Re: [ccp4bb] Basic Anomalous Scattering Theory

[William G. Scott]

> On Mar 11, 2015, at 11:13 AM, Keller, Jacob  wrote:
> 
> Somebody sent me a pdf with such a mind-picture which describes the origin of 
> the anomalous effect as arising from broken centrosymmetry of the anomalous 
> atoms themselves:
> 
> (p. 8) "Under normal conditions, electron distributions within atoms are 
> centrosymmetric...Under conditions of anomalous scattering, electrons are 
> perturbed from their centrosymmetric distributions; electrons are jumping 
> between orbitals. The breakdown of centrosymmetry in the scattering atoms is 
> reflected in a loss of centrosymmetry in the pattern of scattered X-ray 
> intensities."

I think this is wrong.  Unless I am mistaken, the diffraction pattern is 
centrosymmetric simply as a consequence of the electron density (and therefore 
the potential) being a purely real function.  If the function is complex (i.e., 
including that small perturbation I spoke of this morning), then the 
diffraction pattern will no longer be centrosymmetric.  

Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

[Guenter Fritz]

Hi Stephen,

we crystallized several proteins in a glove box placed in a room with 
air condition. We checked the temperature all the times and found that 
is had been quite stable.


For one protein that crystallized only at 4 deg C, we setup the 
crystallization plate within the glove box and put it into an gas-tight 
plexiglass box (built by our workshop). The box with the crystallization 
plate was then incubated at 4degC.


Best, Guenter


Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email and any attachments may contain confidential, copyright and or 
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Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

Thanks for all of the responses, there seems to be no real consensus, but I 
have discovered:

1 There are several small incubators supplied by - Molecular Dimensions, 
Revsci, Centeo

2 Good air conditioning may negate the need for an incubator.

3 A Glove box may not even be necessary depending on the samples sensitivity to 
oxygen.
 
Certainly plenty for me to think about before I commit to buying a glove box.

Yet again the bulletin board proves to be a great source of information for all 
things crystallographic!

Best wishes,

Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of  
[stephen.c...@rc-harwell.ac.uk]
Sent: 11 March 2015 10:17
To: ccp4bb
Subject: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email 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 authorized 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 this email.

Any views or opinions presented are solely those of the author and do not 
necessarily represent those of the Research Complex at Harwell.

There is no guarantee that this email 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.

We use an electronic filing system. Please send electronic versions of 
documents, unless paper is specifically requested.

This email may have a protective marking, for an explanation, please see:
http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm.

Re: [ccp4bb] Basic Anomalous Scattering Theory

[Keller, Jacob]

Several have mentioned James to me, and I have had and read a copy for quite 
some time. I find, however, that the descriptions are more mathematical than 
mind-pictures. While math is more agnostic, I find mind-pictures more 
prognostic and helpful to me. But I absolutely love James.

Somebody sent me a pdf with such a mind-picture which describes the origin of 
the anomalous effect as arising from broken centrosymmetry of the anomalous 
atoms themselves:

(p. 8) "Under normal conditions, electron distributions within atoms are 
centrosymmetric...Under conditions of anomalous scattering, electrons are 
perturbed from their centrosymmetric distributions; electrons are jumping 
between orbitals. The breakdown of centrosymmetry in the scattering atoms is 
reflected in a loss of centrosymmetry in the pattern of scattered X-ray 
intensities."

http://ocw.mit.edu/courses/chemistry/5-069-crystal-structure-analysis-spring-2010/lecture-notes/anomal_hand1_rev.pdf

I had never heard this description before. In a way, this makes great sense and 
connects the anomalous effect with fluorescence: when one measures anomalous 
datasets, one is measuring an actively-fluorescing protein crystal. When the 
atoms fluoresce, they look different to the incident x-ray beam. This also 
explains why we use fluorescence scans to determine edges empirically. But I am 
not sure whether all of the dots connect yet; for example, there are many 
(mostly?) non-centrosymmetric electron distributions in the crystal, and these 
do not create an anomalous effect.

JPK




-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, 
Jacob
Sent: Wednesday, March 11, 2015 12:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Basic Anomalous Scattering Theory

Dear Crystallographers,

I have had only a vague understanding of what specific things are happening 
with shell electrons at anomalous edges. Specifically, for example, to what 
energy of electron-transition does the x-ray k-edge correspond in terms of 
orbitals, and is that transition energy actually equal to the energy of the 
photon, suggesting that the photon is absorbed (or disappears?) in elevating 
the electron? I don't think we say it is absorbed, so how does the energy come 
back out, from the electron's falling back down, right? So then there's a new 
photon created, or the same one comes back out? Where was it? 

