilities
>>-> Reindex Reflections
>>Define Transformation Matrix by entering reflection transformation
>>h=l k=h l=k
>> 
>> 
>>Ethan
>> 
>> 
>> > I have a script file that
>> > runs with scalepack but was wondering if there is an easier way
>>to do it
>> > with HKL2000 gui mode.
>> > thanks,
>> > Shya
>> >
>> 
>>--
>>Ethan A Merritt
>>Biomolecular Structure Center, K-428 Health Sciences Bldg
>>University of Washington, Seattle 98195-7742
>> 
>> 
>> 
>> 
>> --
>> ***
>> Jacob Pearson Keller
>> Northwestern University
>> Medical Scientist Training Program
>> email: j-kell...@northwestern.edu 
>> ***

Re: [ccp4bb] P21221 to P21212 conversion

[Phil Jeffrey]

The program that does the indexing in HKL is Denzo.  Denzo doesn't care 
about the space group.  It cares about the point group (cf. Ethan's 
point) and the cell dimensions, because it integrates the data without 
regard to the symmetry expressed in the intensities - however it does 
take notice of the restrictions placed on cell dimensions by point 
groups.  Denzo therefore picks primitive orthorhombic cells in a

Scalepack scales the integrated data but does not reindex the data if 
you tell it the space group is P22121.  Therefore unit cell choice in 
HKL is by default driven by cell edge size.  Scalepack has the ability 
to reindex the data, for those of us that like to work in P21212 rather 
than P22121.


On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt
> Scaling is done in a point group, not a space group.

My quibble with this statement is that the output reflection data from 
Scalepack differs depending on what space group you tell it, since 
systematic absences along h00, 0k0 and 00l in P2x2x2x are not written 
out.  The number of reflections affected is quite small, of course.



Phil Jeffrey
Princeton




On 5/7/12 4:48 PM, Jacob Keller wrote:

Is it true that HKL adopts the naming convention of putting the screw
axes first and then naming amailto:merr...@u.washington.edu>> wrote:

On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
 > Hi all,
 > I was wondering if anyone knows how to convert the P21221 to P21212
 > spacegroup in HKL2000. I scaled the data set in P21212 in HKL
2000 but I
 > got a correct MR solution in P21221 spacegroup.

Shya:

Scaling is done in a point group, not a space group.

The point group P222 contains both space groups P2(1)22(1) and
P2(1)2(1)2,
so your original scaling is correct in either case.

It is not clear from your query which of two things happened:

1) The MR solution kept the same a, b, and c axis assignments but made a
different call on whether each axis did or did not correspond to a
2(1) screw.
In this case you don't need to do anything to your files. Just make sure
that you keep the new space group as you go forward into refinement.

2) The MR solution kept the orginal screw-axis identifications but
permuted the axes to the standard setting (non-screw axis is
labelled "c").
In this case you will need to construct a file containing the permuted
indices. For example, the reflection originally labeled (h=1 k=2
l=3) is now
(h=3 k=1 l=2). There are several programs that can help you do this,
including the HKL2000 GUI. But you do not need to go back into HKL
if you don't want to. You could, for example, use the ccp4i GUI to
select
-> Reflection Data Utilities
-> Reindex Reflections
Define Transformation Matrix by entering reflection transformation
h=l k=h l=k


Ethan


 > I have a script file that
 > runs with scalepack but was wondering if there is an easier way
to do it
 > with HKL2000 gui mode.
 > thanks,
 > Shya
 >

--
Ethan A Merritt
Biomolecular Structure Center, K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742




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

Re: [ccp4bb] P21221 to P21212 conversion

[Felix Frolow]

Forget to tell that all is done in the menu Macros : - (
FF
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On May 7, 2012, at 23:48 , Jacob Keller wrote:

> Is it true that HKL adopts the naming convention of putting the screw axes 
> first and then naming a a automatically be p21212?
> 
> JPK
> 
> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt  
> wrote:
> On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> > Hi all,
> > I was wondering if anyone knows how to convert the P21221 to P21212
> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> > got a correct MR solution in P21221 spacegroup.
> 
> Shya:
> 
> Scaling is done in a point group, not a space group.
> 
> The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
> so your original scaling is correct in either case.
> 
> It is not clear from your query which of two things happened:
> 
> 1) The MR solution kept the same a, b, and c axis assignments but made a
> different call on whether each axis did or did not correspond to a 2(1) screw.
> In this case you don't need to do anything to your files.  Just make sure
> that you keep the new space group as you go forward into refinement.
> 
> 2) The MR solution kept the orginal screw-axis identifications but
> permuted the axes to the standard setting (non-screw axis is labelled "c").
> In this case you will need to construct a file containing the permuted
> indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is now
> (h=3 k=1 l=2).  There are several programs that can help you do this,
> including the HKL2000 GUI.   But you do not need to go back into HKL
> if you don't want to.  You could, for example, use the ccp4i GUI to
> select
> -> Reflection Data Utilities
>   -> Reindex Reflections
>  Define Transformation Matrix by entering reflection transformation
>  h=l k=h l=k
> 
> 
>Ethan
> 
> 
> > I have a script file that
> > runs with scalepack but was wondering if there is an easier way to do it
> > with HKL2000 gui mode.
> > thanks,
> > Shya
> >
> 
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> University of Washington, Seattle 98195-7742
> 
> 
> 
> -- 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] P21221 to P21212 conversion

[Felix Frolow]

HKL or most probably SCALEPACK know nothing above point group if you do not 
tell it.
But even in GUI one can use "hkl matrix" with the needed transformation matrix 
:-\  
What is nice about it that it will never let you to change the handedness of 
the data, so anomalous signal is safe…
FF
Dr Felix Frolow   
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On May 7, 2012, at 23:48 , Jacob Keller wrote:

> Is it true that HKL adopts the naming convention of putting the screw axes 
> first and then naming a a automatically be p21212?
> 
> JPK
> 
> On Mon, May 7, 2012 at 3:33 PM, Ethan Merritt  
> wrote:
> On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> > Hi all,
> > I was wondering if anyone knows how to convert the P21221 to P21212
> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> > got a correct MR solution in P21221 spacegroup.
> 
> Shya:
> 
> Scaling is done in a point group, not a space group.
> 
> The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
> so your original scaling is correct in either case.
> 
> It is not clear from your query which of two things happened:
> 
> 1) The MR solution kept the same a, b, and c axis assignments but made a
> different call on whether each axis did or did not correspond to a 2(1) screw.
> In this case you don't need to do anything to your files.  Just make sure
> that you keep the new space group as you go forward into refinement.
> 
> 2) The MR solution kept the orginal screw-axis identifications but
> permuted the axes to the standard setting (non-screw axis is labelled "c").
> In this case you will need to construct a file containing the permuted
> indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is now
> (h=3 k=1 l=2).  There are several programs that can help you do this,
> including the HKL2000 GUI.   But you do not need to go back into HKL
> if you don't want to.  You could, for example, use the ccp4i GUI to
> select
> -> Reflection Data Utilities
>   -> Reindex Reflections
>  Define Transformation Matrix by entering reflection transformation
>  h=l k=h l=k
> 
> 
>Ethan
> 
> 
> > I have a script file that
> > runs with scalepack but was wondering if there is an easier way to do it
> > with HKL2000 gui mode.
> > thanks,
> > Shya
> >
> 
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> University of Washington, Seattle 98195-7742
> 
> 
> 
> -- 
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> email: j-kell...@northwestern.edu
> ***

Re: [ccp4bb] P21221 to P21212 conversion

[Jacob Keller]

