Re: [ccp4bb] DTU vs DTT ?

[Engin Özkan]

Hi Emmanuel,

The monomer library is great, but it is also created by scientists (and 
computers) that can err. I remember a few cases (some reported on the 
ccp4bb) where mistakes were revealed and later fixed. Just because 
something is in the monomer library that does absolve anyone from not 
checking their ligands. And that still should not decrease our 
appreciation of scientists for the selfless task of creating the monomer 
library that ships with ccp4 and phenix.


Another great resource is Gerard Kleywegt's HIC-UP. In the cases where I 
suspected the monomer library of having errors, HIC-UP had the right 
geometry. In this case, "Get Monomer"ing DTT and downloading HIC-UP's 
DTT give me opposite chiralities on C3. I don't know which one is true, 
but I suspect HIC-UP is. You can download restraint files through links 
(to prodrg, I believe) from HIC-UP.


Cheers,
Engin

On 11/25/10 8:38 AM, Linda Schuldt wrote:

Hi Emmanuel,

it is hard for me to imagine that Coot has the wrong stereoisomer. So what
I think might have happend is the following:
You have imported the correct DTT, but when you have fitted the molecule
into the map you might have distorted the sterochemistry at the C3 atom.
And then it was refined like that. I had once a similar observation with
MPD. Did you insert the three DTT molecules individually, or did you "copy
and paste" the same molecule around (which might explain why you have it
for all three)?
And why don´t you just import DTT into Coot and check yourself if it has
the correct sterochemistry. This is a fast and easy way to find out if
something is wrong in your Coot library.
Unless you have very high resolution which clearly shows that you have DTU
instead of DTT, I would change your coordinate file to DTT and send it
again to the pdb.

Hope this helps you.

Best wishes,
Linda


Emmanuel Saridakis schrieb:

Dear All,

Possibly a trivial question but your experience would be much appreciated:

I recently submitted a structure to PDB containing 3 DTT (dithiothreitol)
molecules, or so I thought. The molecules had been imported and fitted
with Coot using the Get Monomer... instruction with the code DTT. The
Annotator responded, quite rightly as it turns out, as follows:

Please note DTT in your coordinates has been changed to DTU since it

has incorrect stereochemistry as DTT.
Please review the stereochemistory in the attached validation report
summary.

You can send me corrected stereochemistry for DTT if you want it
changed back.

DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
C4 H10 O2 S2

DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
C4 H10 O2 S2

So, is the "DTT" monomer of Coot in fact its stereoisomer known as
dithioerythritol? Should I import the correct DTT from elsewhere and
re-refine or is there something else behind this?

Thanks a lot for any suggestions!


Emmanuel





--
Engin Özkan
Post-doctoral Scholar
Laboratory of K. Christopher Garcia
Howard Hughes Medical Institute
Dept of Molecular and Cellular Physiology
279 Campus Drive, Beckman Center B173
Stanford School of Medicine
Stanford, CA 94305
ph: (650)-498-7111

Re: [ccp4bb] Tough 'shell' on disturbed drop

[Jürgen Bosch]

Often you can also avoid this skin formation by adding a bit of your reservoir 
solution first to make it a larger droplet.
Then the microtools as mentioned earlier or just two loops
Jürgen 

..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Nov 25, 2010, at 6:45, Rick  wrote:

> Dear CCP4
> 
> I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 
> 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen 
> orthophospate and glycine). The loop i used had been washed more than 10 
> times with deionised water (so assumed as 'clean'). The crystals had grown at 
> 17degreesC, and looped out probably just below room temperature (~20-23 
> degreesC). When transferred to 5% glycerol cryo-buffer the crystal 
> disintegrated (maybe due to glycerol being an unfavourable addition to the 
> mother-liquor). When i looked back at the original cluster-containing drop, a 
> very tough shell had formed over the surface of the drop, from which chunks 
> could be dug out...the nearest analogy is maybe like when you blow-torch 
> sugar on top of creme brulee, and have to crack it with your spoon. The 
> crystals within had also disintegrated. Any clues to what might have caused 
> this very tough shell to form, and maybe how to deal with it? 
> 
> Much appreciated
> 
> Rick Salmon

Re: [ccp4bb] DTU vs DTT ?

[MARTYN SYMMONS]

Just a footnote on the MPD which you mention - correct me if I am wrong but I 
think we buy it as a racemic mix and so you are just as likely to get the 4R 
which is ligand name MRD.

That is in 189 entries in the PDB; 

http://www.ebi.ac.uk/pdbe-srv/view/search?Chemical_code=MRD

compared with 4S-MPD 

which wins out at 585 entries

http://www.ebi.ac.uk/pdbe-srv/view/search?Chemical_code=MPD


cheers,
   Martyn 

Martyn Symmons
Cambridge



--- On Thu, 25/11/10, Linda Schuldt  wrote:

> From: Linda Schuldt 
> Subject: Re: [ccp4bb] DTU vs DTT ?
> To: CCP4BB@JISCMAIL.AC.UK
> Date: Thursday, 25 November, 2010, 16:38
> Hi Emmanuel,
> 
> it is hard for me to imagine that Coot has the wrong
> stereoisomer. So what
> I think might have happend is the following:
> You have imported the correct DTT, but when you have fitted
> the molecule
> into the map you might have distorted the sterochemistry at
> the C3 atom.
> And then it was refined like that. I had once a similar
> observation with
> MPD. Did you insert the three DTT molecules individually,
> or did you "copy
> and paste" the same molecule around (which might explain
> why you have it
> for all three)?
> And why don´t you just import DTT into Coot and check
> yourself if it has
> the correct sterochemistry. This is a fast and easy way to
> find out if
> something is wrong in your Coot library.
> Unless you have very high resolution which clearly shows
> that you have DTU
> instead of DTT, I would change your coordinate file to DTT
> and send it
> again to the pdb.
> 
> Hope this helps you.
> 
> Best wishes,
> Linda
> 
> 
> Emmanuel Saridakis schrieb:
> > Dear All,
> >
> > Possibly a trivial question but your experience would
> be much appreciated:
> >
> > I recently submitted a structure to PDB containing 3
> DTT (dithiothreitol)
> > molecules, or so I thought. The molecules had been
> imported and fitted
> > with Coot using the Get Monomer... instruction with
> the code DTT. The
> > Annotator responded, quite rightly as it turns out, as
> follows:
> >
> > Please note DTT in your coordinates has been changed
> to DTU since it
> >>> has incorrect stereochemistry as DTT.
> >>> Please review the stereochemistory in the
> attached validation report
> >>> summary.
> >>>
> >>> You can send me corrected stereochemistry for
> DTT if you want it
> >>> changed back.
> >>>
> >>> DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
> >>> C4 H10 O2 S2
> >>>
> >>> DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
> >>> C4 H10 O2 S2
> >
> > So, is the "DTT" monomer of Coot in fact its
> stereoisomer known as
> > dithioerythritol? Should I import the correct DTT from
> elsewhere and
> > re-refine or is there something else behind this?
> >
> > Thanks a lot for any suggestions!
> >
> >
> > Emmanuel
> >
> >
>

Re: [ccp4bb] DTU vs DTT ?

