Re: [ccp4bb] electron density close Histidine side chain

[Jon Cooper]

Hello, do you have any negative difference density for the current position of 
the His side chain? If so, it may be in two confirmations. Just a thought.

Best wishes, Jon Cooper, E-mail: jon.b.coo...@protonmail.com

 Original Message 
On 20 Jul 2020, 17:16, samer halabi wrote:

> Hello all,
> I have few blobs in an MHC II structure I am working on, especially opposite 
> to Histidine as in the accompanying screenshot, that I am confused about.
>
> In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
> Whatever ligand I am fitting in I am getting a clash (overlap -1.029), which 
> makes me think whether there is a covalent bond forming between Histidine and 
> other molecule. Perhaps by oxidation.
>
> I would greatly appreciate if you can advice me about it, whether there is 
> some kind of ligand I can try to fit and if this is something that occurs in 
> some structures.
> Thank you.
> Best regards,
> Samer
>
> ---
>
> 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] electron density close Histidine side chain

[Roger Rowlett]

Almost certainly a metal ion, possibly Ni(2+) if a Ni-NTA column was used
for purification. Ni-N bond lengths are typically around 2.0 A. Additional
density is probably coordinated water molecules, which should have similar
Ni-O bond distances around 1.9 A. It is fairly common to find adventitious
metal ions (zinc, copper, nickel) bound to His residues.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

email: rrowl...@colgate.edu

On Mon, Jul 20, 2020 at 12:17 PM samer halabi <
30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello all,
> I have few blobs in an MHC II structure I am working on, especially
> opposite to Histidine as in the accompanying screenshot, that I am confused
> about.
>
> In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and
> Glycerol.
> Whatever ligand I am fitting in I am getting a clash (overlap -1.029),
> which makes me think whether there is a covalent bond forming between
> Histidine and other molecule. Perhaps by oxidation.
>
> I would greatly appreciate if you can advice me about it, whether there is
> some kind of ligand I can try to fit and if this is something that occurs
> in some structures.
> Thank you.
> Best regards,
> Samer
>
> --
>
> 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] Sad News

[Robert Stroud]

I am deeply saddened to hear of Ward’s passing. He was a friend to many of us 
and a tremendous supporter of structural biology. I knew him since his time at 
Michigan. He championed the ways through applications of new innovations 
throughout the era of synchrotron technologies and increasing awareness of the 
possibilities they brought with them. As NIH program director he was always a 
source of insight and support for prospects and possibilities. 
Robert Stroud
str...@msg.ucsf.edu
415 987 7535



