Re: [ccp4bb] Help for Twin Refinement in Refmac5 / CCP4i2

[Parthasarathy Sampathkumar]

Dear All,

Please you may ignore my previous email. TWIN refinement in Refmac5 /
CCP4i2 worked well once used "HKLOUT_0-observed_data_asIMEAN.mtz" from the
data-reduction job. I am slowly getting used to the finer-aspects of
CCP4i2. Previously, I used to have both Intensities and Amplitudes in a
single MTZ file :-)

Thanks,
Partha

On Mon, Jan 25, 2021 at 9:47 PM Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear All,
>
> I have not had much experience in refining structure using data from
> twinned crystals and has started using CCP4i2 very recently only.
>
> Here is a background:
> antigen-Fab crystal structure determined by MR first in P4(3)2(1)2 space
> group with 1-complex molecule in the asymmetric unit (ASU). Later, I
> reprocessed the data in P4(3) performed MR to search for the 2nd molecule
> and refined the structure using Refmac5 without the "twin" keyword. With
> the sequence fully modeled for the two complex molecules in the ASU current
> Rcryst = 27.8%and Rfree = 33.2%. Unit cell = "59.9, 59.9, 404.5, 90.0,
> 90.0, 90.0".
>
> Twin fraction estimates from Britton plot = 0.47 and from H-test = 0.43,
> as reported by AIMLESS. Then, attempted TWIN refinement in Refmac5 /
> CCP4i2 by adding the keyword "twin" in the advanced options. However,
> getting following error in the log-file:
>
>  Error==> array size in mtz_refl_read_int
> 
>  Refmac:  Problem with array sizes
>
> Does Refmac5 expects intensities here?!!
> Any help is greatly appreciated.
>
> Best Wishes,
> Partha
>
>
>
>
>



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[ccp4bb] Help for Twin Refinement in Refmac5 / CCP4i2

[Parthasarathy Sampathkumar]

Dear All,

I have not had much experience in refining structure using data from
twinned crystals and has started using CCP4i2 very recently only.

Here is a background:
antigen-Fab crystal structure determined by MR first in P4(3)2(1)2 space
group with 1-complex molecule in the asymmetric unit (ASU). Later, I
reprocessed the data in P4(3) performed MR to search for the 2nd molecule
and refined the structure using Refmac5 without the "twin" keyword. With
the sequence fully modeled for the two complex molecules in the ASU current
Rcryst = 27.8%and Rfree = 33.2%. Unit cell = "59.9, 59.9, 404.5, 90.0,
90.0, 90.0".

Twin fraction estimates from Britton plot = 0.47 and from H-test = 0.43, as
reported by AIMLESS. Then, attempted TWIN refinement in Refmac5 / CCP4i2 by
adding the keyword "twin" in the advanced options. However, getting
following error in the log-file:

 Error==> array size in mtz_refl_read_int

 Refmac:  Problem with array sizes

Does Refmac5 expects intensities here?!!
Any help is greatly appreciated.

Best Wishes,
Partha



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Re: [ccp4bb] Characterising potential drug-binding pockets

[Jon Cooper]

Hello Sir,

Just confirming PPI is Protein-Protein Interactions rather than Proton Pump 
Inhibitors?

Anyway, I knew someone who used UCSF Chimera a lot for that sort of thing.

Hope this helps. Jon.C.