Further, I also have heard that the emerging anomalous/resonance photons are of 
the same wavelength as the incident radiation, but usually there is something 
lost in transitions (even non-fluorescence ones) I thought? Has it ever been 
definitively shown that the anomalous photons are of the same energy as the 
incident radiation?

In the case of L-edges, why are there three separate edges? Further, if the 
resonance occurs when the energies are equal, why does resonance occur at 
energies greater than the edge? I don't think this happens in other resonance 
phenomena, or does it? If projects a middle-C-tone into a piano, do all of the 
lower notes resonate as well, according to the Kramers-Kronig relation? I think 
it may actually happen in the mammalian cochlea's travelling wave, but is it 
completely general to resonance phenomena?

Just interested, and have wondered these things for a long time in the 
background of my mind...

Jacob Keller


***
Jacob Pearson Keller, PhD
Looger Lab/HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] Basic Anomalous Scattering Theory

[William G. Scott]

Hi Jacob:

If you can find a copy of R. W. James, THE CRYSTALLINE STATE VOL II: THE 
OPTICAL PRINCIPLES OF THE DIFFRACTION OF X-RAYS. Hardcover – 1965, this gives a 
very good (albeit primarily classical) description.  Blundell and Johnson also 
has a good, slightly more modern, description, which I think is derived in part 
from James’s treatment. (Someone has swiped both books from me, so I can’t 
double-check.)  Having said that, I find an approach based on quantum 
scattering theory more intuitive.  Everything we normally deal with is elastic 
scattering within the first-order Born approximation, which means a single 
photon collides exactly once with a billiard ball-like spherically symmetric 
hard-sphere electron cloud, neither gaining nor losing energy.  Using the 
Poisson equation, you can then show that the electron density is the FT of the 
(completely real) potential.  To treat absorption within this framework, an 
imaginary term is added to the potential as a small perturbation.  

Briefly, in the first case, a photon elastically scatters, and in the second 
case, there is a probability of absorption. (An absorbed photon, absorbed in 
the conventional way , in which an electron is promoted to an excited state, 
will be re-emitted as a new photon having less energy, which is how we are able 
to do fluorescent XAFS to find the absorption edge when collecting data.)

Sorry, I will have to stop here to go teach something else I am equally 
unqualified to speak about — enzymology.  Hopefully this will be something to 
get a start with.

Bill










William G. Scott
Director, Program in Biochemistry and Molecular Biology
Professor, Department of Chemistry and Biochemistry
and The Center for the Molecular Biology of RNA
University of California at Santa Cruz
Santa Cruz, California 95064
USA

phone:  +1-831-459-5367 (office)

email:wgsc...@ucsc.edu

> On Mar 11, 2015, at 9:57 AM, Keller, Jacob  wrote:
> 
> Dear Crystallographers,
> 
> I have had only a vague understanding of what specific things are happening 
> with shell electrons at anomalous edges. Specifically, for example, to what 
> energy of electron-transition does the x-ray k-edge correspond in terms of 
> orbitals, and is that transition energy actually equal to the energy of the 
> photon, suggesting that the photon is absorbed (or disappears?) in elevating 
> the electron? I don't think we say it is absorbed, so how does the energy 
> come back out, from the electron's falling back down, right? So then there's 
> a new photon created, or the same one comes back out? Where was it? 
> 
> Further, I also have heard that the emerging anomalous/resonance photons are 
> of the same wavelength as the incident radiation, but usually there is 
> something lost in transitions (even non-fluorescence ones) I thought? Has it 
> ever been definitively shown that the anomalous photons are of the same 
> energy as the incident radiation?
> 
> In the case of L-edges, why are there three separate edges? Further, if the 
> resonance occurs when the energies are equal, why does resonance occur at 
> energies greater than the edge? I don't think this happens in other resonance 
> phenomena, or does it? If projects a middle-C-tone into a piano, do all of 
> the lower notes resonate as well, according to the Kramers-Kronig relation? I 
> think it may actually happen in the mammalian cochlea's travelling wave, but 
> is it completely general to resonance phenomena?
> 
> Just interested, and have wondered these things for a long time in the 
> background of my mind...
> 
> Jacob Keller
> 
> 
> ***
> Jacob Pearson Keller, PhD
> Looger Lab/HHMI Janelia Research Campus
> 19700 Helix Dr, Ashburn, VA 20147
> email: kell...@janelia.hhmi.org
> ***

Re: [ccp4bb] Basic Anomalous Scattering Theory

[Bernhard Rupp (Hofkristallrat a.D.)]