> On Jul 18, 2020, at 4:36 AM, Sweet, Robert 
> <27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> I'm writing to acknowledge the passing of Ward Smith during the weekend of 5 
> July. Ward got his PhD with Martha Ludwig at U. of Michigan, and then came to 
> UCLA in 1977 to join Dave Eisenberg’s group as a postdoc. During the course 
> of things, he met Cheryl Janson, a Paul Boyer postdoc, and they were married 
> in 1980.  During his time at UCLA Ward became expert in operation of the 
> Xuong/Hamlin/Nielsen multi-wire system at UCSD and tutored the UCLA users in 
> its use. In 1985 Ward and Cheryl left UCLA and went to Monsanto in St. Louis, 
> where Ward worked as a structural biologist. In 1987 they went to Agouron 
> Pharmaceuticals in San Diego. And then in 1995 went cross-country to 
> SmithKline Beecham (which became Glaxo SmithKline, merging with 
> GlaxoWellcome). 
> 
> Ward was very involved with getting IMCA set up as a functional facility for 
> pharmaceuticals at Argonne as SmithKline's representative. This experience 
> gave him significant credibility in synchrotron macromolecular 
> crystallography, and in 2003 he joined the GM/CA-CAT beamlines at the APS to 
> help Bob Fischetti and others construct that excellent facility. During this 
> time Cheryl worked at Shamrock Structures.
> 
> Ward moved to the NIH headquarters in 2007. There he took some responsibility 
> for the Protein Structure Initiative, also playing an important role in 
> supporting NIH synchrotron facilities. In 2010 he became the branch chief for 
> the Structural Genomics and Proteomics Technology Branch in the Division of 
> Cell Biology and Biophysics.  He remained in that position through 2017. At 
> the 2018 NIGMS re-organization Ward went to the Biophysics, Biomedical 
> Technology, and Computational Biosciences division as the branch chief for 
> the Biomedical Technology Branch. 
> 
> Ward helped oversee the big NIH-funded, $45 M construction of three major 
> beamlines at NSLS-II, a project called ABBIX that ran 2011-2017. In 2017 he 
> became program director for NIH support of structural biology beamlines at 
> NSLS-II and other DOE synchrotrons. 
> 
> Many knew Ward for his always calm, reasoned demeanor; he was unflappable, 
> resilient, and friendly. He was well read and devoted to his family.  
> 
> 
>  Robert M. Sweet   E-Dress:  sw...@bnl.gov
>  Scientific Advisor, CBMS: The Center for BioMolecular
>Structure at NSLS-II
>  Photon Sciences and Biology Dept
>  Brookhaven Nat'l Lab.
>  Upton, NY  11973 U.S.A.
>  Phones: 631 344 3401  (Office)
>631 338 7302  (Mobile)
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing 
> list hosted by www.jiscmail.ac.uk, terms & conditions are available at 
> https://www.jiscmail.ac.uk/policyandsecurity/



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[ccp4bb] Graduate Student Position (MSc or PhD)

[Gerd Prehna]

The Prehna Lab is looking to recruit an outstanding graduate student (MSc or 
PhD position) to join the Department of Microbiology at the University of 
Manitoba, Canada (https://home.cc.umanitoba.ca/~prehnag/). 

Our lab studies how bacteria communicate with their hosts, how they communicate 
with each other, and how they communicate with other micro-organisms. 

Project: Quorum sensing in Streptococcus
The supported graduate student position will study the natural defense 
mechanisms that Streptococcus pyogenes uses to defend itself from 
anti-bacterial peptides. S. pyogenes is a major human pathogen responsible for 
Strep. Throat and flesh-eating disease that uses quorum sensing to coordinate 
gene expression within a human host. This social coordination is responsible 
for the up-regulation of enzymes that modify the bacterial cell wall and cell 
membrane to resist toxic anti-bacterial peptides secreted by the human host and 
competing bacteria. This project is aimed at understanding the biochemistry and 
structural biology of these defense enzymes in S. pyogenes with the goal of 
designing novel antibiotics. 

Qualifications

Applicants should possess s Bachelor’s degree in the life sciences or a related 
discipline, and be interested in both biochemistry and microbiology. Some 
experience in molecular biology as well as protein expression and purification 
is desired but not required. Training in protein purification, X-ray 
crystallography, and NMR spectroscopy will be provided. This project has the 
potential for training in cryo-EM. English language skills, the ability to work 
in a team, willingness to learn, and good organizational skills are required.

Application

Applications should include: 
1. The applicant's CV
2. A one-page summary of their motivation for pursing graduate studies
3. Contact details for at least two references

Please send application materials to:

gerd.pre...@umanitoba.ca 

The starting dates are Fall 2020 or January 2021.

The University of Manitoba (http://umanitoba.ca/) is located in Winnipeg, 
Manitoba and is Western Canada’s first University. The University has large 
core-group of structural biology and biochemical researchers between the 
departments of Microbiology (http://www.sci.umanitoba.ca/microbiology/) and 
Chemistry (The Manitoba Group in Protein Structure and Function), access to the 
Canadian Light Source synchrotron, and comprehensive molecular biophysics 
instrumentation (500 and 600 MHz NMR, in-house XRD, ITC, CD, DSC, nanoDSF, AUC, 
DLS, SEC-MALS, and MST). Winnipeg is also home to the National Microbiology 
Laboratory, a leader in infectious disease research and collaborator with the 
Department of Microbiology.