[Phil Evans]

 
DTT.cif in $CLIBD_MON has explicit chiralities, assuming these are right

 DTT  chir_01  C2 C1 O2 C3positiv
 DTT  chir_02  C3 C2 O3 C4negativ

On 25 Nov 2010, at 17:11, Paul Emsley wrote:

> Credit where it's due, Coot's "Get Monomer" is just a wrapper for LIBCHECK.
> 
> If you look at the restraints for DTT after Get Monomer, you will notice that 
> chiral centres are both marked as "both".  Using the restraints editor (or 
> otherwise) change them to "positive" (C2) and "negative" (C3) and everything 
> will be fine (you may need to flip the H2 and H3 hydrogens if you have them).
> 
> If you wanted to use something other than LIBCHECK, you could search the 
> SBase monomers and prodrgify the result (search using the substring 
> "dithiobutane" (and "mercaptobutane" for DTU) incidentally).
> 
> Paul
> 
> 
> On 25/11/10 16:38, Linda Schuldt wrote:
>> Hi Emmanuel,
>> 
>> it is hard for me to imagine that Coot has the wrong stereoisomer. So what
>> I think might have happend is the following:
>> You have imported the correct DTT, but when you have fitted the molecule
>> into the map you might have distorted the sterochemistry at the C3 atom.
>> And then it was refined like that. I had once a similar observation with
>> MPD. Did you insert the three DTT molecules individually, or did you "copy
>> and paste" the same molecule around (which might explain why you have it
>> for all three)?
>> And why don´t you just import DTT into Coot and check yourself if it has
>> the correct sterochemistry. This is a fast and easy way to find out if
>> something is wrong in your Coot library.
>> Unless you have very high resolution which clearly shows that you have DTU
>> instead of DTT, I would change your coordinate file to DTT and send it
>> again to the pdb.
>> 
>> Hope this helps you.
>> 
>> Best wishes,
>> Linda
>> 
>> 
>> Emmanuel Saridakis schrieb:
>>   
>>> Dear All,
>>> 
>>> Possibly a trivial question but your experience would be much appreciated:
>>> 
>>> I recently submitted a structure to PDB containing 3 DTT (dithiothreitol)
>>> molecules, or so I thought. The molecules had been imported and fitted
>>> with Coot using the Get Monomer... instruction with the code DTT. The
>>> Annotator responded, quite rightly as it turns out, as follows:
>>> 
>>> Please note DTT in your coordinates has been changed to DTU since it
>>> 
> has incorrect stereochemistry as DTT.
> Please review the stereochemistory in the attached validation report
> summary.
> 
> You can send me corrected stereochemistry for DTT if you want it
> changed back.
> 
> DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
> C4 H10 O2 S2
> 
> DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
> C4 H10 O2 S2
> 
>>> So, is the "DTT" monomer of Coot in fact its stereoisomer known as
>>> dithioerythritol? Should I import the correct DTT from elsewhere and
>>> re-refine or is there something else behind this?
>>> 
>>> Thanks a lot for any suggestions!
>>> 
>>> 
>>> Emmanuel
>>> 
>>> 
>>> 

Re: [ccp4bb] DTU vs DTT ?

[Paul Emsley]

Credit where it's due, Coot's "Get Monomer" is just a wrapper for LIBCHECK.

If you look at the restraints for DTT after Get Monomer, you will notice 
that chiral centres are both marked as "both".  Using the restraints 
editor (or otherwise) change them to "positive" (C2) and "negative" (C3) 
and everything will be fine (you may need to flip the H2 and H3 
hydrogens if you have them).


If you wanted to use something other than LIBCHECK, you could search the 
SBase monomers and prodrgify the result (search using the substring 
"dithiobutane" (and "mercaptobutane" for DTU) incidentally).


Paul


On 25/11/10 16:38, Linda Schuldt wrote:

Hi Emmanuel,

it is hard for me to imagine that Coot has the wrong stereoisomer. So what
I think might have happend is the following:
You have imported the correct DTT, but when you have fitted the molecule
into the map you might have distorted the sterochemistry at the C3 atom.
And then it was refined like that. I had once a similar observation with
MPD. Did you insert the three DTT molecules individually, or did you "copy
and paste" the same molecule around (which might explain why you have it
for all three)?
And why don´t you just import DTT into Coot and check yourself if it has
the correct sterochemistry. This is a fast and easy way to find out if
something is wrong in your Coot library.
Unless you have very high resolution which clearly shows that you have DTU
instead of DTT, I would change your coordinate file to DTT and send it
again to the pdb.

Hope this helps you.

Best wishes,
Linda


Emmanuel Saridakis schrieb:
   

Dear All,

Possibly a trivial question but your experience would be much appreciated:

I recently submitted a structure to PDB containing 3 DTT (dithiothreitol)
molecules, or so I thought. The molecules had been imported and fitted
with Coot using the Get Monomer... instruction with the code DTT. The
Annotator responded, quite rightly as it turns out, as follows:

Please note DTT in your coordinates has been changed to DTU since it
 

has incorrect stereochemistry as DTT.
Please review the stereochemistory in the attached validation report
summary.

You can send me corrected stereochemistry for DTT if you want it
changed back.

DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
C4 H10 O2 S2

DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
C4 H10 O2 S2
 

So, is the "DTT" monomer of Coot in fact its stereoisomer known as
dithioerythritol? Should I import the correct DTT from elsewhere and
re-refine or is there something else behind this?

Thanks a lot for any suggestions!


Emmanuel


 

Re: [ccp4bb] Tough 'shell' on disturbed drop

[Ed Pozharski]

If it's on a glass coverslip, another good trick is to (carefully) cut
through the skin around the crystal with a razor blade.  With some
practice, one manages not to get the crystal entangled in the skin.