Sent from ProtonMail mobile

 Original Message 
On 25 Jan 2021, 19:02, Sergei Strelkov wrote:

> Dear everyone,
>
> In a drug discovery project where our aim is to interfere with some PPIs, we 
> could obtain binding of drug-like fragments in several potentially 
> interesting pockets on our target. We would like to make a projection on how 
> promising these individual pockets are. One way of doing this is through the 
> Sitemap program (Halgren, T. A. Identifying and characterizing binding sites 
> and assessing druggability. J. Chem. Inf. Model 49, 377–389 (2009)). Are 
> there other tools around to do this? In particular, we would like to have 
> accurate numbers for the pocket volume, surface, no. of H-bond donors and 
> acceptors, average hydrophobicity, etc etc.
>
> Thank you,
>
> Sergei
>
> Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of 
> Pharmaceutical Sciences, K
>
> U
>
> Leuven O, Campus Gasthuisberg, Herestraat 49 bus 822, 3000 Leuven, Belgium 
> Phone: +32 16 33 08 45,
>
> m
>
> obile: +32 486 29 41 32 Lab pages:
> [http://pharm.kuleuven.be/Biocrystallography](http://pharm.kuleuven.be/anafar)
>
> ---
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Characterising potential drug-binding pockets

[Bernhard Rupp]

ICM PocketFinder might fit the bill (not free but academic license)

eg in

https://pubmed.ncbi.nlm.nih.gov/20977231/

 

Best, BR

 

From: CCP4 bulletin board  On Behalf Of Sergei
Strelkov
Sent: Monday, January 25, 2021 11:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Characterising potential drug-binding pockets

 

Dear everyone,

 

In a drug discovery project where our aim is to interfere with some PPIs, we
could obtain binding of drug-like fragments in several potentially
interesting pockets on our target. We would like to make a projection on how
promising these individual pockets are. One way of doing this is through the
Sitemap program (Halgren, T. A. Identifying and characterizing binding sites
and assessing druggability. J. Chem. Inf. Model 49, 377-389 (2009)). Are
there other tools around to do this? In particular, we would like to have
accurate numbers for the pocket volume, surface, no. of H-bond donors and
acceptors, average hydrophobicity, etc etc. 

 

Thank you,

Sergei

 

 

 

Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of
Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49
bus 822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41
32 Lab pages:  
http://pharm.kuleuven.be/Biocrystallography

 

  _  

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 =1 




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[ccp4bb] Characterising potential drug-binding pockets

[Sergei Strelkov]

Dear everyone,


In a drug discovery project where our aim is to interfere with some PPIs, we 
could obtain binding of drug-like fragments in several potentially interesting 
pockets on our target. We would like to make a projection on how promising 
these individual pockets are. One way of doing this is through the Sitemap 
program (Halgren, T. A. Identifying and characterizing binding sites and 
assessing druggability. J. Chem. Inf. Model 49, 377-389 (2009)). Are there 
other tools around to do this? In particular, we would like to have accurate 
numbers for the pocket volume, surface, no. of H-bond donors and acceptors, 
average hydrophobicity, etc etc.


Thank you,

Sergei




Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of 
Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 
822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab 
pages: 
http://pharm.kuleuven.be/Biocrystallography



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[ccp4bb] ccp4bb PhD position oppurtunity

[Thatipally Mounika]

Dear Dr Xinlai Cheng,

My name is Mounika, have completed Masters in Biotechnology in the 2019
from Hyderabad India. I am writing this mail regarding any PhD positions
offered in your lab. I feel that pursuing PhD in onco biology would help me
reach my dream and also be among one who could try giving a solution to
cancer. I have read that your research interest area is also cancer
therapy. I have also read your article on the anti-tumor properties of gold
which made me think about the various ways to address cancer. I would love
to start working on a project in your lab, if possible. The below attached
is my resume and a scientific write up. I look forward to hear from you.

Thanking you,

Mounika Thatipally.

  mounika resume-pdf.pdf


  Standard operating procedure- pdf.pdf




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[ccp4bb] Post-doc opportunity - Structural biology of AdhE spirosomes

[Mads Gabrielsen]

I would like to draw your attention to the position below.
Please contact Andrew Roe or Olwyn Byron if interested.

RE: 3-year BBSRC funded project, University of Glasgow, Scotland, UK.
 