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Re: [ccp4bb] electron density close Histidine side chain

[David Briggs]

Hi Samer,

Did you use a His tag/Ni-NTA during purification? Sometimes Ni2+ ions leach off 
the Ni-NTA -  maybe the two "ears" are  accompanying waters?

Ni-His co-ordination distance is pretty short
(2-2.2Å - table 3 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872550/#!po=0.69) and might 
account for your bump when you model in a ligand.

Good luck!

Dave

--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs


From: CCP4 bulletin board  on behalf of Nils Marechal 
<4954a024d277-dmarc-requ...@jiscmail.ac.uk>
Sent: Monday, July 20, 2020 5:27:37 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] electron density close Histidine side chain

‌Dear Samer,

What king of cryo-protectant did you use ?

Such a bent density, with that size, looks like an ethylene-diol.

Best regards,

Nils Marechal

De : "samer halabi" <30c2162795b2-dmarc-requ...@jiscmail.ac.uk>
A : CCP4BB@JISCMAIL.AC.UK
Envoyé: lundi 20 Juillet 2020 18:17
Objet : [ccp4bb] electron density close Histidine side chain

Hello all,
I have few blobs in an MHC II structure I am working on, especially opposite to 
Histidine as in the accompanying screenshot, that I am confused about.

In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
Whatever ligand I am fitting in I am getting a clash (overlap -1.029), which 
makes me think whether there is a covalent bond forming between Histidine and 
other molecule. Perhaps by oxidation.

I would greatly appreciate if you can advice me about it, whether there is some 
kind of ligand I can try to fit and if this is something that occurs in some 
structures.
Thank you.
Best regards,
Samer



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1





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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] Sad News

[Debanu Das]

It is very saddening to hear this news. It took me a couple of days to wrap
my head around it and write.

Thank you for this obituary Bob, which captures so eloquently the breadth
of all of Ward's remarkable engagements and contributions.

I interacted with him a few times over the course of the Protein Structure
Initiative structural genomics programs at the BSGC and JCSG, and more
recently over the last couple of years as part of current endeavors. His
efforts contributed very significantly to everything I have been doing for
the past 15 years too. It is a tremendous loss but a life very well lived
with many important contributions to remember.

Best regards,
Debanu
--
Debanu Das,
Accelero Biostructures

On Sun, Jul 19, 2020 at 4:55 PM James Holton  wrote:

> Ward was the Program Official for all my grants!  My life will not be
> the same without him.  He was always so supportive and helpful with
> advice on how to navigate the sometimes convoluted system that is the
> NIH.  From the Protein Structure Initiative to today his hard work has
> made possible my entire scientific career.
>
> "missed" doesn't seem to cover it. May your rest be a peaceful one, Ward.
>
> -James Holton
> MAD Scientist
>
> On 7/18/2020 4:36 AM, Sweet, Robert wrote:
> > I'm writing to acknowledge the passing of Ward Smith during the weekend
> of 5 July. Ward got his PhD with Martha Ludwig at U. of Michigan, and then
> came to UCLA in 1977 to join Dave Eisenberg’s group as a postdoc. During
> the course of things, he met Cheryl Janson, a Paul Boyer postdoc, and they
> were married in 1980.  During his time at UCLA Ward became expert in
> operation of the Xuong/Hamlin/Nielsen multi-wire system at UCSD and tutored
> the UCLA users in its use. In 1985 Ward and Cheryl left UCLA and went to
> Monsanto in St. Louis, where Ward worked as a structural biologist. In 1987
> they went to Agouron Pharmaceuticals in San Diego. And then in 1995 went
> cross-country to SmithKline Beecham (which became Glaxo SmithKline, merging
> with GlaxoWellcome).
> >
> > Ward was very involved with getting IMCA set up as a functional facility
> for pharmaceuticals at Argonne as SmithKline's representative. This
> experience gave him significant credibility in synchrotron macromolecular
> crystallography, and in 2003 he joined the GM/CA-CAT beamlines at the APS
> to help Bob Fischetti and others construct that excellent facility. During
> this time Cheryl worked at Shamrock Structures.
> >
> > Ward moved to the NIH headquarters in 2007. There he took some
> responsibility for the Protein Structure Initiative, also playing an
> important role in supporting NIH synchrotron facilities. In 2010 he became
> the branch chief for the Structural Genomics and Proteomics Technology
> Branch in the Division of Cell Biology and Biophysics.  He remained in that
> position through 2017. At the 2018 NIGMS re-organization Ward went to the
> Biophysics, Biomedical Technology, and Computational Biosciences division
> as the branch chief for the Biomedical Technology Branch.
> >
> > Ward helped oversee the big NIH-funded, $45 M construction of three
> major beamlines at NSLS-II, a project called ABBIX that ran 2011-2017. In
> 2017 he became program director for NIH support of structural biology
> beamlines at NSLS-II and other DOE synchrotrons.
> >
> > Many knew Ward for his always calm, reasoned demeanor; he was
> unflappable, resilient, and friendly. He was well read and devoted to his
> family.
> >
> > 
> >Robert M. Sweet   E-Dress:  sw...@bnl.gov
> >Scientific Advisor, CBMS: The Center for BioMolecular
> >  Structure at NSLS-II
> >Photon Sciences and Biology Dept
> >Brookhaven Nat'l Lab.
> >Upton, NY  11973 U.S.A.
> >Phones: 631 344 3401  (Office)
> >  631 338 7302  (Mobile)
> > 
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
> > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
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This 

Re: [ccp4bb] electron density close Histidine side chain

[Nils Marechal]

‌Dear 
Samer,

What king of cryo-protectant did you use ?

Such a bent density, with that size, looks like an ethylene-diol.

Best regards,

Nils Marechal


De : "samer 
halabi" 30c2162795b2-dmarc-requ...@jiscmail.ac.uk
A : CCP4BB@JISCMAIL.AC.UK
Envoyé: lundi 20 Juillet 2020 18:17
Objet : [ccp4bb] electron density close Histidine side chain




Helloall,
I have few blobs in 
an MHC II structure I am working on, especially opposite to Histidine as in the 
accompanying screenshot, that I am confused about.

In the crystal 
conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
Whatever ligand I am 
fitting in I am getting a clash (overlap -1.029), which makes me think whether 
there is a covalent bond forming between Histidine and other molecule. Perhaps 
by oxidation.

I would greatly 
appreciate if you can advice me about it, whether there is some kind of ligand 
I can try to fit and if this is something that occurs in some 
structures.
Thankyou.
Bestregards,
Samer





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link:
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Re: [ccp4bb] electron density close Histidine side chain

[Dale Tronrud]

   If there is a covalent link, maybe sending a sample off to mass spec
would be a good idea.  That would remove some of the guesswork.

Dale Tronrud

On 7/20/2020 9:16 AM, samer halabi wrote:
> Hello all,
> I have few blobs in an MHC II structure I am working on, especially
> opposite to Histidine as in the accompanying screenshot, that I am
> confused about.
> 
> In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and
> Glycerol.
> Whatever ligand I am fitting in I am getting a clash (overlap -1.029),
> which makes me think whether there is a covalent bond forming between
> Histidine and other molecule. Perhaps by oxidation.
> 
> I would greatly appreciate if you can advice me about it, whether there
> is some kind of ligand I can try to fit and if this is something that
> occurs in some structures.
> Thank you.
> Best regards,
> Samer
> 
> 
> 
> 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] AW: [EXTERNAL] chirality with electron diffraction

[Jessica Bruhn]

Hi Herman and Tim,

Thank you Tim for a comprehensive answer! Personally, we are collecting
data using the rotation method (traditional "microED" style data
collection) and processing our data with DIALS, which cannot perform the
dynamical refinement Tim discussed. We therefore cannot determine the
absolute configuration based strictly on the intensities.