Is it true that HKL adopts the naming convention of putting the screw axes
first and then naming awrote:

> On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> > Hi all,
> > I was wondering if anyone knows how to convert the P21221 to P21212
> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> > got a correct MR solution in P21221 spacegroup.
>
> Shya:
>
> Scaling is done in a point group, not a space group.
>
> The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
> so your original scaling is correct in either case.
>
> It is not clear from your query which of two things happened:
>
> 1) The MR solution kept the same a, b, and c axis assignments but made a
> different call on whether each axis did or did not correspond to a 2(1)
> screw.
> In this case you don't need to do anything to your files.  Just make sure
> that you keep the new space group as you go forward into refinement.
>
> 2) The MR solution kept the orginal screw-axis identifications but
> permuted the axes to the standard setting (non-screw axis is labelled "c").
> In this case you will need to construct a file containing the permuted
> indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is
> now
> (h=3 k=1 l=2).  There are several programs that can help you do this,
> including the HKL2000 GUI.   But you do not need to go back into HKL
> if you don't want to.  You could, for example, use the ccp4i GUI to
> select
> -> Reflection Data Utilities
>   -> Reindex Reflections
>  Define Transformation Matrix by entering reflection transformation
>  h=l k=h l=k
>
>
>Ethan
>
>
> > I have a script file that
> > runs with scalepack but was wondering if there is an easier way to do it
> > with HKL2000 gui mode.
> > thanks,
> > Shya
> >
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> University of Washington, Seattle 98195-7742
>



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

Re: [ccp4bb] P21221 to P21212 conversion

[Ethan Merritt]

On Monday, May 07, 2012 01:42:58 pm Shya Biswas wrote:
> Hi,
> My case is old b is changed to c (scenario 2 as you explained) or hkl is
> changed to hlk. Thanks for the help

hkl -> hkl  gives an inverted coordinate system.  You don't want that.

Ethan


> 
> Shya
> 
> On Mon, May 7, 2012 at 4:33 PM, Ethan Merritt wrote:
> 
> > On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> > > Hi all,
> > > I was wondering if anyone knows how to convert the P21221 to P21212
> > > spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> > > got a correct MR solution in P21221 spacegroup.
> >
> > Shya:
> >
> > Scaling is done in a point group, not a space group.
> >
> > The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
> > so your original scaling is correct in either case.
> >
> > It is not clear from your query which of two things happened:
> >
> > 1) The MR solution kept the same a, b, and c axis assignments but made a
> > different call on whether each axis did or did not correspond to a 2(1)
> > screw.
> > In this case you don't need to do anything to your files.  Just make sure
> > that you keep the new space group as you go forward into refinement.
> >
> > 2) The MR solution kept the orginal screw-axis identifications but
> > permuted the axes to the standard setting (non-screw axis is labelled "c").
> > In this case you will need to construct a file containing the permuted
> > indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is
> > now
> > (h=3 k=1 l=2).  There are several programs that can help you do this,
> > including the HKL2000 GUI.   But you do not need to go back into HKL
> > if you don't want to.  You could, for example, use the ccp4i GUI to
> > select
> > -> Reflection Data Utilities
> >   -> Reindex Reflections
> >  Define Transformation Matrix by entering reflection transformation
> >  h=l k=h l=k
> >
> >
> >Ethan
> >
> >
> > > I have a script file that
> > > runs with scalepack but was wondering if there is an easier way to do it
> > > with HKL2000 gui mode.
> > > thanks,
> > > Shya
> > >
> >
> > --
> > Ethan A Merritt
> > Biomolecular Structure Center,  K-428 Health Sciences Bldg
> > University of Washington, Seattle 98195-7742
> >
> 

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

Re: [ccp4bb] saxs on xtals

[Colin Nave]

Anna
Interesting.

Yes, the cryo-em might be the way to go to see if some structures (i.e. not 
just spheres) within the protein shell are aligned. 

The SAXS study does make some sense. If the magnetic particles have some 
alignment this should manifest itself in the SAXS pattern, with the precise 
effects depending on the correlation length (how far the alignment extends). 
This really requires SAXS, not WAXS which would not be sensitive to the long 
range effects you wish to see. 

As you suggest, it would also be good fun (and perhaps even informative) to do 
this on single crystals. A coherent x-ray beam, using CDI or ptychography 
(search for these terms or contact me if you want some details) could be 
employed if you restrict yourself to crystals of a few microns in size. One can 
even play around with absorption edges/anomalous dispersion/magnetic resonance 
(for single crystals or powders) as I understand the interest is in magnetic 
phenomena. Making resonant soft x-ray scattering measurements at the oxygen K 
edge have been used for studying magnetite. The polarisation of the x-rays 
could be exploited. Our neutron friends can also study magnetic effects but I 
don't know about sample size requirements. Doing the measurements in a strong 
magnetic field might be interesting.

If the samples are easy to prepare, I would start from collecting the lowest 
hkl reflections from single crystals to check you are not missing anything. 
Then a low angle SAXS pattern as suggested by your collaborators. After this 
consider the other suggestions which are rather speculative and not really 
routine. 

Good luck

Colin


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Murray, 
James W
Sent: 07 May 2012 19:25
To: ccp4bb
Subject: Re: [ccp4bb] saxs on xtals

Dear Anna, 

I once modified CNS to refine two solvent regions of ferritin, one inside and 
one outside the shell. Perhaps this can be done in Phenix now. If you want to 
locate magnetite particles in this way, you should collect data to as low a 
resolution as you can (may need to move backstop), as this is where the solvent 
has most effect. I would also collect control data with apo and holo ferritin 
to compare.

However a much easier way may be to directly visualise the particles in cryoEM. 
I have seen very nice micrographs of particles inside ferritin (can't remember 
ref now).

best wishes

James

--
Dr. James W. Murray
David Phillips Research  Fellow
Division of Molecular Biosciences
Imperial College, LONDON
Tel: +44 (0)20 759 48895

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anna anna 
[marmottalb...@gmail.com]
Sent: Monday, May 07, 2012 5:30 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] saxs on xtals

Dear all,
I'd like some suggestions/opinions about the sense of an experiment proposed by 
a collaborator expert in saxs.
In few words, he wants to collect SAXS data on a suspension of protein xtals to 
investigate "low resolution periodicity" of the xtal (more details below).
The experiment requires a very huge number of xtals to obtain the circles 
typical of saxs and it is very time-consuming to me (I know nothing about saxs, 
I have only to prepare the sample). I proposed to measure a single rotating 
xtal (like in XRD) but he told they don't have a goniometer on saxs beamline.
Here is my concern: does it make sense to measure many xtals together? Don't we 
lose information with respect to single xtal? And, most of all, what can I see 
by saxs that I can't see by waxs??
Sorry for the almost off-topic question but I think that only someone who knows 
both the techniques can help me!!