Post-doctoral project with Profs Olwyn Byron and Andrew Roe
A new twist on drug design: AdhE spirosomes as cross species anti-virulence
targets 

In our quest to develop new compounds to block a range of infections we have
been studying a bacterial protein called AdhE. AdhE is important because
when it is deleted from pathogenic E. coli (str. O157:H7) it causes an
attenuation of virulence and a reduction in expression of the major system
used to attach to host cells. AdhE oligomerises in vivo and in vitro to form
long (15-120 nm) filaments, called spirosomes, that can be visualised using
electron microscopy. These are assumed to be an important aspect of the
enzymatic efficiency of the protein. Importantly we have solved the
high-resolution structure of the protein and identified lead compounds that
bind and inhibit spirosome formation. However, there are several outstanding
questions that we propose to address, thereby enhancing our understanding of
how AdhE spirosomes work. This basic knowledge will help us to translate our
work in the longer-term and to develop specific inhibitors that might
function against a range of Gram-negative pathogens. Specifically, we seek
answers to the following questions:
 
1.  Why are AdhE spirosomes formed and how is this regulated?
2.  Do AdhE spirosomes channel substrates?
3.  Where is the binding site for the lead compounds?
4.  Does AdhE influence expression of T3SSs in other species?
 
To address these questions we will use SAXS, AUC (University of Glasgow) and
molecular dynamics simulations (in collaboration with Prof Syma Khalid
(University of Southampton)). We will also aim to determine where compounds
bind on spirosomes by performing cryo-EM studies in collaboration with Prof
Ji-Joon Song (KAIST, Korea). To widen our understanding of the relevance for
other pathogens, we will generate adhE deletions in other Gram-negative
species and perform virulence-based assays with each pathogen.
 
The work requires a motivated protein biochemist/structural biologist who is
keen to take on an exciting project. Informal enquiries to Olwyn or Andrew:
 
olwyn.by...@glasgow.ac.uk  and
andrew@glasgow.ac.uk 
 

Mads Gabrielsen, MSc, PhD
Protein Crystallographer / Biophysicist
MVLS Structural Biology and Biophysical Characterisation Facility

Room B4-13
Joseph Black Building
University of Glasgow
GLASGOW
G12 8QQ
UK

+44 141 330 6447

mads.gabriel...@glasgow.gla.ac.uk





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[ccp4bb] Call open for beamtime at DESY beamline P11, PETRA III, Hamburg

[Hakanpää , Johanna]

Dear all,

We would like to inform you that the Call for Proposals (regular proposals) is 
now open for DESY beamline P11 at the PETRA III synchrotron in Hamburg.
This call relates to remote and onsite access during the run period 
August-December 2021.

The deadline for submission is
--
1 March 2021 until midnight local Hamburg time (UTC+1)
--

Proposals are to be submitted via https://door.desy.de

Preparation guidelines and templates for the proposals can be found here:
https://photon-science.desy.de/users_area/user_guide/write_a_proposal/

Beamline P11 is a versatile beamline dedicated to protein crystallography with 
a sample changer in unipuck format.

We offer various focusing modes with beam size from 5 x 10 um (flux 1 × 10^13 
ph/s at 12 keV at the sample position) to 300 x 300 um. Broad energy range from 
5.5 - 28 keV and Eiger2 16M detector. Fast sample changer cycle (20 s) with 
storage capacity for 23 unipucks (368 samples) and remote access.

For further information please see 
http://photon-science.desy.de/facilities/petra_iii/beamlines/p11_bio_imaging_and_diffraction

On behalf of the P11 team,
Johanna



Johanna Hakanpää, PhD

Scientist, Beamline P11 (PETRA III)
Deutsches Elektronen-Synchrotron DESY
Notkestrasse 85
22607 Hamburg
Germany

Phone: +49 40 8998 9 5756
Email: johanna.hakan...@desy.de



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[ccp4bb] [Job post] RA position in RNA biochemistry/structural biology

[Likai Liu]