A look at the term scheme might help to understand the absorption edge
structure - pp 286 and Figure 6-30 BMC.

Best, BR

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Keller, Jacob
Sent: Wednesday, March 11, 2015 9:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Basic Anomalous Scattering Theory

Dear Crystallographers,

I have had only a vague understanding of what specific things are happening
with shell electrons at anomalous edges. Specifically, for example, to what
energy of electron-transition does the x-ray k-edge correspond in terms of
orbitals, and is that transition energy actually equal to the energy of the
photon, suggesting that the photon is absorbed (or disappears?) in elevating
the electron? I don't think we say it is absorbed, so how does the energy
come back out, from the electron's falling back down, right? So then there's
a new photon created, or the same one comes back out? Where was it? 

Further, I also have heard that the emerging anomalous/resonance photons are
of the same wavelength as the incident radiation, but usually there is
something lost in transitions (even non-fluorescence ones) I thought? Has it
ever been definitively shown that the anomalous photons are of the same
energy as the incident radiation?

In the case of L-edges, why are there three separate edges? Further, if the
resonance occurs when the energies are equal, why does resonance occur at
energies greater than the edge? I don't think this happens in other
resonance phenomena, or does it? If projects a middle-C-tone into a piano,
do all of the lower notes resonate as well, according to the Kramers-Kronig
relation? I think it may actually happen in the mammalian cochlea's
travelling wave, but is it completely general to resonance phenomena?

Just interested, and have wondered these things for a long time in the
background of my mind...

Jacob Keller


***
Jacob Pearson Keller, PhD
Looger Lab/HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] Offtopic: Software to closely visualize interacting partnets in protein complex

[Tim]

Hi,
Molprobity is also a nice software to do this kind of analysis - if you 
use it as implemented in phenix you also get good visualization in coot.
I also asked the pymol community to create an implementation for pymol, 
but I did not follow if somebody took that up.


Also this 'protein interactions calculation' server is very neat:
http://pic.mbu.iisc.ernet.in/

/Tim


On 10/03/15 11:25, Debasish Kumar Ghosh wrote:

Dear All,

Apologies for this little off-topic inquiry. I want to closely visualize the 
interacting residues in an multimeric protein complex to understand the nature 
of interactions. Is there any good software to give this information with good 
clarity.
Any suggestion is highly appreciated.

Thanks,
Best !!!

Debasish Kumar Ghosh

CSIR- Junior Research Fellow (PhD Scholar)
C/o: Dr. Akash Ranjan
Computational and Functional Genomics Group
Centre for DNA Fingerprinting and Diagnostics
Hyderabad, INDIA

Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html

[ccp4bb] Protein purification and crystallization position available in the Fesik Lab at Vanderbilt University

[Phan, Jason]

An opening is available in the laboratory of Dr. Stephen Fesik 
(http://www.mc.vanderbilt.edu/fesiklab) for the Research Associate position in 
structural biology and drug discovery. The candidate must possess a degree in 
biochemistry or a related field with 1-2 years of experience in protein 
purification. Experience in protein crystallography is a plus but not required. 
The ideal candidate should be courteous, responsible, organized, hard-working, 
and be able to multi-task and communicate efficiently.  The successful 
candidate will be trained initially but will work independently or with limited 
supervision on assigned duties thereafter. Please send CV or resume to 
jason.p...@vanderbilt.edu

Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

[Hruza, Alan]

Steve,

I used a very sophisticated apparatus  to grow oxygen sensitive iNOS crystals 
at room temperature.

Take a zip lock bag and cut a hole in one corner to insert a tube flowing 
nitrogen from your house source.
Place plate inside zip lock bag and zip close. ( drawback is plate setup not in 
anaerobic environment )
Bag will pressurize and the purge of oxygen worked in my crystal system.  

Good luck,
Alan

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Sent: Wednesday, March 11, 2015 6:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email and any attachments may contain confidential, copyright and or 
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Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

[Conn Mallett]

Dear Steve,

There is a model of small incubator that fits through the large portal of a 
Belle Technology rigid glovebox -  as an aside, that’s a good box for 
crystallisation and crystal manipulation since you can get the 
microscope-mounted side box with a dewar port for rapid and pretty efficient 
cryo-cooling.  - I don’t work for Belle and don’t even know for sure if they 
are still in business..