Some detail for who is intrigued by my story:
we prepared doped magnetite nanoparticles using ferritin as bioreactor. I 
crystallized this spheres filled with metal and solved the structure at 3.7A 
but I can see only the protein shell while there is no density inside, even if 
I know that the nanoparticles are there. A simple explanation is that the 
particles are free to move in the cavity(note that the diameter of the 
nanoparticle is shorter then the inner diameter of the protein shell), ie are 
disordered, and do not contribute to diffraction, in fact, to my knowledge, 
nobody have ever seen the metal core inside ferritin or dps proteins. However, 
since they are magnetic particles they must "see" each other through the 
protein wall, ie they can't be completely free to move in the cavity. Maybe, 
but this is just my opinion, I don't see the particle because the "period of 
the particle" in the xtal is different/longer than the period of the protein 
shell.
Anyway, we are interested in the relative distance between the magnetic 
particles in the xtal to study the effects of magnetostatic interactions in 
nanoparticles 3D arrays. We are going to do this by saxs since, they told me, 
lower resolution is useful in studying this long range periodicity (th

Re: [ccp4bb] P21221 to P21212 conversion

[Matthew Franklin]

On 5/7/12 4:09 PM, Shya Biswas wrote:

Hi all,
I was wondering if anyone knows how to convert the P21221 to P21212 
spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but 
I got a correct MR solution in P21221 spacegroup. I have a script file 
that runs with scalepack but was wondering if there is an easier way 
to do it with HKL2000 gui mode.

thanks,
Shya

Hi Shya -

Under the Scale tab of HKL2000, you'll see a button near the bottom 
labeled "Reindex".  Clicking this brings up a dialog box with the 
reindexing conventions appropriate to your spacegroup.  For P 
orthorhombic, there are only two choices: hkl -> klh, or hkl -> lhk.  If 
you have P21221, and want to go to P21212, you want hkl ->lhk.


HOWEVER, there seems to be a bug in this particular reindexing (or maybe 
the options are written confusingly).  You want to choose the "wrong" 
option (number 2 in the list presented), as the two options seem to be 
reversed.  You'll know that you got it right by inspection of the 
scaling log file - look at the systematic absence table at the bottom.  
Also check that the unit cell axes were permuted in the correct way - 
you want your old b axis to be your new c axis.


Once you select the correct reindexing (make sure you apply it to all 
datasets being scaled at the same time), you click "Reindex" in the 
popup dialog, and this triggers another round of scaling with the 
reindexing included.  This reindexing is "sticky" - you don't need to 
select it again for subsequent rounds of scaling.


Hope that helps - feel free to contact me if you want more explanation.

- Matt


--
Matthew Franklin, Ph. D.
Senior Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(212) 939-0660 ext. 9374

Re: [ccp4bb] P21221 to P21212 conversion

[Shya Biswas]

Hi,
My case is old b is changed to c (scenario 2 as you explained) or hkl is
changed to hlk. Thanks for the help

Shya

On Mon, May 7, 2012 at 4:33 PM, Ethan Merritt wrote:

> On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> > Hi all,
> > I was wondering if anyone knows how to convert the P21221 to P21212
> > spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> > got a correct MR solution in P21221 spacegroup.
>
> Shya:
>
> Scaling is done in a point group, not a space group.
>
> The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
> so your original scaling is correct in either case.
>
> It is not clear from your query which of two things happened:
>
> 1) The MR solution kept the same a, b, and c axis assignments but made a
> different call on whether each axis did or did not correspond to a 2(1)
> screw.
> In this case you don't need to do anything to your files.  Just make sure
> that you keep the new space group as you go forward into refinement.
>
> 2) The MR solution kept the orginal screw-axis identifications but
> permuted the axes to the standard setting (non-screw axis is labelled "c").
> In this case you will need to construct a file containing the permuted
> indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is
> now
> (h=3 k=1 l=2).  There are several programs that can help you do this,
> including the HKL2000 GUI.   But you do not need to go back into HKL
> if you don't want to.  You could, for example, use the ccp4i GUI to
> select
> -> Reflection Data Utilities
>   -> Reindex Reflections
>  Define Transformation Matrix by entering reflection transformation
>  h=l k=h l=k
>
>
>Ethan
>
>
> > I have a script file that
> > runs with scalepack but was wondering if there is an easier way to do it
> > with HKL2000 gui mode.
> > thanks,
> > Shya
> >
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> University of Washington, Seattle 98195-7742
>

Re: [ccp4bb] P21221 to P21212 conversion

[Ethan Merritt]

On Monday, May 07, 2012 01:09:25 pm Shya Biswas wrote:
> Hi all,
> I was wondering if anyone knows how to convert the P21221 to P21212
> spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
> got a correct MR solution in P21221 spacegroup. 

Shya:

Scaling is done in a point group, not a space group.

The point group P222 contains both space groups P2(1)22(1) and P2(1)2(1)2,
so your original scaling is correct in either case.

It is not clear from your query which of two things happened: 

1) The MR solution kept the same a, b, and c axis assignments but made a 
different call on whether each axis did or did not correspond to a 2(1) screw.
In this case you don't need to do anything to your files.  Just make sure
that you keep the new space group as you go forward into refinement.

2) The MR solution kept the orginal screw-axis identifications but 
permuted the axes to the standard setting (non-screw axis is labelled "c").
In this case you will need to construct a file containing the permuted
indices.  For example, the reflection originally labeled  (h=1 k=2 l=3) is now
(h=3 k=1 l=2).  There are several programs that can help you do this,
including the HKL2000 GUI.   But you do not need to go back into HKL
if you don't want to.  You could, for example, use the ccp4i GUI to
select
-> Reflection Data Utilities
   -> Reindex Reflections
  Define Transformation Matrix by entering reflection transformation
  h=l k=h l=k


Ethan


> I have a script file that
> runs with scalepack but was wondering if there is an easier way to do it
> with HKL2000 gui mode.
> thanks,
> Shya
> 

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

[ccp4bb] P21221 to P21212 conversion

[Shya Biswas]

Hi all,
I was wondering if anyone knows how to convert the P21221 to P21212
spacegroup in HKL2000. I scaled the data set in P21212 in HKL 2000 but I
got a correct MR solution in P21221 spacegroup. I have a script file that
runs with scalepack but was wondering if there is an easier way to do it
with HKL2000 gui mode.
thanks,
Shya

Re: [ccp4bb] saxs on xtals

[Edward Snell]

Hi Anna,

There has been some nice single crystal SAXS work done, check out J. Mol. Biol 
(1998) 284, 1439-1452 "Imaging RNA and Dynamic Protein Segments with 
Low-resolution Virus Crystallography: Experimental Design, Data Processing and 
Implications of Electron Density Maps" by Tsuruta et al. There is the 
capability to study single crystals on SAXS lines - in this case 4-2 at SSRL.  
However if you desperately need to use crystals, you may be better served with 
a single one by moving the beamstop back, choosing a lower energy and trying to 
collect all the low resolution diffraction information from a single crystal. 
You can truncate the high resolution data and see if you start to see 
information from the  particles if there is some ordering. Even better if you 
can compare it from a crystal without them if it's in the same space group.

Have you thought about the possibility of anomalous SAXS with and without doped 
ferritin?

Note that you may be inducing interparticle effects which will complicate the 
interpretation of any SAXS data, solution or suspended crystals.

Cheers,,

Eddie

Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Senior Scientist, Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
Telepathy: 42.2 GHz

Heisenberg was probably here!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of anna anna
Sent: Monday, May 07, 2012 12:30 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] saxs on xtals

Dear all,
I'd like some suggestions/opinions about the sense of an experiment proposed by 
a collaborator expert in saxs.
In few words, he wants to collect SAXS data on a suspension of protein xtals to 
investigate "low resolution periodicity" of the xtal (more details below).
The experiment requires a very huge number of xtals to obtain the circles 
typical of saxs and it is very time-consuming to me (I know nothing about saxs, 
I have only to prepare the sample). I proposed to measure a single rotating 
xtal (like in XRD) but he told they don't have a goniometer on saxs beamline.
Here is my concern: does it make sense to measure many xtals together? Don't we 
lose information with respect to single xtal? And, most of all, what can I see 
by saxs that I can't see by waxs??
Sorry for the almost off-topic question but I think that only someone who knows 
both the techniques can help me!!