Hello,

We are looking for a motivated RA to join our Structural Biology Team at 
Waltham, MA! The successful candidate would have rich experience working with 
different biomolecules and is interested in using NMR to aid drug design. 
Please apply by sending a cover letter and resumé to 
structural.biology.j...@skyhawktx.com

https://05174277-077d-4bd1-b8cf-4aa0f6343f4c.filesusr.com/ugd/cb0357_3761b0f8d8e84ca8b926c32a1b06147b.pdf

Thank you,

Likai Liu, PhD



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Re: [ccp4bb] [Job post] RA position in RNA biochemistry

[Likai Liu]

Please see the post below

https://05174277-077d-4bd1-b8cf-4aa0f6343f4c.filesusr.com/ugd/cb0357_3761b0f8d8e84ca8b926c32a1b06147b.pdf



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[ccp4bb] [Job post] RA position in RNA biochemistry

[Likai Liu]

Hello,

We are looking for a motivated RA to join our team at Waltham, MA! The 
successful candidate would have rich experience working with different 
biomolecules and is interested in using NMR to aid drug design. Please apply by 
sending a cover letter and resumé to structural.biology.j...@skyhawktx.com

Thank you,

Likai Liu, PhD


Li-Kai Liu, Sr. Scientist, Structural Biology | Skyhawk Therapeutics, Inc. | 35 
Gatehouse Drive, Waltham MA


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[ccp4bb] iNEXT-Discovery First Annual Scientific Meeting

[a . perrakis]

Dear all,

We are happy to announce the  "iNEXT-Discovery First Annual Scientific Meeting".

This online Zoom-event will take place on February 16th, 2021

The program, with presentations from iNEXT-Discovery partners and external 
speakers, is open for everybody with interest in modern state-of-the-art 
structural biology technologies and applications.

The first part of the program (11:00 - 13:00 CET) is geared towards new 
developments within iNEXT-Discovery.
It is followed by an open discussions and information session.
The afternoon (16:30 - 18:00) session features invited speakers on a variety of 
subjects, and anopen discussion with the speakers after 18:00!

A session with several short "Flash Talks” is also available, where we welcome 
anyone, but in particular users of iNEXT-Discovery facilities, to submit an 
abstract for considerations

Details of the program and a link for registration can be found here:
https://inext-discovery.eu/events/inext-discovery-1st-annual-scientific-meeting/

The registration link is also here:
https://zoom.us/webinar/register/5516115668255/WN_wY3vRzVoQn6eX2Gh5MeYQQ


On behalf of the Organizing Team,

Tassos

Prof. Dr. Anastassis Perrakis, PhD
Group leader, Dept. of Biochemistry, Netherlands Cancer Institute
Professor of Macromolecular Structures, University of Utrecht
Oncode Institute Investigator
Coordinator iNEXT-Discovery H2020











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[ccp4bb] 3-year postdoctoral position in biophysics at the Institute for Structural Biology, Grenoble, France.

[Dominique Bourgeois]

3-year postdoctoral position in biophysics at the Institute for Structural 
Biology, Grenoble, France. 



Investigating structural dynamics of green-to-red photoconvertible fluorescent 
proteins for the design of ultrastable variants. 



The recruited candidate will investigate the structural dynamics of 
green-to-red photoconvertible fluorescent proteins used in Single Molecule 
Localization Microscopy (SMLM) using NMR, kinetic X-ray crystallography and 
single-molecule imaging. The goal will be to engineer variants of enhanced 
photo stability to improve the quality of quantitative SMLM data. 