On Thu, 2010-11-25 at 16:03 +, Frederic VELLIEUX wrote:
> Hi,
> 
> In our hands, the crystallisation droplets of glycosomal pyruvate
> phosphate dikinase had a 'skin' of what I thought was denatured
> protein at the surface of every crystallisation droplet. We had to
> learn to use the crystal microtools (such as a microknife, or a
> micro-needle can't remember what we have - sold by Hampton Research
> and I do not own shares in this company) to be able to cut this skin
> and drag it to the side of the droplet before being able to suck out
> the crystals. A bit like dissection under the binoculars.
> 
> Fred.
> 
> > Message du 25/11/10 15:56
> > De : "Rick" 
> > A : CCP4BB@JISCMAIL.AC.UK
> > Copie à : 
> > Objet : [ccp4bb] Tough 'shell' on disturbed drop
> > 
> > Dear CCP4
> > 
> > I looped a v.thin rod emerging from a cluster of v.thin rods
> that grew in 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic
> acid, sodium dihydrogen orthophospate and glycine). The loop i
> used had been washed more than 10 times with deionised water
> (so assumed as 'clean'). The crystals had grown at 17degreesC,
> and looped out probably just below room temperature (~20-23
> degreesC). When transferred to 5% glycerol cryo-buffer the
> crystal disintegrated (maybe due to glycerol being an
> unfavourable addition to the mother-liquor). When i looked
> back at the original cluster-containing drop, a very tough
> shell had formed over the surface of the drop, from which
> chunks could be dug out...the nearest analogy is maybe like
> when you blow-torch sugar on top of creme brulee, and have to
> crack it with your spoon. The crystals within had also
> disintegrated. Any clues to what might have caused this very
> tough shell to form, and maybe how to deal with it? 
> > 
> > Much appreciated
> > 
> > Rick Salmon

Re: [ccp4bb] DTU vs DTT ?

[Linda Schuldt]

Hi Emmanuel,

it is hard for me to imagine that Coot has the wrong stereoisomer. So what
I think might have happend is the following:
You have imported the correct DTT, but when you have fitted the molecule
into the map you might have distorted the sterochemistry at the C3 atom.
And then it was refined like that. I had once a similar observation with
MPD. Did you insert the three DTT molecules individually, or did you "copy
and paste" the same molecule around (which might explain why you have it
for all three)?
And why don´t you just import DTT into Coot and check yourself if it has
the correct sterochemistry. This is a fast and easy way to find out if
something is wrong in your Coot library.
Unless you have very high resolution which clearly shows that you have DTU
instead of DTT, I would change your coordinate file to DTT and send it
again to the pdb.

Hope this helps you.

Best wishes,
Linda


Emmanuel Saridakis schrieb:
> Dear All,
>
> Possibly a trivial question but your experience would be much appreciated:
>
> I recently submitted a structure to PDB containing 3 DTT (dithiothreitol)
> molecules, or so I thought. The molecules had been imported and fitted
> with Coot using the Get Monomer... instruction with the code DTT. The
> Annotator responded, quite rightly as it turns out, as follows:
>
> Please note DTT in your coordinates has been changed to DTU since it
>>> has incorrect stereochemistry as DTT.
>>> Please review the stereochemistory in the attached validation report
>>> summary.
>>>
>>> You can send me corrected stereochemistry for DTT if you want it
>>> changed back.
>>>
>>> DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
>>> C4 H10 O2 S2
>>>
>>> DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
>>> C4 H10 O2 S2
>
> So, is the "DTT" monomer of Coot in fact its stereoisomer known as
> dithioerythritol? Should I import the correct DTT from elsewhere and
> re-refine or is there something else behind this?
>
> Thanks a lot for any suggestions!
>
>
> Emmanuel
>
>

[ccp4bb] DTU vs DTT ?

[Emmanuel Saridakis]

Dear All,

Possibly a trivial question but your experience would be much appreciated:

I recently submitted a structure to PDB containing 3 DTT (dithiothreitol) 
molecules, or so I thought. The molecules had been imported and fitted with 
Coot using the Get Monomer... instruction with the code DTT. The Annotator 
responded, quite rightly as it turns out, as follows:

Please note DTT in your coordinates has been changed to DTU since it
>> has incorrect stereochemistry as DTT.
>> Please review the stereochemistory in the attached validation report
>> summary.
>>
>> You can send me corrected stereochemistry for DTT if you want it
>> changed back.
>>
>> DTU (2R,3S)-1,4-disulfanylbutane-2,3-diol
>> C4 H10 O2 S2
>>
>> DTT (2R,3R)-1,4-disulfanylbutane-2,3-diol
>> C4 H10 O2 S2

So, is the "DTT" monomer of Coot in fact its stereoisomer known as 
dithioerythritol? Should I import the correct DTT from elsewhere and re-refine 
or is there something else behind this?

Thanks a lot for any suggestions!


Emmanuel

Re: [ccp4bb] Tough 'shell' on disturbed drop

[Frederic VELLIEUX]

Hi,

In our hands, the crystallisation droplets of glycosomal pyruvate phosphate 
dikinase had a 'skin' of what I thought was denatured protein at the surface of 
every crystallisation droplet. We had to learn to use the crystal microtools 
(such as a microknife, or a micro-needle can't remember what we have - sold by 
Hampton Research and I do not own shares in this company) to be able to cut 
this skin and drag it to the side of the droplet before being able to suck out 
the crystals. A bit like dissection under the binoculars.

Fred.

> Message du 25/11/10 15:56
> De : "Rick" 
> A : CCP4BB@JISCMAIL.AC.UK
> Copie à : 
> Objet : [ccp4bb] Tough 'shell' on disturbed drop
> 
> Dear CCP4
> 
> I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 
> 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen 
> orthophospate and glycine). The loop i used had been washed more than 10 
> times with deionised water (so assumed as 'clean'). The crystals had grown at 
> 17degreesC, and looped out probably just below room temperature (~20-23 
> degreesC). When transferred to 5% glycerol cryo-buffer the crystal 
> disintegrated (maybe due to glycerol being an unfavourable addition to the 
> mother-liquor). When i looked back at the original cluster-containing drop, a 
> very tough shell had formed over the surface of the drop, from which chunks 
> could be dug out...the nearest analogy is maybe like when you blow-torch 
> sugar on top of creme brulee, and have to crack it with your spoon. The 
> crystals within had also disintegrated. Any clues to what might have caused 
> this very tough shell to form, and maybe how to deal with it? 
> 
> Much appreciated
> 
> Rick Salmon

Re: [ccp4bb] Tough 'shell' on disturbed drop

[David Schuller]

The shell may be denatured protein. Remove the protein from the 
experiment and the problem will likely go away.