Some detail for who is intrigued by my story:
we prepared doped magnetite nanoparticles using ferritin as bioreactor. I 
crystallized this spheres filled with metal and solved the structure at 3.7A 
but I can see only the protein shell while there is no density inside, even if 
I know that the nanoparticles are there. A simple explanation is that the 
particles are free to move in the cavity(note that the diameter of the 
nanoparticle is shorter then the inner diameter of the protein shell), ie are 
disordered, and do not contribute to diffraction, in fact, to my knowledge, 
nobody have ever seen the metal core inside ferritin or dps proteins. However, 
since they are magnetic particles they must "see" each other through the 
protein wall, ie they can't be completely free to move in the cavity. Maybe, 
but this is just my opinion, I don't see the particle because the "period of 
the particle" in the xtal is different/longer than the period of the protein 
shell.
Anyway, we are interested in the relative distance between the magnetic 
particles in the xtal to study the effects of magnetostatic interactions in 
nanoparticles 3D arrays. We are going to do this by saxs since, they told me, 
lower resolution is useful in studying this long range periodicity (the 
diameter of ferritin is about 120A) but it seems fool to me using a suspension 
of so many xtals to obtain a scattering curve while I could collect diffraction 
images from a single xtal!!! I know that saxs is used when you don't have xtals 
but if you have xtals, ie your system is ordered, xtallography is much more 
powerful!!

Another question: how can I handle my diffraction data at 3.7A resolution to 
"look for" nanoparticles? Should I try a lower symmetry? Maybe the anomalous 
signal? Have you any reference for a similar case?

Thank you very much!!

anna

Re: [ccp4bb] effective iodide conc. for SAD data

[Pete Meyer]

James Holton wrote:


 In general, a Bijvoet ratio of 3% or so is needed to solve a structure (the 
current world record is 0.5% and lots of multiplicity).  The above web page 
will also tell you how many crystals you need if you type in their size in all 
three dimensions. but this estimate assumes that you don't have high 
concentrations of heavy metals in your solution!  So, if it says you can get 
away with one crystal but you know your have a dose-doubling concentration of 
something, then you're going to need to average data from two crystals, etc.



I don't know of any systematic studies, but in my experience if you've 
got weak anomalous signal you're better off with multi-crystal phasing 
than multi-crystal merging.


Pete

Re: [ccp4bb] saxs on xtals

[Murray, James W]

Dear Anna, 

I once modified CNS to refine two solvent regions of ferritin, one inside and 
one outside the shell. Perhaps this can be done in Phenix now. If you want to 
locate magnetite particles in this way, you should collect data to as low a 
resolution as you can (may need to move backstop), as this is where the solvent 
has most effect. I would also collect control data with apo and holo ferritin 
to compare.

However a much easier way may be to directly visualise the particles in cryoEM. 
I have seen very nice micrographs of particles inside ferritin (can't remember 
ref now).

best wishes

James

--
Dr. James W. Murray
David Phillips Research  Fellow
Division of Molecular Biosciences
Imperial College, LONDON
Tel: +44 (0)20 759 48895

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anna anna 
[marmottalb...@gmail.com]
Sent: Monday, May 07, 2012 5:30 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] saxs on xtals

Dear all,
I'd like some suggestions/opinions about the sense of an experiment proposed by 
a collaborator expert in saxs.
In few words, he wants to collect SAXS data on a suspension of protein xtals to 
investigate "low resolution periodicity" of the xtal (more details below).
The experiment requires a very huge number of xtals to obtain the circles 
typical of saxs and it is very time-consuming to me (I know nothing about saxs, 
I have only to prepare the sample). I proposed to measure a single rotating 
xtal (like in XRD) but he told they don't have a goniometer on saxs beamline.
Here is my concern: does it make sense to measure many xtals together? Don't we 
lose information with respect to single xtal? And, most of all, what can I see 
by saxs that I can't see by waxs??
Sorry for the almost off-topic question but I think that only someone who knows 
both the techniques can help me!!


Some detail for who is intrigued by my story:
we prepared doped magnetite nanoparticles using ferritin as bioreactor. I 
crystallized this spheres filled with metal and solved the structure at 3.7A 
but I can see only the protein shell while there is no density inside, even if 
I know that the nanoparticles are there. A simple explanation is that the 
particles are free to move in the cavity(note that the diameter of the 
nanoparticle is shorter then the inner diameter of the protein shell), ie are 
disordered, and do not contribute to diffraction, in fact, to my knowledge, 
nobody have ever seen the metal core inside ferritin or dps proteins. However, 
since they are magnetic particles they must "see" each other through the 
protein wall, ie they can't be completely free to move in the cavity. Maybe, 
but this is just my opinion, I don't see the particle because the "period of 
the particle" in the xtal is different/longer than the period of the protein 
shell.
Anyway, we are interested in the relative distance between the magnetic 
particles in the xtal to study the effects of magnetostatic interactions in 
nanoparticles 3D arrays. We are going to do this by saxs since, they told me, 
lower resolution is useful in studying this long range periodicity (the 
diameter of ferritin is about 120A) but it seems fool to me using a suspension 
of so many xtals to obtain a scattering curve while I could collect diffraction 
images from a single xtal!!! I know that saxs is used when you don't have xtals 
but if you have xtals, ie your system is ordered, xtallography is much more 
powerful!!

Another question: how can I handle my diffraction data at 3.7A resolution to 
"look for" nanoparticles? Should I try a lower symmetry? Maybe the anomalous 
signal? Have you any reference for a similar case?

Thank you very much!!

anna

Re: [ccp4bb] saxs on xtals

[Jacob Keller]

It might be that the "bulk solvent correction" is nullifying the interior
of the ferritin structure, and there should be a way to tell the refinement
software not to treat the interior as solvent. Perhaps then you might find
your Fe? Also, I would think there should be some powder-like diffraction
rings in the background of the ferritin crystal diffraction corresponding
to the scattering from the jumbled but crystalline Fe in the inside--did
you see any extra rings in the background of your original diffraction
data? Not sure what the Fe-Fe distance is in this case in particular, but
there should be a ring (or rings) corresponding to that (those)
distance(s)

JPK

On Mon, May 7, 2012 at 11:30 AM, anna anna  wrote:

> Dear all,
> I'd like some suggestions/opinions about the sense of an experiment
> proposed by a collaborator expert in saxs.
> In few words, he wants to collect SAXS data on a suspension of protein
> xtals to investigate "low resolution periodicity" of the xtal (more details
> below).
> The experiment requires a very huge number of xtals to obtain the circles
> typical of saxs and it is very time-consuming to me (I know nothing about
> saxs, I have only to prepare the sample). I proposed to measure a single
> rotating xtal (like in XRD) but he told they don't have a goniometer on
> saxs beamline.
> Here is my concern: does it make sense to measure many xtals together?
> Don't we lose information with respect to single xtal? And, most of all,
> what can I see by *s*axs that I can't see by* w*axs??
> Sorry for the almost off-topic question but I think that only someone who
> knows both the techniques can help me!!
>
>
> Some detail for who is intrigued by my story:
> we prepared doped magnetite nanoparticles using ferritin as bioreactor. I
> crystallized this spheres filled with metal and solved the structure at
> 3.7A but I can see only the protein shell while there is no density inside,
> even if I know that the nanoparticles are there. A simple explanation is
> that the particles are free to move in the cavity(note that the diameter of
> the nanoparticle is shorter then the inner diameter of the protein shell),
> ie are disordered, and do not contribute to diffraction, in fact, to my
> knowledge, nobody have ever seen the metal core inside ferritin or dps
> proteins. However, since they are magnetic particles they must "see" each
> other through the protein wall, ie they can't be completely free to move in
> the cavity. Maybe, but this is just my opinion, I don't see the particle
> because the "period of the particle" in the xtal is different/longer than
> the period of the protein shell.
> Anyway, we are interested in the relative distance between the magnetic
> particles in the xtal to study the effects of magnetostatic interactions in
> nanoparticles 3D arrays. We are going to do this by saxs since, they told
> me, lower resolution is useful in studying this long range periodicity (the
> diameter of ferritin is about 120A) but it seems fool to me using a
> suspension of so many xtals to obtain a scattering curve while I could
> collect diffraction images from a single xtal!!! I know that saxs is used
> when you don't have xtals but if you have xtals, ie your system is ordered,
> xtallography is much more powerful!!
>
> Another question: how can I handle my diffraction data at 3.7A resolution
> to "look for" nanoparticles? Should I try a lower symmetry? Maybe the
> anomalous signal? Have you any reference for a similar case?
>
> Thank you very much!!
>
> anna
>
>
>
>
>
>