Fluorescent proteins (FPs) are the markers of choice for many applications in 
advanced fluorescence microscopy. Yet, super-resolution techniques such as 
PhotoActivation Localization Microscopy (PALM) and single-particle-tracking 
PALM (sptPALM) have not yet reached maturity, notably due to the non-ideal 
photophysical performance of the used FP labels, in particular green-to-red 
photoconvertible fluorescent proteins (PCFPs). This project aims at enhancing 
the photostability of PCFPs by combining investigations of their structural 
dynamics in realistic environmental conditions with large-scale engineering and 
screening at the single-molecule (SM) level. The recruited candidate will 
concentrate on mechanistic investigations of PCFPs by NMR and crystallography. 
Notably, a state-of-the-art NMR toolbox has been recently developed at the 
Institute, specifically adapted to the study of fluorescent proteins. Applying 
and developing this NMR toolbox, in combination with crystallography and 
optical spectroscopy, will be central to the project and will be used to 
suggest relevant mutation sites to engineer enhanced PCFPs. The FP engineering 
part of the work will be performed in collaboration with our partners in 
Bordeaux (J.B. Sibarita, M. Sainlos), in the context of a recently awarded 
grant by the French “ANR”. Overall, the candidate will seek to provide key 
mechanistic insights into the “dark side” of PCFPs, which has remained largely 
overlooked thus far due to its intricate nature, pushing the knowledge of FPs 
beyond the state-of-the-art. 



Related publications: 

De Zitter et al , Nature Meth. , (2019) 16, 707-710; Christou et al, Biophys. 
J. , (2019), 117, 1-14 ; E. De Zitter et al, J. Am. Chem. Soc. , (2020), 142, 
10978–10988 



Grenoble is situated in the middle of the beautiful French Alps, and the IBS 
provides a unique environment for state-of-the-art integrated cellular and 
structural biology ( [ http://www.ibs.fr/ | http://www.ibs.fr/ ] ). 



Candidates should have a strong background in biophysics. Experience in 
multidimensional NMR spectroscopy, X-ray protein crystallography and good 
knowledge of biochemistry and fluorescence microscopy will be required. 
Additional expertise in molecular biology, cell biology, super-resolution 
microscopy, single-molecule imaging and fluorescent proteins will be key 
advantages. 



The position will be financed for 2+1 years by a recently obtained ANR grant ( 
[ https://anr.fr/en/ | https://anr.fr/en/ ] ) and IBS funding already 
available. 



Applications are now open. Please send a complete CV, a motivation letter, and 
at least two reference letters to Dominique Bourgeois ( [ 
mailto:dominique.bourge...@ibs.fr | dominique.bourge...@ibs.fr ] ), before 
February 28, 2021 . 

Candidates must also feel an application at CNRS: [ 
https://emploi.cnrs.fr/Offres/CDD/UMR5075-JERGAU-004/Default.aspx?lang=EN | 
https://emploi.cnrs.fr/Offres/CDD/UMR5075-JERGAU-004/Default.aspx?lang=EN ] 




-- 
Dominique BOURGEOIS 

** 
PIXEL Team 
Institut de Biologie Structurale J-P. Ebel 
IBS 
71 AVENUE DES MARTYRS 
CS 10090 
38044 GRENOBLE CEDEX 9 
FRANCE 
Tel: 33-(0)4 57 42 86 44 
http://www.ibs.fr/research/research-groups/dynamics-and-kinetics-of-molecular/pixel/
 
** 



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Re: [ccp4bb] Question about tncs and SAD phasing

[Clemens Vonrhein]

On top of what Eleanor mentioned, looking for about 160 S atoms with
anomalous signal to 3.9A will be VERY hard. If you manage actually
find those sites (or a good subset of them), the next density
modification step can also be very hard: jumping over that 3.5-4A
hurdle to get interpretable density right to your high reslution limit
is often tricky. But ... with your high level of NCS you have a chance
if you can get hold of some NCS operators to start with. For an
example of 20-fold NCS and phasing to 6A see [1] (and GETAX is in
CCP4, although without an interface in i2).

Do you get the feeling from your substructure solution trials that
those sites are actually correct? Most of the stats at 3.9A
(e.g. CCall/CCweak from SHELX) will not be very useful to judge the
quality of a potential solution. What does the (relative) occupancy of
all those sites look like? You don't want to see the first site with
occ=1.0 and then a big jump with all other sites coming in at
occ=0.3-0.4 or lower ... that wouldn't make a lot of sense. Also: do
you see some kind of distinction between successful and unsuccecssful
trials?