On 11/25/10 09:45, Rick wrote:

Dear CCP4

I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 
29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen 
orthophospate and glycine). The loop i used had been washed more than 10 times 
with deionised water (so assumed as 'clean'). The crystals had grown at 
17degreesC, and looped out probably just below room temperature (~20-23 
degreesC). When transferred to 5% glycerol cryo-buffer the crystal 
disintegrated (maybe due to glycerol being an unfavourable addition to the 
mother-liquor). When i looked back at the original cluster-containing drop, a 
very tough shell had formed over the surface of the drop, from which chunks 
could be dug out...the nearest analogy is maybe like when you blow-torch sugar 
on top of creme brulee, and have to crack it with your spoon. The crystals 
within had also disintegrated. Any clues to what might have caused this very 
tough shell to form, and maybe how to deal with it?

Much appreciated

Rick Salmon



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

Re: [ccp4bb] Tough 'shell' on disturbed drop

[Artem Evdokimov]

Hi,

You're working with a very 'rich' crystallization condition. It probably was
supersaturated or close to super-saturated with respect to something, and
that something crashed out on the surface (where liquid contacted air)
forming a crust. Your loop (while perfectly clean) can also be the source of
nucleation. Just a mere act of opening a drop can cause phenomena like this
one. How old was your drop (older partially evaporated drops tend to do this
more often)?

Sometimes it's possible to slow this crust formation down by coating the
drop with a film of oil, as soon as you open the reservoir. I would also
recommend avoiding complex buffers like that one unless there's literally no
other way to grow your crystals - there's a lot of inherent trouble
(especially with phosphate).

Artem

On Thu, Nov 25, 2010 at 8:45 AM, Rick  wrote:

> Dear CCP4
>
> I looped a v.thin rod emerging from a cluster of v.thin rods that grew in
> 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen
> orthophospate and glycine). The loop i used had been washed more than 10
> times with deionised water (so assumed as 'clean'). The crystals had grown
> at 17degreesC, and looped out probably just below room temperature (~20-23
> degreesC). When transferred to 5% glycerol cryo-buffer the crystal
> disintegrated (maybe due to glycerol being an unfavourable addition to the
> mother-liquor). When i looked back at the original cluster-containing drop,
> a very tough shell had formed over the surface of the drop, from which
> chunks could be dug out...the nearest analogy is maybe like when you
> blow-torch sugar on top of creme brulee, and have to crack it with your
> spoon. The crystals within had also disintegrated. Any clues to what might
> have caused this very tough shell to form, and maybe how to deal with it?
>
> Much appreciated
>
> Rick Salmon

[ccp4bb] Tough 'shell' on disturbed drop

[Rick]

Dear CCP4

I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 
29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen 
orthophospate and glycine). The loop i used had been washed more than 10 times 
with deionised water (so assumed as 'clean'). The crystals had grown at 
17degreesC, and looped out probably just below room temperature (~20-23 
degreesC). When transferred to 5% glycerol cryo-buffer the crystal 
disintegrated (maybe due to glycerol being an unfavourable addition to the 
mother-liquor). When i looked back at the original cluster-containing drop, a 
very tough shell had formed over the surface of the drop, from which chunks 
could be dug out...the nearest analogy is maybe like when you blow-torch sugar 
on top of creme brulee, and have to crack it with your spoon. The crystals 
within had also disintegrated. Any clues to what might have caused this very 
tough shell to form, and maybe how to deal with it? 

Much appreciated

Rick Salmon

Re: [ccp4bb] Protein sequencing service?

[Mark Brooks]

Jeff Keen is good, but just to add to the list "Cambridge Peptides"
http://www.cambridgepeptides.com/proteinsequencing.html based in Birmingham
(!), is fast and efficient from either membrane or directly from gel slices.

Yours,

Mark

On 24 September 2010 16:46, Rex Palmer  wrote:

>  Can anyone please recommend a UK protein sequencing service. Our protein
> is dimeric with reported molecuar weight of about 85kDa.Our funds are
> somewhat limited.
>
> Thanks in advance.
>
> Rex Palmer
> Birkbeck College, London
>
>
> RexPalmer2010.homestead.com 
>

Re: [ccp4bb] Protein sequencing service?

[Tom Murray-Rust]

I would second that suggestion, I just had some bands sequenced to
identify internal cleavage sites within a protein and got very clear
sequence back.

Cheers

Tom

On Thu, Nov 25, 2010 at 11:20 AM, MARTYN SYMMONS
 wrote:
> I have had excellent service from Mike Weldon (m.a.wel...@bioc.cam.ac.uk) at 
> PNAC University of Cambridge Biochemistry. He will accept outside orders 
> subject to capacity being available.
>
> http://www.bioc.cam.ac.uk/pnac/
>
> Cheers
>      Martyn
>
> Martyn Symmons
> EBI - Cambridge
>>
>> On 24/09/2010 4:46, Rex Palmer wrote:
>> > Can anyone please recommend a UK protein sequencing
>> service. Our protein
>> > is dimeric with reported molecuar weight of about
>> 85kDa.Our funds are
>> > somewhat limited.
>> > Thanks in advance.
>> > Rex Palmer
>> > Birkbeck College, London
>> > RexPalmer2010.homestead.com
>>
>> --         Andreas Förster,
>> Research Associate
>>         Paul Freemont & Xiaodong
>> Zhang Labs
>> Department of Biochemistry, Imperial College London
>>             http://www.msf.bio.ic.ac.uk
>>
>



-- 
Skype: tom.murray.rust
+44 7970 480 601 (UK Number)
+61 41535 4486 (Aussie Mobile)

Re: [ccp4bb] Protein sequencing service?