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

Re: [ccp4bb] saxs on xtals

[Lucas]

2012/5/7 David Schuller :
> That sounds like powder diffraction.

That was also my impression. There are at least two groups doing
interesting things on this subject - Andy Fitch/Irene Margiolaki at
ESRF and Robert von Dreele at APS. I've contacted the first group
after meeting Andy Fitch in a XRD symposium some years ago (2005, I
think) and they were very kind to provide many hints on sample
preparation, which seems to be the hardest thing to do in those cases.
We are tempted to think "oh, so do I simply need to harvest a whole
lot of small crystals in a loop and everything will be fine?", but it
is a bit trickier than that. I am pretty sure they will be able to
provide some hints to Anna's case, which seems very similar to a
powder diffraction experiment.

Lucas Bleicher

Re: [ccp4bb] saxs on xtals

[David Schuller]

That sounds like powder diffraction.

On 05/07/12 12:30, anna anna wrote:

Dear all,
I'd like some suggestions/opinions about the sense of an experiment 
proposed by a collaborator expert in saxs.
In few words, he wants to collect SAXS data on a suspension of protein 
xtals to investigate "low resolution periodicity" of the xtal (more 
details below).
The experiment requires a very huge number of xtals to obtain the 
circles typical of saxs and it is very time-consuming to me (I know 
nothing about saxs, I have only to prepare the sample). I proposed to 
measure a single rotating xtal (like in XRD) but he told they don't 
have a goniometer on saxs beamline.
Here is my concern: does it make sense to measure many xtals together? 
Don't we lose information with respect to single xtal? And, most of 
all, what can I see by /s/axs that I can't see by/w/axs??
Sorry for the almost off-topic question but I think that only someone 
who knows both the techniques can help me!!



Some detail for who is intrigued by my story:
we prepared doped magnetite nanoparticles using ferritin as 
bioreactor. I crystallized this spheres filled with metal and solved 
the structure at 3.7A but I can see only the protein shell while there 
is no density inside, even if I know that the nanoparticles are there. 
A simple explanation is that the particles are free to move in the 
cavity(note that the diameter of the nanoparticle is shorter then the 
inner diameter of the protein shell), ie are disordered, and do not 
contribute to diffraction, in fact, to my knowledge, nobody have ever 
seen the metal core inside ferritin or dps proteins. However, since 
they are magnetic particles they must "see" each other through the 
protein wall, ie they can't be completely free to move in the cavity. 
Maybe, but this is just my opinion, I don't see the particle because 
the "period of the particle" in the xtal is different/longer than the 
period of the protein shell.
Anyway, we are interested in the relative distance between the 
magnetic particles in the xtal to study the effects of magnetostatic 
interactions in nanoparticles 3D arrays. We are going to do this by 
saxs since, they told me, lower resolution is useful in studying this 
long range periodicity (the diameter of ferritin is about 120A) but it 
seems fool to me using a suspension of so many xtals to obtain a 
scattering curve while I could collect diffraction images from a 
single xtal!!! I know that saxs is used when you don't have xtals but 
if you have xtals, ie your system is ordered, xtallography is much 
more powerful!!


Another question: how can I handle my diffraction data at 3.7A 
resolution to "look for" nanoparticles? Should I try a lower symmetry? 
Maybe the anomalous signal? Have you any reference for a similar case?


Thank you very much!!

anna








--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

[ccp4bb] saxs on xtals

[anna anna]

Dear all,
I'd like some suggestions/opinions about the sense of an experiment
proposed by a collaborator expert in saxs.
In few words, he wants to collect SAXS data on a suspension of protein
xtals to investigate "low resolution periodicity" of the xtal (more details
below).
The experiment requires a very huge number of xtals to obtain the circles
typical of saxs and it is very time-consuming to me (I know nothing about
saxs, I have only to prepare the sample). I proposed to measure a single
rotating xtal (like in XRD) but he told they don't have a goniometer on
saxs beamline.
Here is my concern: does it make sense to measure many xtals together?
Don't we lose information with respect to single xtal? And, most of all,
what can I see by *s*axs that I can't see by* w*axs??
Sorry for the almost off-topic question but I think that only someone who
knows both the techniques can help me!!


Some detail for who is intrigued by my story:
we prepared doped magnetite nanoparticles using ferritin as bioreactor. I
crystallized this spheres filled with metal and solved the structure at
3.7A but I can see only the protein shell while there is no density inside,
even if I know that the nanoparticles are there. A simple explanation is
that the particles are free to move in the cavity(note that the diameter of
the nanoparticle is shorter then the inner diameter of the protein shell),
ie are disordered, and do not contribute to diffraction, in fact, to my
knowledge, nobody have ever seen the metal core inside ferritin or dps
proteins. However, since they are magnetic particles they must "see" each
other through the protein wall, ie they can't be completely free to move in
the cavity. Maybe, but this is just my opinion, I don't see the particle
because the "period of the particle" in the xtal is different/longer than
the period of the protein shell.
Anyway, we are interested in the relative distance between the magnetic
particles in the xtal to study the effects of magnetostatic interactions in
nanoparticles 3D arrays. We are going to do this by saxs since, they told
me, lower resolution is useful in studying this long range periodicity (the
diameter of ferritin is about 120A) but it seems fool to me using a
suspension of so many xtals to obtain a scattering curve while I could
collect diffraction images from a single xtal!!! I know that saxs is used
when you don't have xtals but if you have xtals, ie your system is ordered,
xtallography is much more powerful!!

Another question: how can I handle my diffraction data at 3.7A resolution
to "look for" nanoparticles? Should I try a lower symmetry? Maybe the
anomalous signal? Have you any reference for a similar case?

Thank you very much!!

anna

Re: [ccp4bb] NCS on ligands

[Rajesh Kumar]

Dear Pavel,
I would try both options and see how it works.Thanks for time
RegardsRajesh

Date: Sat, 5 May 2012 13:39:42 -0700
Subject: Re: [ccp4bb] NCS on ligands
From: pafon...@gmail.com
To: ccp4...@hotmail.com
CC: CCP4BB@jiscmail.ac.uk

Hi Rajesh,
I guess they could (in most programs), but the question is whether they should? 
Can't you try both options to see which one works best in your specific case? 
Pavel


On Sat, May 5, 2012 at 6:02 AM, Rajesh Kumar  wrote:

Dear all,

Is ligands and metals could also be NCS restrained during the refinement. I 
have Na+ and SO4 in my model which are NCS related.