If there is absolutely nothing that gives you a feeling for a
successful substructure solution (even after trying with different
resolution limits, NSIT parameters, programs etc), I would abandon
that S-SAD approach and go for something with a bigger signal (and
definitely think about more than just one wavelength if at all
possible).

What I often find to be crucial in such cases: really careful data
processing (beamstop masking, correct (!) outlier rejection, ice-ring
and damaged pixel handling etc).

Regarding those operators from PROFESSS (I like that program a lot):
they seem suspiciously close to 90/45/180 and 90/90/180, which always
makes me suspicious.

Not sure any of the above actually helps ... ;-)

Cheers

Clemens

[1] http://scripts.iucr.org/cgi-bin/paper?S0907444902004614




On Sat, Jan 23, 2021 at 11:45:31AM +, Andrew Lovering wrote:
> Dear All,
> 
> 
> Having some fun with a marginal SAD S phasing case that you may be able to 
> help me on.
> 
> My primary question is "does strong tNCS artefactually inflate FOM scores"? I 
> have a solution with FOM 0.48 to 3.5Ang or so and the map isn't as good as 
> I'd expect (in either hand).
> 
> 
> -more detail if this helps:
> 
> We have collected 3 sweeps at 1.77A wavelength, Rpim is good 2% or less, 
> xtriage Z-score is 2, aimless suggests anom signal to 3.9A, as does shelxc, 
> no signs of radiation damage, anom multiplicity is 40, cc anom 0.4 overall
> 
> 
> Cell is P41212/P43212, 146.9 146.9 335.1 diffracts to 2Ang or so which 
> suggests 50% solvent = 22 copies in asu!
> 
> 
> each copy should have 2 disulphides and 6 extra S in Met. I am using dsul 
> keyword in shelxd. I ask for a variety of sites, usually gets 240+, there is 
> also 300mM Mg and 20mM As in conditions (site completion in phaser  with Mg 
> or As doesnt really help)
> 
> 
> When we look at native patterson we see one major off origin peak at 0.5 0.5 
> 0.173 fractional, 25% height of origin
> 
> -and a smaller peak at 0.35 0.35 0.5 10% of origin
> 
> 
> I have tried everything. Changing phasing limits, programme, cutting back 
> data range, number of sites. But I can get a decent sites pdb from 
> shelx/crank2 which popped into phenix autosol gives stats that wrongly make 
> me think I am close (fom 0.4-0.5, score 30+, skew 0.1+). My model has no 
> molrep possibilities and likely no alpha helical content
> 
> 
> If I run the substructure sites through profess, I get two nice operators:
> 
> -(op1) polar 90 -44 179.9 trans -72 -73 -444.6 rotation order 8, N atoms 
> paired 74 from 16 of input atoms
> 
> -(op2) polar 90 -87 180 trans -147 -9 -863 rotation order 2 paired 8 atoms 
> from 8 of input
> 
> (profess notes these two sit 43 degrees apart)
> 
> 
> I can see that op1 has a translational component that is my tNCS vector (-0.5 
> -0.5 and 0.17 away from 0.5)and that likely I have 4 fold operator that 
> sits over my crystallographic 2-fold, which is 45 degree from another 2 fold 
> (self rotation function attached..). I would welcome a better description of 
> this 
> 
> Using these operators (or indeed a pure tNCS only operator) in dm gives good 
> correlation but again no map improvement. Is it worth playing with nmol?
> 
> 
> Any suggestions welcome, before we hit this with some higher electron 
> derivatives!
> 
> 
> Thanks
> 
> Andy
> 
> 
> 
> 
> 
> 
> From: >
> Sent: January 23, 2021 11:08 AM
> To: Andrew Lovering (Biosciences)
> Subject: Srf
> 
> 
> 
> 
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