[MARTYN SYMMONS]

I have had excellent service from Mike Weldon (m.a.wel...@bioc.cam.ac.uk) at 
PNAC University of Cambridge Biochemistry. He will accept outside orders 
subject to capacity being available.

http://www.bioc.cam.ac.uk/pnac/

Cheers
  Martyn 

Martyn Symmons
EBI - Cambridge
> 
> On 24/09/2010 4:46, Rex Palmer wrote:
> > Can anyone please recommend a UK protein sequencing
> service. Our protein
> > is dimeric with reported molecuar weight of about
> 85kDa.Our funds are
> > somewhat limited.
> > Thanks in advance.
> > Rex Palmer
> > Birkbeck College, London
> > RexPalmer2010.homestead.com
> 
> --         Andreas Förster,
> Research Associate
>         Paul Freemont & Xiaodong
> Zhang Labs
> Department of Biochemistry, Imperial College London
>             http://www.msf.bio.ic.ac.uk
>

Re: [ccp4bb] unusual diffraction spots

[Mark J van Raaij]

Dear Hubing,
please don't discard a structure just because the Rfree > 30%, or approve a 
structure just because the Rfree < 30%. Other quality parameters like relative 
absence of clashes and Ramachandran outliers are much more important. Perhaps 
even more important is whether the structure gives an important new insight - 
even a structure with mediocre overall quality parameters may yield a clear, 
defendable, interesting feature.
Of course, this is not to say one should not try to get better crystals and 
data where at all possible...but perhaps what you wanted to discover about your 
protein is already in your present structure.
Mark van Raaij


On 25 Nov 2010, at 06:09, Hubing Lou wrote:

> 
> 
> 
> To further clarify things, the data was collected at a synchrotron beamline 
> with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The 
> detector type was MarCCD.
> The crystal was multiple-faced trigonal (space group P3121) the size was 
> about 0.1*0.1*0.15mm. The exposure time was 2s for each image. 
> 
> I am currently refining the structure, however the Rfree stays above 30%. A 
> close inspection shows at high resolution shell the spots become rod shaped. 
> As I said we are preparing new constructs with N-terminal his-tag cleaved. 
> But any other good suggestions out there might be helpful to avoid future 
> frustration.
> 
> Thanks,
> Hubing
> 
> On Wed, Nov 24, 2010 at 10:26 PM,  wrote:
> I think there may be two effects going on here:
> 
>  
> I think the “ears” on the round spots which also feature on the more rod 
> shaped spots if you look closely could be related to a misalignment of the 
> beamline optics.
> 
>  
> I think the change in spot shape from round to rod shaped is due to the 
> crystal quality.
> 
>  
> Do the “ears” only feature on this image of this crystal or do they appear on 
> other images? If the ear effect is a one off then that would tend to suggest 
> it isn’t a beamline optic effect.
> 
>  
> Liz
> 
>  
> Dr. Liz Duke
> 
> Principal Beamline Scientist
> 
> Diamond Light Source
> 
> Harwell Science and Innovation Campus
> 
> Chilton
> 
> OX11 0DE
> 
> UK
> 
>  
> Tel. 01235 778057
> 
> Mob. 07920 138148
> 
>  
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing 
> Lou
> Sent: 24 November 2010 14:09
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] unusual diffraction spots
> 
>  
> Dear CCP4BBer, 
> 
> I recently collected a dataset at synchrotron. The diffraction was quite 
> anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What 
> unusual is in the diffraction image (see the attached file), clearly at low 
> resolution there were some spots with tails ("two ears") and at the high 
> resolution shell the spots turned to be rod-shaped.  Please, can anyone 
> explain how this could be? Is this related to the anisotropy? The protein was 
> N-terminal his6-tagged, we  are currently preparing new samples with the 
> His-tag removed. But any other suggestions are also very welcomed.
> 
> Regards,
> 
> Hubing
> 
> 
>  
> -- 
> 
> This e-mail and any attachments may contain confidential, copyright and or 
> privileged material, and are for the use of the intended addressee only. If 
> you are not the intended addressee or an authorised recipient of the 
> addressee please notify us of receipt by returning the e-mail and do not use, 
> copy, retain, distribute or disclose the information in or attached to the 
> e-mail.
> Any opinions expressed within this e-mail are those of the individual and not 
> necessarily of Diamond Light Source Ltd. 
> Diamond Light Source Ltd. cannot guarantee that this e-mail or any 
> attachments are free from viruses and we cannot accept liability for any 
> damage which you may sustain as a result of software viruses which may be 
> transmitted in or with the message.
> Diamond Light Source Limited (company no. 4375679). Registered in England and 
> Wales with its registered office at Diamond House, Harwell Science and 
> Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
>  
> 
> 
> 

Re: [ccp4bb] Can LSQKAB calculate RMSTAB for two pre-superposed structures?

[Eleanor Dodson]

Oh dear - Yes you are right

There is an old program called compar
See http://www.ccp4.ac.uk/dist/html/compar.html

Here is the example script. You need the same residue numbering in each, 
so if the sequence of the 2 coordinate files is different you will need 
to run CHAINSAW to renumber one pdb to match the other.


Then use the LSQKAB output PDB moved to fit the first 100 residues, and 
you will get a list for all the residues present

Eleanor

#!/bin/sh

set -e

#  Tabulates differences between 2 or 3 sets of coordinates,
#   against atom type and B value and residue number.
# COMPARISON OF COORDINATES - ON XYZIN1 XYZIN2  AND PROVISIONALLY XYZIN3.
#CARD 1 - TITLE
#CARD 2 - NSETS - NUMBER OF CORD SETS FOR COMPARISON (2 OR 3.)
#CARD 3 - DELXYZ DELB FOR MONITORING

#  RMSTAB is written out - useful for SQUID
#  A temporary file called TMPOUT is written.

compar  \
  XYZIN1 $CEXAM/rnase/rnase.pdb \
  XYZIN2 $CCP4_SCR/rnase_out.pdb \
  RMSTAB $CCP4_SCR/rnase.rms\
  << END-compar
comparing rnase coordinates before and after refinement
2
3.0 16
END-compar
#

On 11/24/2010 05:54 PM, Huiying Li wrote:

Thanks for your reply, Eleanor.

The problem I had with LSQKAB is that it ONLY outputs the RMSD for the
residue range the superpose calculation based upon. What I wanted is to
superpose the two structures with the N-terminal 100 residues, but
calculate the RMSD for the entire structure (400 residues). Can LSQKAB
do that?