Thanks for the help

Warm regards

Rajesh

Sent from my iPhone
  

Re: [ccp4bb] effective iodide conc. for SAD data

[James Holton]

Jacob,

The main reason why it is not common practice to saturate every crystal 
with every heavy metal under the sun is radiation damage.  X-ray 
absorption increases very rapidly with atomic number (third power), so 
on the order of 100 mM of "heavy atom" is usually enough to cut your 
crystal's useful life in half.  It doesn't matter if the heavy atom is 
"in the lattice" or not.  At 12 keV the photoelectron cascade crashes 
through about 3 microns worth of organic matter before finally coming to 
rest (Cole, 1969).


The program RADDOSE (Murray et al. 2004, 2005, Paithankar et al 2009, 
2010) was written to calculate expected crystal lifetime given your 
buffer concentrations, etc.  For example, with a 50% solvent crystal, 
250 mM iodide in the solvent channels is "dose doubling" (life 
halving).  That is, the number of photons you will get scattered into 
spots before the crystal is dead will be half that which you would get 
with no iodine.  This is because the iodine-soaked crystal is absorbing 
twice as much energy per incident photon, but scattering at pretty much 
the same rate with or without the iodine.  500 mM iodine will cut the 
"useful life" to 1/3 of what it would be with no iodine, and 1 M will 
cut it to 1/5th.  (250/([iodine]+250)).  For Cs, 200 mM is the 
dose-doubling buffer concentration.  For Rb it is 1 M.  For Cl, it is 
2.4 M, and for fluoride there is no concentration that doubles the dose 
because water absorbs more than fouoride.  This is why I recommend using 
"low-Z" buffers in crystallography whenever possible.


I have listed dose-doubling concentrations of a few common elements on 
the last page of this document:

http://bl831.als.lbl.gov/damage_rates.pdf
and a fairly comprehensive "look up table" is here:
http://bl831.als.lbl.gov/~jamesh/pickup/dose_doubler_solc50.txt

The dose doubling concentration does depend on the wavelength.  The 
"worst" one at 1 A is tungsten (57 mM), but this is not all that 
different from tantalum (62 mM) or mercury (74 mM), or gold (76 mM), or 
uranium (105 mM).  So, by and large ~100 mM concentration of anything 
"heavy" will double the "dose per scattered photon".  Note that this is 
the concentration of the atoms, not molecules.  The dose-doubling 
concentration of Ta6Br12 clusters is 9.5 mM at 1 A.


As for the original poster's question, 20 mM iodide disrupting your 
crystallization is actually a good sign that it's binding, but the bad 
news is that it appears your crystals don't like that.  Nevertheless it 
could be worth a shot, you can estimate your expected Bijvoet ratio 
assuming one site per molecule using this little web app I wrote:

http://bl831.als.lbl.gov/xtalsize.html
 In general, a Bijvoet ratio of 3% or so is needed to solve a structure 
(the current world record is 0.5% and lots of multiplicity).  The above 
web page will also tell you how many crystals you need if you type in 
their size in all three dimensions. but this estimate assumes that you 
don't have high concentrations of heavy metals in your solution!  So, if 
it says you can get away with one crystal but you know your have a 
dose-doubling concentration of something, then you're going to need to 
average data from two crystals, etc.


Good luck!

-James Holton
MAD Scientist

On 5/3/2012 8:29 AM, Jacob Keller wrote:
I have wondered for a long time now why it is not standard practice 
for all crystallization protein stocks to contain either Br- or I- 
ions instead of Cl-, even for cationic buffers like TRIS, which could 
be titrated with HBr or HI to get in the 10+ mM range. Also, one could 
use Cs or Rb for the cations (and titrate anionic buffers with the 
respective hydroxides). What's there to lose? The gain is obviously 
the possible anomalous signal (always helpful), and one might pick up 
additional interesting and possibly physiologically-relevant halide or 
alkali metal sites. Seems that structural genomics people might 
standardize this into the pipeline as well, and thereby potentially 
cut out SeMet protein production in many if not most cases.


JPK

On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth > wrote:


Hi Rajesh,
it can be a bit all over the place:
For quick soaks, we typically use 500mM-1000mM. A good starting
point might be to simply replace the NaCl concentration in your
protein buffer. By some serendipity we also managed to solve a
structure by I/S SAD after a 1mM NaI soak. One iodide had found
its way into a nice binding pocket.
For co-crystallization, mostly 200mM should be fine.
Another approach could be to supplement your cryo buffer with
iodide, replacing NaCl. NaI is highly soluble in ethylene glycol.

Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836

Good luck!
Cheers,
Jan
--
Jan Abendroth
Emerald Bio
Seattle / Bainbridge Island WA, USA
home: Jan.Abendroth_at_gmail.com 
work: JAb

Re: [ccp4bb] detergent or protein

[R. M. Garavito]

Anita,

To answer a lot of your questions, you need to provide some more information 
about your protein, at least some of its biophysical characteristics (size, 
oligomeric state, an integral membrane protein, etc.).  Just because you are 
using detergents does not mean all your problems are detergent related. Also 
you need to provide a bit more information about the conditions where you see 
"crystals," particularly about  the presence of divalent cations and/or 
phosphate and the detergent concentrations.

Lots of things will crystallize: contaminating proteins, small molecules, and 
(although not as often) detergents.  The fact that you see "crystals" under 
conditions with quite different detergents suggests that the crystals are not 
detergents (unless you are very unlucky).  For example, C10E6 is a well 
characterized and studied detergent that has no apparent crystalline phase in 
aqueous solution.  Thus, your hexagonal crystals are interesting.

My own bias is that I don't trust what the Izit dye suggests, because its 
partitioning is not exclusively dependent on protein.  If your conditions don't 
have ammonium ions or free amines (i.e., no Tris buffer, ammonium sulfate, 
etc.), try adding a microliter droplet of glutaraldehyde next to your protein 
drop.  The  glutaraldehyde will vapor diffuse into the protein droplet and 
protein crystals will become fixed via the lysines.  They won't dissolve in 
buffer; they will also turn a bit yellow from the Schiff's bases being formed.  
Works great on membrane proteins.

It is possible that you can get different crystal habits using different 
detergents, but providing more information about the conditions of the hits and 
the detergent concentrations would make it easier to give you suggestions.  
Were you actually using 5% DDM in the drop or using a 5% DDM stock? With many 
precipitants, high detergent concentrations results in substantial phase 
separation.  Do you see this?

When evaluated suspected "hits" from screens on a membrane protein, I first 
think of inorganic compounds, particularly the dreaded struvite (NH4MgPO4), 
which occurs at very low [PO4], but high ammonium and magnesium concentrations. 
 

I next consider the quality of the protein I am producing and the 
reproducibility of the purification scheme.  Sometimes a "too pure" protein 
prep won't crystallize, while a "less pure" prep does fine.  Contaminating 
proteins, particularly those that copurify with Ni-chelation resins (see 
Bolanos-Garcia and Davies, Biochimica et Biophysica Acta 1760 (2006) 1304–1313 
and Structural Genomics Consortium NATURE METHODS 5(2) 135, 2008) can also 
crystallize at concentrations below 1 mg/mL.  Your protein could appear to be 
95% pure, but but a 1-2% contaminant could be crystallizing, albeit poorly.

A strategy might be 1) to repeat with different protein preps to demonstrate 
reproducibility and 2) to tweak some of your conditions to explore a wider 
environmental space.  Then evaluate whether your protein preparation protocol 
needs optimization.  While a problem, don't focus to much on the detergent a 
sole source of your problems.