We can of course determine the relative stereochemistry (as is possible
with X-rays). Basically if you know the handedness of one site, you can use
it to infer the chirality of all of the other chiral centers in the
molecule. We have been suggesting to our clients that they attempt
co-crystallization with small chiral molecules which can then serve as the
chirality reference. We have had one client successfully accomplish this
for one compound, but hopefully others will attempt this as well. I
personally have minimal experience with small molecule crystallization
techniques, but some reading suggested that first searching the CSD for
compounds that tend to co-crystallize with your compound of interest may be
a good way to start.

And though not every structure will be able to help weigh in on chirality,
there is still a lot of value in these structures for those in the
chemistry field. People working in solid formulations are especially
interested in ED structures because unlike (most) protein
crystallographers, they are actually very interested in the lattice and the
forces that drive packing. Additionally, using an ED structure to simulate
a powder diffraction pattern can be helpful for those optimizing
large-scale synthesis reactions as they can now use X-ray powder
diffraction and a comparison with the simulated pattern to determine the
relative purity of their sample or to detect small amounts of contaminants.
The list goes on.

And as Tim said, hopefully further advances allow for more
straightforward/routine determination of the absolute stereochemistry.

Cheers,
Jessica



On Mon, Jul 20, 2020 at 5:34 AM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Thank you Tim, I had the same impression.
> I am afraid that our chemists will have to wait a little longer before
> they routinely can get absolute configurations from electron diffraction.
>
> Best Herman
>
> -Ursprüngliche Nachricht-
> Von: Tim Gruene 
> Gesendet: Montag, 20. Juli 2020 14:21
> An: Schreuder, Herman /DE 
> Cc: CCP4 bulletin board 
> Betreff: Re: [EXTERNAL] chirality with electron diffraction
>
> EXTERNAL : Real sender is  tim.gru...@univie.ac.at
>
>
>
> Dear Herman,
>
> no, I don't think this is routine, yet!
> First of all, instrument manufacturers need to catch up. What's on the
> market is quite behind what is possible - although, I don't have the
> instrument I'd love to have, but at least the meta data transfer to the
> image headers only works when you work with a native CCD camera (stone age,
> I know), not with a hybrid pixel detector.
>
> Secondly, Lukas Palatinus' software is, as far as I know, not yet
> compatible with the rotation method, i.e. when you use XDS or DIALS or
> mosflm, etc, you cannot use his software for dynamic refinement. I also
> understand that dynamic refinement is quite time consuming. Lukas is
> working on this, though.
>
> Best wishes,
> Tim
>
> On Mon, 20 Jul 2020 10:36:49 +
> "Schreuder, Herman /DE"  wrote:
>
> > Hi Tim,
> >
> > thank you for your reply. The 1998 Schenk paper is "new" to me, I had
> > seen this one:
> > https://science.sciencemag.org/content/sci/364/6441/667.full.pdf The
> > background of my question was about the status in practice: Is it
> > possible to routinely determine the absolute configuration of small
> > molecules by electron diffraction, or is it something that in theory
> > can be done, but only difficult in practice?
> >
> > Best,
> > Herman
> >
> >
> >
> > -Ursprüngliche Nachricht-
> > Von: Tim Gruene 
> > Gesendet: Montag, 20. Juli 2020 11:03
> > An: CCP4 bulletin board ; Schreuder, Herman /DE
> >  Betreff: [EXTERNAL] chirality with
> > electron diffraction
> >
> > EXTERNAL : Real sender is  tim.gru...@univie.ac.at
> >
> >
> >
> > Dear Herman,
> >
> > The absolute configuration can be determined, although very
> > differently from X-ray ccrystallography.
> >
> > So far, two different experimental approaches have been published:
> > Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890
> >
> > and Brazda et al (2019), 10.1126/science.aaw2560
> >
> > The former is based on imaging, the latter is based on dynamical
> > refinement: Jansen, Tang, Zandbergen, Schenk (1998),
> > https://doi.org/10.1107/S0108767397010489
> >
> > There is a very interesting paper by Burmester and  Schroeder Scanning
> > Microscopy Vol. 11, 1997 (Pages 323-334). According to this paper, the
> > anomalous signal in ED is actually stronger than in X-ray
> > crystallography. As far as I know, this has not been pursued further.
> >
> > Best wishes,
> > Tim
> >
> > P.S.: This is a response to your 