Best regards,
Huiying


Huiying Li, Ph. D
Department of Molecular Biology and Biochemistry
Natural Sciences I, Rm 2443
University of California at Irvine
Irvine, CA 92697, USA
Tel: 949-824-4322(or -1953); Fax: 949-824-3280
email: h...@uci.edu

On Wed, 24 Nov 2010, Eleanor Dodson wrote:


Yes - I think there is a button on the GUI to click?
Eleanor

On 11/24/2010 01:58 AM, Huiying Li wrote:

Another question on RMSD:

I have two structures of the same protein superposed with the LSQ
Superpose in Coot by matching the first ~100 residues of the N-terminal
domain. Now I'd like to calculate the pair-wise RMSD for the entire
pre-superposed structures (~400 residues). Can LSQKAB output RMSTAB for
the two input PDB files without doing its own FIT/MATCH calculation?

Thanks,
Huiying

___
Huiying Li, Ph. D
Department of Molecular Biology and Biochemistry
Natural Sciences I, Rm 2443
University of California at Irvine
Irvine, CA 92697, USA
Tel: 949-824-4322(or -1953); Fax: 949-824-3280
email: h...@uci.edu

Re: [ccp4bb] unusual diffraction spots

[elizabeth . duke]

In the past I've collected data on crystals with hollow ends and the
spots were round and had no ears. As it was 15years ago, collecting on
the old SRS my recollection of beam and crystal relative size is a
little hazy but I think they were fairly well matched (both 200um). 

 

Dr. Liz Duke

Principal Beamline Scientist

Diamond Light Source

Harwell Science and Innovation Campus

Chilton

OX11 0DE

UK

 

Tel. 01235 778057

Mob. 07920 138148

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Harry Powell
Sent: 25 November 2010 09:54
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots

 

Hi folks

 

I'm wondering if the "ears" may be due to hollow ends of the rod-shaped
crystals? Hollow ends are more common than you might imagine (especially
in rods), and it's fairly easy to see how they could give rise to these
ears...

 

On 25 Nov 2010, at 09:09, Colin Nave wrote:





Dear all

Herman might be correct in this case but spot shapes can be affected by
imperfactions in the crystal rather than the crystal shape.

Some types of imperfections (e.g. strain) manifest themselves more for
higher resolution data. They are still there for the low resolution
data, but buried by the overall instrument (detector, beamline)
resolution. An interesting thing to do is to examine spot shapes at
different detector distances making sure the beam divergence doesn't
dominate. . In this case one might find that the low resolution spots
also have this behavior (ears etc.). 

 

Of course it is probably too late to do this.

 

Regards

  Colin



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
herman.schreu...@sanofi-aventis.com
Sent: 25 November 2010 08:28
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots

Dear Hubing,

 

since your crystal is smaller than the beam, the shape of your
spots will be the shape of your crystal as viewed from the spot position
on the detector. This means that if your crystal has a rod shape, spots
at certain detector positions will have a rod shape. If your crystal has
ears, this may explain the ears you see in your diffraction pattern.

 

Different spot shapes at different detector regions are normally
not a problem since most processing programs use different local
profiles for different regions of the detector. Your high Rfree is not
caused by the different spot shapes, but must have other causes which
may be anisotropy of your data, ice rings, disorder of your protein etc.


 

If you do not like different spot shapes, you must collimate
your beam to be smaller than your crystal, but again, this is not the
cause of your high Rfree (and frustration)!

 

Good luck with your refinement!

Herman

 





From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk]
On Behalf Of Hubing Lou
Sent: Thursday, November 25, 2010 6:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots

  



To further clarify things, the data was collected at a
synchrotron beamline with collimator size ~130*40(um*square), beam
divergence ~0.3*0.1mRad. The detector type was MarCCD.
The crystal was multiple-faced trigonal (space group
P3121) the size was about 0.1*0.1*0.15mm. The exposure time was 2s for
each image. 

I am currently refining the structure, however the Rfree
stays above 30%. A close inspection shows at high resolution shell the
spots become rod shaped. As I said we are preparing new constructs with
N-terminal his-tag cleaved. But any other good suggestions out there
might be helpful to avoid future frustration.

Thanks,
Hubing

On Wed, Nov 24, 2010 at 10:26 PM,
 wrote:

I think there may be two effects going on here: 

 

I think the "ears" on the round spots which also feature
on the more rod shaped spots if you look closely could be related to a
misalignment of the beamline optics.

 

I think the change in spot shape from round to rod
shaped is due to the crystal quality. 

 

Do the "ears" only feature on this image of this crystal
or do they appear on other images? If the ear effect is a one off then
that would tend to suggest it isn't a beamline optic effect. 

 

Liz

 

Dr. Liz Duke

Principal Beamline Scientist

Diamond Light Source

Harwell Science and Innovation Campus

Chilton

OX11 0DE

UK

 

Tel. 01235 77

Re: [ccp4bb] unusual diffraction spots

[Harry Powell]

Hi folks

I'm wondering if the "ears" may be due to hollow ends of the rod-shaped 
crystals? Hollow ends are more common than you might imagine (especially in 
rods), and it's fairly easy to see how they could give rise to these ears...

On 25 Nov 2010, at 09:09, Colin Nave wrote:

> Dear all
> Herman might be correct in this case but spot shapes can be affected by 
> imperfactions in the crystal rather than the crystal shape.
> Some types of imperfections (e.g. strain) manifest themselves more for higher 
> resolution data. They are still there for the low resolution data, but buried 
> by the overall instrument (detector, beamline) resolution. An interesting 
> thing to do is to examine spot shapes at different detector distances making 
> sure the beam divergence doesn't dominate. . In this case one might find that 
> the low resolution spots also have this behavior (ears etc.).
>  
> Of course it is probably too late to do this.
>  
> Regards
>   Colin
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of 
> herman.schreu...@sanofi-aventis.com
> Sent: 25 November 2010 08:28
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] unusual diffraction spots
> 
> Dear Hubing,
>  
> since your crystal is smaller than the beam, the shape of your spots will be 
> the shape of your crystal as viewed from the spot position on the detector. 
> This means that if your crystal has a rod shape, spots at certain detector 
> positions will have a rod shape. If your crystal has ears, this may explain 
> the ears you see in your diffraction pattern.
>  
> Different spot shapes at different detector regions are normally not a 
> problem since most processing programs use different local profiles for 
> different regions of the detector. Your high Rfree is not caused by the 
> different spot shapes, but must have other causes which may be anisotropy of 
> your data, ice rings, disorder of your protein etc.
>  
> If you do not like different spot shapes, you must collimate your beam to be 
> smaller than your crystal, but again, this is not the cause of your high 
> Rfree (and frustration)!
>  
> Good luck with your refinement!
> Herman
> 
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing 
> Lou
> Sent: Thursday, November 25, 2010 6:10 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] unusual diffraction spots
> 
> 
> 
> 
> To further clarify things, the data was collected at a synchrotron beamline 
> with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The 
> detector type was MarCCD.
> The crystal was multiple-faced trigonal (space group P3121) the size was 
> about 0.1*0.1*0.15mm. The exposure time was 2s for each image. 
> 
> I am currently refining the structure, however the Rfree stays above 30%. A 
> close inspection shows at high resolution shell the spots become rod shaped. 
> As I said we are preparing new constructs with N-terminal his-tag cleaved. 
> But any other good suggestions out there might be helpful to avoid future 
> frustration.
> 
> Thanks,
> Hubing
> 
> On Wed, Nov 24, 2010 at 10:26 PM,  wrote:
> I think there may be two effects going on here:
> 
>  
> I think the “ears” on the round spots which also feature on the more rod 
> shaped spots if you look closely could be related to a misalignment of the 
> beamline optics.
> 
>  
> I think the change in spot shape from round to rod shaped is due to the 
> crystal quality.
> 
>  
> Do the “ears” only feature on this image of this crystal or do they appear on 
> other images? If the ear effect is a one off then that would tend to suggest 
> it isn’t a beamline optic effect.
> 
>  
> Liz
> 
>  
> Dr. Liz Duke
> 
> Principal Beamline Scientist
> 
> Diamond Light Source
> 
> Harwell Science and Innovation Campus
> 
> Chilton
> 
> OX11 0DE
> 
> UK
> 
>  
> Tel. 01235 778057
> 
> Mob. 07920 138148
> 
>  
> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing 
> Lou
> Sent: 24 November 2010 14:09
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] unusual diffraction spots
> 
>  
> Dear CCP4BBer, 
> 
> I recently collected a dataset at synchrotron. The diffraction was quite 
> anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang. What 
> unusual is in the diffraction image (see the attached file), clearly at low 
> resolution there were some spots with tails ("two ears") and at the high 
> resolution shell the spots turned to be rod-shaped.  Please, can anyone 
> explain how this could be? Is this related to the anisotropy? The protein was 
>N-terminal his6-tagged, we  are currently preparing new samples with 
> the His-tag removed. But any other suggestions are also very welcomed.
> 
> Regards,
> 
> Hubing
> 
> 
>  
> -- 
> 
> This e-mail and any attachments may contain confidential, copyright and or 
> privileged material, and are for the use of the intended addressee only. If 
> you are not the intended addressee or an authorised recipient of th

Re: [ccp4bb] unusual diffraction spots

[Colin Nave]

Dear all
Herman might be correct in this case but spot shapes can be affected by
imperfactions in the crystal rather than the crystal shape.
Some types of imperfections (e.g. strain) manifest themselves more for
higher resolution data. They are still there for the low resolution
data, but buried by the overall instrument (detector, beamline)
resolution. An interesting thing to do is to examine spot shapes at
different detector distances making sure the beam divergence doesn't
dominate. . In this case one might find that the low resolution spots
also have this behavior (ears etc.). 
 
Of course it is probably too late to do this.
 
Regards
  Colin


From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
herman.schreu...@sanofi-aventis.com
Sent: 25 November 2010 08:28
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots



Dear Hubing,
 
since your crystal is smaller than the beam, the shape of your
spots will be the shape of your crystal as viewed from the spot position
on the detector. This means that if your crystal has a rod shape, spots
at certain detector positions will have a rod shape. If your crystal has
ears, this may explain the ears you see in your diffraction pattern.
 
Different spot shapes at different detector regions are normally
not a problem since most processing programs use different local
profiles for different regions of the detector. Your high Rfree is not
caused by the different spot shapes, but must have other causes which
may be anisotropy of your data, ice rings, disorder of your protein etc.

 
If you do not like different spot shapes, you must collimate
your beam to be smaller than your crystal, but again, this is not the
cause of your high Rfree (and frustration)!
 
Good luck with your refinement!
Herman




From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk]
On Behalf Of Hubing Lou
Sent: Thursday, November 25, 2010 6:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots







To further clarify things, the data was collected at a
synchrotron beamline with collimator size ~130*40(um*square), beam
divergence ~0.3*0.1mRad. The detector type was MarCCD.
The crystal was multiple-faced trigonal (space group
P3121) the size was about 0.1*0.1*0.15mm. The exposure time was 2s for
each image. 

I am currently refining the structure, however the Rfree
stays above 30%. A close inspection shows at high resolution shell the
spots become rod shaped. As I said we are preparing new constructs with
N-terminal his-tag cleaved. But any other good suggestions out there
might be helpful to avoid future frustration.

Thanks,
Hubing


On Wed, Nov 24, 2010 at 10:26 PM,
 wrote:


I think there may be two effects going on here: 

 

I think the "ears" on the round spots which also
feature on the more rod shaped spots if you look closely could be
related to a misalignment of the beamline optics.

 

I think the change in spot shape from round to
rod shaped is due to the crystal quality. 

 

Do the "ears" only feature on this image of this
crystal or do they appear on other images? If the ear effect is a one
off then that would tend to suggest it isn't a beamline optic effect. 

 

Liz

 

Dr. Liz Duke

Principal Beamline Scientist

Diamond Light Source

Harwell Science and Innovation Campus

Chilton

OX11 0DE

UK

 

Tel. 01235 778057

Mob. 07920 138148

 

From: CCP4 bulletin board
[mailto:ccp...@jiscmail.ac.uk] On Behalf Of Hubing Lou
Sent: 24 November 2010 14:09
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unusual diffraction spots

 

Dear CCP4BBer, 

I recently collected a dataset at synchrotron.
The diffraction was quite anisotropic with one direction to 2.1Angstrom
while the other is 3.0Ang. What unusual is in the diffraction image (see
the attached file), clearly at low resolution there were some spots with
tails ("two ears

Re: [ccp4bb] unusual diffraction spots

[John R Helliwell]

Dear Hubing,
Thankyou for the extra details.
The MARCCD to my experience does not show 'bleeding' from pixels at strong
spots. Such effects on a CCD anyway are in a line not like the 'ears' you
have here.
It also does not look like diffuse scattering.
So, since it seems to be an effect visible on very strong spots, I wonder if
there is some texture to your crystal ie in effect a very small degree of
fragmentation on this particular sample.
Best wishes,
John
On Thu, Nov 25, 2010 at 5:09 AM, Hubing Lou  wrote:

>
>
>
> To further clarify things, the data was collected at a synchrotron beamline
> with collimator size ~130*40(um*square), beam divergence ~0.3*0.1mRad. The
> detector type was MarCCD.
> The crystal was multiple-faced trigonal (space group P3121) the size was
> about 0.1*0.1*0.15mm. The exposure time was 2s for each image.
>
> I am currently refining the structure, however the Rfree stays above 30%. A
> close inspection shows at high resolution shell the spots become rod shaped.
> As I said we are preparing new constructs with N-terminal his-tag cleaved.
> But any other good suggestions out there might be helpful to avoid future
> frustration.
>
> Thanks,
> Hubing
>
>  On Wed, Nov 24, 2010 at 10:26 PM,  wrote:
>
>>I think there may be two effects going on here:
>>
>>
>>
>> I think the “ears” on the round spots which also feature on the more rod
>> shaped spots if you look closely could be related to a misalignment of the
>> beamline optics.
>>
>>
>>
>> I think the change in spot shape from round to rod shaped is due to the
>> crystal quality.
>>
>>
>>
>> Do the “ears” only feature on this image of this crystal or do they appear
>> on other images? If the ear effect is a one off then that would tend to
>> suggest it isn’t a beamline optic effect.
>>
>>
>>
>> Liz
>>
>>
>>
>> Dr. Liz Duke
>>
>> Principal Beamline Scientist
>>
>> Diamond Light Source
>>
>> Harwell Science and Innovation Campus
>>
>> Chilton
>>
>> OX11 0DE
>>
>> UK
>>
>>
>>
>> Tel. 01235 778057
>>
>> Mob. 07920 138148
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of
>> *Hubing Lou
>> *Sent:* 24 November 2010 14:09
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] unusual diffraction spots
>>
>>
>>
>> Dear CCP4BBer,
>>
>> I recently collected a dataset at synchrotron. The diffraction was quite
>> anisotropic with one direction to 2.1Angstrom while the other is 3.0Ang.
>> What unusual is in the diffraction image (see the attached file), clearly at
>> low resolution there were some spots with tails ("two ears") and at the high
>> resolution shell the spots turned to be rod-shaped.  Please, can anyone
>> explain how this could be? Is this related to the anisotropy? The protein
>> was N-terminal his6-tagged, we  are currently preparing new samples with
>> the His-tag removed. But any other suggestions are also very welcomed.
>>
>> Regards,
>>
>> Hubing
>>
>>
>>
>> --
>>
>> This e-mail and any attachments may contain confidential, copyright and or
>> privileged material, and are for the use of the intended addressee only. If
>> you are not the intended addressee or an authorised recipient of the
>> addressee please notify us of receipt by returning the e-mail and do not
>> use, copy, retain, distribute or disclose the information in or attached to
>> the e-mail.
>> Any opinions expressed within this e-mail are those of the individual and
>> not necessarily of Diamond Light Source Ltd.
>> Diamond Light Source Ltd. cannot guarantee that this e-mail or any
>> attachments are free from viruses and we cannot accept liability for any
>> damage which you may sustain as a result of software viruses which may be
>> transmitted in or with the message.
>> Diamond Light Source Limited (company no. 4375679). Registered in England
>> and Wales with its registered office at Diamond House, Harwell Science and
>> Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
>>
>>
>>
>


-- 
Professor John R Helliwell DSc

Re: [ccp4bb] unusual diffraction spots

[Herman . Schreuder]

Dear Hubing,
 
since your crystal is smaller than the beam, the shape of your spots
will be the shape of your crystal as viewed from the spot position on
the detector. This means that if your crystal has a rod shape, spots at
certain detector positions will have a rod shape. If your crystal has
ears, this may explain the ears you see in your diffraction pattern.
 
Different spot shapes at different detector regions are normally not a
problem since most processing programs use different local profiles for
different regions of the detector. Your high Rfree is not caused by the
different spot shapes, but must have other causes which may be
anisotropy of your data, ice rings, disorder of your protein etc. 
 
If you do not like different spot shapes, you must collimate your beam
to be smaller than your crystal, but again, this is not the cause of
your high Rfree (and frustration)!
 
Good luck with your refinement!
Herman




From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On
Behalf Of Hubing Lou
Sent: Thursday, November 25, 2010 6:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] unusual diffraction spots







To further clarify things, the data was collected at a
synchrotron beamline with collimator size ~130*40(um*square), beam
divergence ~0.3*0.1mRad. The detector type was MarCCD.
The crystal was multiple-faced trigonal (space group P3121) the
size was about 0.1*0.1*0.15mm. The exposure time was 2s for each image. 

I am currently refining the structure, however the Rfree stays
above 30%. A close inspection shows at high resolution shell the spots
become rod shaped. As I said we are preparing new constructs with
N-terminal his-tag cleaved. But any other good suggestions out there
might be helpful to avoid future frustration.

Thanks,
Hubing


On Wed, Nov 24, 2010 at 10:26 PM, 
wrote:


I think there may be two effects going on here: 

 

I think the "ears" on the round spots which also feature
on the more rod shaped spots if you look closely could be related to a
misalignment of the beamline optics.

 

I think the change in spot shape from round to rod
shaped is due to the crystal quality. 

 

Do the "ears" only feature on this image of this crystal
or do they appear on other images? If the ear effect is a one off then
that would tend to suggest it isn't a beamline optic effect. 

 

Liz

 

Dr. Liz Duke

Principal Beamline Scientist

Diamond Light Source

Harwell Science and Innovation Campus

Chilton

OX11 0DE

UK

 

Tel. 01235 778057

Mob. 07920 138148

 

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk]
On Behalf Of Hubing Lou
Sent: 24 November 2010 14:09
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unusual diffraction spots

 

Dear CCP4BBer, 

I recently collected a dataset at synchrotron. The
diffraction was quite anisotropic with one direction to 2.1Angstrom
while the other is 3.0Ang. What unusual is in the diffraction image (see
the attached file), clearly at low resolution there were some spots with
tails ("two ears") and at the high resolution shell the spots turned to
be rod-shaped.  Please, can anyone explain how this could be? Is this
related to the anisotropy? The protein was N-terminal his6-tagged, we
are currently preparing new samples with the His-tag removed. But any
other suggestions are also very welcomed.

Regards,

Hubing 


 

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