Cheers,

Michael



R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On May 6, 2012, at 7:45 AM, anita p wrote:

> Hi All,
> Thanks for your advices. I tried staining with izit dye, please have a look 
> at the attached image. The granules are all stained. 
> The detergent used here is Chaps (as an additive to crystallization condition)
> Does it look like protein ?? 
> If yes, please do advice on methods to improve this.
> 
> 
> I found granules in some other conditions as well and they didn't stain with 
> izit. So probably they were of detergent... Plz suggest
> Thanks in advance for suggestions
> regards
> Anita
> 
> 
> On Fri, May 4, 2012 at 11:43 PM, John K Lee  wrote:
> Hi Anita,
> 
> One question that comes up immediately is why you are using DDM @ 5% 
> concentration.  If it's a typo, understandable. I figure most of us use DDM 
> at 0.05-0.1% concentration, and I've used as low as 0.015%.
> 
> Also, as others have pointed out, until you shoot it, you don't know. But if 
> it's hexaganol with very smooth/non-sharp edges, many of us have seen it 
> using DDM.
> 
> -john
> 
> 
> On May 4, 2012, at 7:48 AM, anita p wrote:
> 
> > Hi All,
> > I would like to have your expert advice on crystals.
> > I am using detergents as 5% (w/v) of DDM 0.4ul in a 4ul (protein + 
> > condition + detergent). The precipitant is 28% peg 20K
> > After 1 day I am able to see little plates of irregular shape .
> >
> > I am able to see some needles if I change into MEGA-10.
> >
> > I am abl

Re: [ccp4bb] Hydrogens added when using Phenix Readyset for ligands

[Pavel Afonine]

Hi Chris,

- there is Phenix mailing list for Phenix-related questions. Please check
http://www.phenix-online.org/

- the data resolution of 2.5A does not mean hydrogen atoms are not present
in the crystal. There are methods to account for them. For details see (and
references therein):

"On contribution of hydrogen atoms to X-ray scattering"
http://www.phenix-online.org/newsletter/

and around pages 41-42 here:
http://www.phenix-online.org/presentations/latest/pavel_phenix_refine.pdf

- if there are reasons for not using H atoms (such as crude initial model,
for instance), you can always trivially remove them:

phenix.reduce model_with_H.pdb -trim > model_no_H.pdb

All the best,
Pavel


On Mon, May 7, 2012 at 6:07 AM, Christopher Browning <
christopher.brown...@epfl.ch> wrote:

> Hi,
>
> I've generally used PRODRG to create paramater files for any ligands I
> add during refinement with CCP4 and/or PHENIX. I've been trying READYSET
> from PHENIX as it greatly helps refining some metal ion positions. But
> when I use READYSET, any ligand I add (in this case EDO or ethylene
> glycol) gets modified to contain hydrogens. My resolution is 2.5,
> definitely not high enough to resolve the hydrogens, so why are they
> added. Should I just leave them there? I have the same problem with
> another structure which has bizarre sugar molecules so they are not
> standard COOT/CCP4/PHENIX small molecules.
>
> As a test, I removed the hydrogens added to the ligands and the refined
> protein coordinates are way different than when the hydrogens are left
> in?
>
>
> Cheers,
>
> Chris
>
>
>
> --
> Dr. Christopher Browning
> Post-Doctor to Prof. Petr Leiman
> EPFL
> BSP-416
> 1015 Lausanne
> Switzerland
> Tel: 0041 (0) 02 16 93 04 40
>

Re: [ccp4bb] Hydrogens added when using Phenix Readyset for ligands

[Christopher Browning]

Hi Tim,

Yeah, riding hydrogens have always been on. I guess I need some more
investigation.

Chris


On Mon, 2012-05-07 at 15:36 +0200, Tim Gruene wrote:
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Hi Chris,
> 
> If  the absence/ presence of hydrogens in the coordinate file made a
> difference in the refinement, I guess some parameter setting for the
> refinement program is awkward. Did you refine with riding hydrogens in
> both cases? Hydrogens can and should be added in riding positions even
> at low resolution, since they do not add to the number of parameters
> but help with anti-bumping restraints.
> 
> Tim
> 
> On 05/07/12 15:07, Christopher Browning wrote:
> > Hi,
> > 
> > I've generally used PRODRG to create paramater files for any
> > ligands I add during refinement with CCP4 and/or PHENIX. I've been
> > trying READYSET from PHENIX as it greatly helps refining some metal
> > ion positions. But when I use READYSET, any ligand I add (in this
> > case EDO or ethylene glycol) gets modified to contain hydrogens. My
> > resolution is 2.5, definitely not high enough to resolve the
> > hydrogens, so why are they added. Should I just leave them there? I
> > have the same problem with another structure which has bizarre
> > sugar molecules so they are not standard COOT/CCP4/PHENIX small
> > molecules.
> > 
> > As a test, I removed the hydrogens added to the ligands and the
> > refined protein coordinates are way different than when the
> > hydrogens are left in?
> > 
> > 
> > Cheers,
> > 
> > Chris
> > 
> > 
> > 
> 
> - -- 
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
> iD8DBQFPp8/jUxlJ7aRr7hoRAl2PAJ0WG7405GO1V96UzzFJd53x7rijwQCfeA1i
> NOGF4DTnAQNGxe6N4mbpXLM=
> =ov1h
> -END PGP SIGNATURE-

-- 
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Switzerland
Tel: 0041 (0) 02 16 93 04 40

Re: [ccp4bb] Hydrogens added when using Phenix Readyset for ligands

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi Chris,

If  the absence/ presence of hydrogens in the coordinate file made a
difference in the refinement, I guess some parameter setting for the
refinement program is awkward. Did you refine with riding hydrogens in
both cases? Hydrogens can and should be added in riding positions even
at low resolution, since they do not add to the number of parameters
but help with anti-bumping restraints.

Tim

On 05/07/12 15:07, Christopher Browning wrote:
> Hi,
> 
> I've generally used PRODRG to create paramater files for any
> ligands I add during refinement with CCP4 and/or PHENIX. I've been
> trying READYSET from PHENIX as it greatly helps refining some metal
> ion positions. But when I use READYSET, any ligand I add (in this
> case EDO or ethylene glycol) gets modified to contain hydrogens. My
> resolution is 2.5, definitely not high enough to resolve the
> hydrogens, so why are they added. Should I just leave them there? I
> have the same problem with another structure which has bizarre
> sugar molecules so they are not standard COOT/CCP4/PHENIX small
> molecules.
> 
> As a test, I removed the hydrogens added to the ligands and the
> refined protein coordinates are way different than when the
> hydrogens are left in?
> 
> 
> Cheers,
> 
> Chris
> 
> 
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPp8/jUxlJ7aRr7hoRAl2PAJ0WG7405GO1V96UzzFJd53x7rijwQCfeA1i
NOGF4DTnAQNGxe6N4mbpXLM=
=ov1h
-END PGP SIGNATURE-

[ccp4bb] Hydrogens added when using Phenix Readyset for ligands

[Christopher Browning]

Hi,

I've generally used PRODRG to create paramater files for any ligands I
add during refinement with CCP4 and/or PHENIX. I've been trying READYSET
from PHENIX as it greatly helps refining some metal ion positions. But
when I use READYSET, any ligand I add (in this case EDO or ethylene
glycol) gets modified to contain hydrogens. My resolution is 2.5,
definitely not high enough to resolve the hydrogens, so why are they
added. Should I just leave them there? I have the same problem with
another structure which has bizarre sugar molecules so they are not
standard COOT/CCP4/PHENIX small molecules.