Anyway…to your question...the incubator is constructed of grey plastic but 
holds temperature nicely - let me ring the guys back at Wake Forest Univ to see 
if they have it still around in the lab and I will send you the manufacturer 
and model number, or maybe they will see this here and post.

Cheers

-Conn

Conn Mallett, PhD
Director of Sales - SCX SBU
Rigaku
9009 New Trails Drive
The Woodlands, TX 77381
Office: +1 281 362 2300 ext. 126
Cell: +1 713 614 6891
FAX: +1 281 364 3628

Subscribe to Rigaku eNewsletters  .

conn.mall...@rigaku.com
www.rigaku.com

On 11Mar, 2015, at 09:11, Martin Montgomery 
mailto:m...@mrc-mbu.cam.ac.uk>> wrote:

Dear Steve,

You should not require an incubator if you have good, stable air con.  We 
routinely grow crystals in plates stored at room temperature in wall mounted 
cabinets in our crystallisation lab.  Another of the groups here have 
experience of growing crystals in an anaerobic box.  Hopefully they will post 
more information.

Regards

MGM


Martin G Montgomery
ATP Synthase Group
MRC Mitochondrial Biology Unit
Wellcome Trust/MRC Building
Cambridge Biomedical Campus
Hills Road
Cambridge
Great Britain
CB2 0XY

www.mrc-mbu.cam.ac.uk






On 11 Mar 2015, at 10:17,  
mailto:stephen.c...@rc-harwell.ac.uk>>  wrote:

Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email 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 authorized 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 this email.

Any views or opinions presented are solely those of the author and do not 
necessarily represent those of the Research Complex at Harwell.

There is no guarantee that this email 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.

We use an electronic filing system. Please send electronic versions of 
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Re: [ccp4bb] Off-topic - Crystallisation in anaerobic glove box

[Martin Montgomery]

Dear Steve,

You should not require an incubator if you have good, stable air con.  We 
routinely grow crystals in plates stored at room temperature in wall mounted 
cabinets in our crystallisation lab.  Another of the groups here have 
experience of growing crystals in an anaerobic box.  Hopefully they will post 
more information.

Regards

MGM


Martin G Montgomery
ATP Synthase Group
MRC Mitochondrial Biology Unit
Wellcome Trust/MRC Building
Cambridge Biomedical Campus
Hills Road 
Cambridge
Great Britain
CB2 0XY

www.mrc-mbu.cam.ac.uk






> On 11 Mar 2015, at 10:17,   
>  wrote:
> 
> Dear CCP4BBer's
> 
> Apologies for the off-topic post, but the CCP4BB seems to be the best place 
> to ask about crystallisation.
> 
> I am looking to set up crystallisation in an anaerobic glove box and wondered 
> how other people did this, specifically the crystallisation stage.  My 
> initial thoughts were to place a small crystallisation incubator inside the 
> box, however the smallest I have come across so far (~27L) is still rather 
> large.  Has anyone come across smaller incubators?  Alternatively are 
> incubators even neccessary if the glove box is placed in a room with good air 
> conditioning and stable temperature control?
> 
> Any recommendations would be very helpful.
> 
> Thanks in advance,
> 
> Steve Carr
> 
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
> 
> This email 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 authorized 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 
> this email.
> 
> Any views or opinions presented are solely those of the author and do not 
> necessarily represent those of the Research Complex at Harwell.
> 
> There is no guarantee that this email 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.
> 
> We use an electronic filing system. Please send electronic versions of 
> documents, unless paper is specifically requested.
> 
> This email may have a protective marking, for an explanation, please see:
> http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm.

[ccp4bb] Off-topic - Crystallisation in anaerobic glove box

Dear CCP4BBer's

Apologies for the off-topic post, but the CCP4BB seems to be the best place to 
ask about crystallisation.

I am looking to set up crystallisation in an anaerobic glove box and wondered 
how other people did this, specifically the crystallisation stage.  My initial 
thoughts were to place a small crystallisation incubator inside the box, 
however the smallest I have come across so far (~27L) is still rather large.  
Has anyone come across smaller incubators?  Alternatively are incubators even 
neccessary if the glove box is placed in a room with good air conditioning and 
stable temperature control?

Any recommendations would be very helpful.

Thanks in advance,

Steve Carr

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717

This email 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 authorized 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 this email.

Any views or opinions presented are solely those of the author and do not 
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There is no guarantee that this email 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.

We use an electronic filing system. Please send electronic versions of 
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This email may have a protective marking, for an explanation, please see:
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