Re: [ccp4bb] electron density close Histidine side chain

[EchelonIV]

Hello,

what immediately comes into mind is phosphohistidine, especially since the
density looks rather large, but the geometry seems a bit off for that but I
cannot judge only from one angle. You could of course try to fit it in and
see how it looks.

Cheers,
Arne

On Mon, Jul 20, 2020 at 6:17 PM samer halabi <
30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello all,
> I have few blobs in an MHC II structure I am working on, especially
> opposite to Histidine as in the accompanying screenshot, that I am confused
> about.
>
> In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and
> Glycerol.
> Whatever ligand I am fitting in I am getting a clash (overlap -1.029),
> which makes me think whether there is a covalent bond forming between
> Histidine and other molecule. Perhaps by oxidation.
>
> I would greatly appreciate if you can advice me about it, whether there is
> some kind of ligand I can try to fit and if this is something that occurs
> in some structures.
> Thank you.
> Best regards,
> Samer
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>


-- 
--
Arne Raasakka
PhD Biochemistry
Department of Biomedicine, University of Bergen
Norway



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[ccp4bb] AW: [EXTERNAL] chirality with electron diffraction

[Schreuder, Herman /DE]

Thank you Tim, I had the same impression. 
I am afraid that our chemists will have to wait a little longer before they 
routinely can get absolute configurations from electron diffraction.

Best Herman

-Ursprüngliche Nachricht-
Von: Tim Gruene  
Gesendet: Montag, 20. Juli 2020 14:21
An: Schreuder, Herman /DE 
Cc: CCP4 bulletin board 
Betreff: Re: [EXTERNAL] chirality with electron diffraction

EXTERNAL : Real sender is  tim.gru...@univie.ac.at   



Dear Herman,

no, I don't think this is routine, yet! 
First of all, instrument manufacturers need to catch up. What's on the market 
is quite behind what is possible - although, I don't have the instrument I'd 
love to have, but at least the meta data transfer to the image headers only 
works when you work with a native CCD camera (stone age, I know), not with a 
hybrid pixel detector.

Secondly, Lukas Palatinus' software is, as far as I know, not yet compatible 
with the rotation method, i.e. when you use XDS or DIALS or mosflm, etc, you 
cannot use his software for dynamic refinement. I also understand that dynamic 
refinement is quite time consuming. Lukas is working on this, though.

Best wishes,
Tim

On Mon, 20 Jul 2020 10:36:49 +
"Schreuder, Herman /DE"  wrote:

> Hi Tim,
> 
> thank you for your reply. The 1998 Schenk paper is "new" to me, I had 
> seen this one:
> https://science.sciencemag.org/content/sci/364/6441/667.full.pdf The 
> background of my question was about the status in practice: Is it 
> possible to routinely determine the absolute configuration of small 
> molecules by electron diffraction, or is it something that in theory 
> can be done, but only difficult in practice?
> 
> Best,
> Herman
> 
> 
> 
> -Ursprüngliche Nachricht-
> Von: Tim Gruene 
> Gesendet: Montag, 20. Juli 2020 11:03
> An: CCP4 bulletin board ; Schreuder, Herman /DE 
>  Betreff: [EXTERNAL] chirality with 
> electron diffraction
> 
> EXTERNAL : Real sender is  tim.gru...@univie.ac.at   
> 
> 
> 
> Dear Herman,
> 
> The absolute configuration can be determined, although very 
> differently from X-ray ccrystallography.
> 
> So far, two different experimental approaches have been published: 
> Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890
> 
> and Brazda et al (2019), 10.1126/science.aaw2560
> 
> The former is based on imaging, the latter is based on dynamical
> refinement: Jansen, Tang, Zandbergen, Schenk (1998),
> https://doi.org/10.1107/S0108767397010489
> 
> There is a very interesting paper by Burmester and  Schroeder Scanning 
> Microscopy Vol. 11, 1997 (Pages 323-334). According to this paper, the 
> anomalous signal in ED is actually stronger than in X-ray 
> crystallography. As far as I know, this has not been pursued further.
> 
> Best wishes,
> Tim
> 
> P.S.: This is a response to your email to Jessica Bruhns in the thread 
> 'quote source inquiry'. This thread has reached an overflow, so I took 
> the liberty to adjust the subject.
> 
> --
> --
> Tim Gruene
> Head of the Centre for X-ray Structure Analysis Faculty of Chemistry 
> University of Vienna
> 
> Phone: +43-1-4277-70202
> 
> GPG Key ID = A46BEE1A



--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University 
of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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Re: [ccp4bb] [EXTERNAL] chirality with electron diffraction

[Tim Gruene]

Dear Herman,

no, I don't think this is routine, yet! 
First of all, instrument manufacturers need to catch up. What's on the
market is quite behind what is possible - although, I don't have the
instrument I'd love to have, but at least the meta data transfer to the
image headers only works when you work with a native CCD camera (stone
age, I know), not with a hybrid pixel detector.

Secondly, Lukas Palatinus' software is, as far as I know, not yet
compatible with the rotation method, i.e. when you use XDS or DIALS or
mosflm, etc, you cannot use his software for dynamic refinement. I also
understand that dynamic refinement is quite time consuming. Lukas is
working on this, though.

Best wishes,
Tim

On Mon, 20 Jul 2020 10:36:49 +
"Schreuder, Herman /DE"  wrote:

> Hi Tim,
> 
> thank you for your reply. The 1998 Schenk paper is "new" to me, I had
> seen this one:
> https://science.sciencemag.org/content/sci/364/6441/667.full.pdf The
> background of my question was about the status in practice: Is it
> possible to routinely determine the absolute configuration of small
> molecules by electron diffraction, or is it something that in theory
> can be done, but only difficult in practice?
> 
> Best,
> Herman
> 
> 
> 
> -Ursprüngliche Nachricht-
> Von: Tim Gruene  
> Gesendet: Montag, 20. Juli 2020 11:03
> An: CCP4 bulletin board ; Schreuder, Herman
> /DE  Betreff: [EXTERNAL] chirality with
> electron diffraction
> 
> EXTERNAL : Real sender is  tim.gru...@univie.ac.at   
> 
> 
> 
> Dear Herman,
> 
> The absolute configuration can be determined, although very
> differently from X-ray ccrystallography.
> 
> So far, two different experimental approaches have been published: 
> Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890
> 
> and Brazda et al (2019), 10.1126/science.aaw2560 
> 
> The former is based on imaging, the latter is based on dynamical
> refinement: Jansen, Tang, Zandbergen, Schenk (1998),
> https://doi.org/10.1107/S0108767397010489
> 
> There is a very interesting paper by Burmester and  Schroeder
> Scanning Microscopy Vol. 11, 1997 (Pages 323-334). According to this
> paper, the anomalous signal in ED is actually stronger than in X-ray
> crystallography. As far as I know, this has not been pursued further.
> 
> Best wishes,
> Tim
> 
> P.S.: This is a response to your email to Jessica Bruhns in the
> thread 'quote source inquiry'. This thread has reached an overflow,
> so I took the liberty to adjust the subject.
> 
> --
> --
> Tim Gruene
> Head of the Centre for X-ray Structure Analysis Faculty of Chemistry
> University of Vienna
> 
> Phone: +43-1-4277-70202
> 
> GPG Key ID = A46BEE1A