As a test, I removed the hydrogens added to the ligands and the refined
protein coordinates are way different than when the hydrogens are left
in?  


Cheers,

Chris



-- 
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Switzerland
Tel: 0041 (0) 02 16 93 04 40

Re: [ccp4bb] ShelXL and Coot

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Kalyan,

the reason why you cannot read in the .res-file is that you don't have
RESI-cards. They are required by coot.

For the same reason your PDB-file does not contain any information
that would allow coot to refine the coordinates.

As far as I know coot does not create restraints from the .res-file,
hence you need to give your structure residue names for which
cif-files exist with constraints.

However, I since you are working on such a small structure at atomic
resolution I suggest you use Christian Huebschle's shelxle instead of
coot as GUI for shelxl refinement.

You find it at
http://ewald.ac.chemie.uni-goettingen.de/shelx/eingabe.php

Best,
Tim

On 05/07/12 05:38, Kalyan wrote:
> Hi George:
> 
> I am facing the same problem reading .pdb file generated from
> shelxl in coot. I have tried inserting the space group name in the
> CRYST1 record but still unable to read the file. COOT is able to
> read the .res and .fcf file and I can see the map but COOT is not
> allowing me to build the molecule from .res file. I am trying to
> refine the solution obtained from direct methods using SHELXS. What
> are the parameters that I need to change in the .res or .pdb file
> to be able to modify and build the molecule in the first place. Do
> I have to replace 'Q' with 'C' or some other changes, and then how
> will I connect the atoms in COOT? The appearance of the part of the
> .pdb file is shown below (space group name is manually inserted).
> 
> CRYST1   21.139   51.900   31.090  90.00 104.69  90.00 P 1 21/a 1
> 1 SCALE1  0.047306  0.00  0.012400   0.00 SCALE2
> 0.00  0.019268  0.00   0.00 SCALE3  0.00
> 0.00  0.033251   0.00 ATOM  1  S1  0
> -0.215  21.413  10.717 1.000  4.73 ATOM  2  S2  0
> 0.551  21.040  12.581 1.000  4.64 ATOM  3  S3  0
> 1.526  18.258   7.542 1.000  5.05 ATOM  4  S4  0
> 0.802  16.539   8.374 1.000  4.95 ATOM  5  S5  0
> 11.171  22.546   8.376 1.000  5.35 ATOM  6  S6  0
> 9.747  22.514   6.903 1.000  5.85 ATOM  7  Q2  0
> -1.517  23.108  -2.742 1.000  0.41 ATOM  8  Q3  0
> -5.977  20.216  -0.493 1.000  1.03 ATOM  9  Q4  0
> 13.309  15.857  11.911 1.000  1.63 ATOM 10  Q6  0
> -2.030  15.341   9.819 1.000  2.42 ATOM 11  Q7  0
> 0.489  22.190   6.739 1.000  2.46 ATOM 12  Q8  0
> -1.247  24.612   5.096 1.000  2.29 ATOM 13  Q9  0
> 4.588  21.769  11.905 1.000  2.41 ATOM 14  Q10 0
> -3.731  19.433  12.713 1.000  2.78 ATOM 15  Q11 0
> 7.498  18.251   9.847 1.000  2.39 ATOM 16  Q12 0
> 3.703  20.519   8.595 1.000  2.22 ATOM 17  Q13 0
> 0.926  24.359  15.407 1.000  2.33
> 
> Thanks,
> 
> Kalyan
> 
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPp5CJUxlJ7aRr7hoRAut9AKCwBvWucGGdz9sfqRnlymWePgjs7gCg2Ewk
BwWxLB0dWfOmj6cr/Shpuqs=
=8jcn
-END PGP SIGNATURE-

Re: [ccp4bb] detergent or protein

[Herman . Schreuder]

Hi Anita,
I do not have much experience with detergents, but spherulites like you
showed often occur with proteins. Unfortunately, these things usually do
not diffract. On the other hand, they occur often in conditions close to
good crystallization conditions, so in your case I would try to optimize
the conditions (fine pH, precipitant screen, additives etc.). It does
not always work though.
I would also wash a number of shperulites and put them on a gel to see
if they contain protein.
 
Best,
Herman 




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of anita p
Sent: Sunday, May 06, 2012 1:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] detergent or protein


Hi All,
Thanks for your advices. I tried staining with izit dye, please
have a look at the attached image. The granules are all stained. 
The detergent used here is Chaps (as an additive to
crystallization condition)
Does it look like protein ?? 
If yes, please do advice on methods to improve this.


I found granules in some other conditions as well and they
didn't stain with izit. So probably they were of detergent... Plz
suggest
Thanks in advance for suggestions
regards
Anita



On Fri, May 4, 2012 at 11:43 PM, John K Lee 
wrote:


Hi Anita,

One question that comes up immediately is why you are
using DDM @ 5% concentration.  If it's a typo, understandable. I figure
most of us use DDM at 0.05-0.1% concentration, and I've used as low as
0.015%.

Also, as others have pointed out, until you shoot it,
you don't know. But if it's hexaganol with very smooth/non-sharp edges,
many of us have seen it using DDM.

-john


On May 4, 2012, at 7:48 AM, anita p wrote:


> Hi All,
> I would like to have your expert advice on crystals.
> I am using detergents as 5% (w/v) of DDM 0.4ul in a
4ul (protein + condition + detergent). The precipitant is 28% peg 20K
> After 1 day I am able to see little plates of
irregular shape .
>
> I am able to see some needles if I change into
MEGA-10.
>
> I am able to see some hexagonal 3D crystals when I
change the precipitant to Ammonium sulphate and the detergent to
Anapoe-C10E6
>
> Is there a possibility to check whether this crystals
are protein or of detergent, even before shooting it ?
> Does the control drop test help ??
>
> awaiting for feedback
> Anita

[ccp4bb]

[Herman . Schreuder]

Dear Ruby,
 
With this information I can give some more concrete hints:
 
- Add the sugar (monosaccharide, disaccharide?) that the lectin is
supposed to bind to your crystallization trials.
- Try different protein concentrations: 10 mg/ml, 100 mg/ml. In the
latter case you may have to dilute your screens.
- Try the same lectin from different plants.
- Do you purify it from the plant? I once purified a plant protein and
had to be very careful for phenol hydroxylases and had to add something
like dithionite before putting the plant in the blender. Adding protease
inhibitors may not hurt as well. You may want to check the integrity of
your protein for proteolytic cuts (giving ragged N- or C-termini) and
for other modifications making your protein heterogeneous. Is it
glycosylated? In that case you may want to try to remove it with some
glycanase.
 
Best,
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of ruby singh
Sent: Saturday, May 05, 2012 8:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb]




On Sat, May 5, 2012 at 12:17 PM, ruby singh
 wrote:


Sir, 
i m sorry for not giving full details of  protein.
its plant lectin protein on which im working currently.
its sequence information is very little. crystallization has become near
to impossible.  the protein is a tetramer( of 28KDa). its purification
is very simple...but for crystallization i tried many things nt working
though(Hampton Reserach-pegion and index/CSS1/JCSG etc..). need some
help. the protein conecentration used was 30mg/ml stock . most of the
crystallization condition shows precipitation. the set up was put at
20C. the protein is very stable in MilliQ water upto 100mg/ml
concentration with no precipitation.

the protein doesnt aggregate..doesnt loose activity even after
boiling at 80C.

getting its crystal is important for my thesis. so need help.