-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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pgprjT4eCQVZI.pgp
Description: OpenPGP digital signature

Re: [ccp4bb] protein oligomer

[Mark J van Raaij]

Dear Shijun,
Just a reminder that the pI calculated from the sequence is not necessarily the 
real pI, and sometimes can very quite a bit due to folding and oligomerization 
of the protein. The first protein I ever expressed had a calculated pI of 9.2 
and a real pI of 7.8 (if I remember correctly), so the difference can be quite 
large. You can determine the real pI by iso-electric focussing. 
Best wishes,
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 20 Jul 2020, at 08:38, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:
> 
> Dear:
> 
> The protein was purified in 4 degree, and the expression level is low, so the 
> aggregation is not by high concentration; the buffer pH is 7.5 which is not 
> colse to the PI 8.6. It should be a dimer when function, but it was 
> aggregated when negative staining. Maybe I could try to add arginine when 
> purification, or do mutantions. anyone has website for prediction the 
> mutation sites of protein?
> 
> Thanks!
> 
> Best,
> 
> shijun
> 
> 
> 
> -原始邮件-
> 发件人:"Nikolay Dobrev" 
> 发送时间:2020-07-19 20:29:49 (星期日)
> 收件人: CCP4BB@JISCMAIL.AC.UK
> 抄送: 
> 主题: Re: [ccp4bb] protein oligomer
> 
> It really depends from the nature of the protein and if is 
> oligomerizing/agregating/forming polymers if any of this is reversible.
> On the other side if you are working with one of the fibril forming protein 
> it will require optimizaiton on its on as they will form naturally polymers.
> 
> Do you observed different specises when you analyze your protein by SEC or if 
> you are able to perfom DLS?
> Additional information regarding your protein will be really helpful for more 
> detalied suggestions how to overcome your protein.
> 
> Best,
> Nikolay
> 
> Nikolay Dobrev 
> Scientific Officer, Protein Expression and Purification Core Facility
> EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
> T +49 6221 387 8633 | M +49 173 684 0532
> twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
> Visit www.embl.org/events  for a complete list of 
> all EMBL events.
> 
> On 19/07/2020 14:15, S. Mohanty wrote:
>> Keep the protein concentration low during purification steps along with 
>> using other anti-aggregation agent/s. Make sure that the pH at which you are 
>> purifying is not close to the pI of the protein. Until completely purified, 
>> all purification steps should be performed in a cold room if it is a soluble 
>> protein.
>> 
>> Smita 
>> 
>> 
>> On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga  
>>  wrote:
>> 
>> 
>> I am not sure what you mean by polymer formation. Presuming that you have 
>> optimized your protein concentration, pH and salt concentration, you could 
>> try arginine as an anti-aggregation agent in your purification (I presume 
>> you do FPLC). Have a look at chaotropic agents used in protein purification, 
>> The answer is generally dependent on the protein/proteins you are trying to 
>> purify and is not necessarily straightforward.
>> 
>> Kinds regards
>> 
>> On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn 
>> > wrote:
>> Dear all:
>> 
>> Any ideas to decrease protein polymer formation? my protein was easy to form 
>> oligomers and precipitation when do purification,I have tried add glycerol 
>> and DTT,both didn't work. Does anyone has experience to avoid it happening. 
>> Thanks!
>> 
>> Best Regards
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
>> -- 
> Nikolay Dobrev 
> Scientific Officer, Protein Expression and Purification Core Facility
> EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
> T +49 6221 387 8633 | M +49 173 684 0532
> twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
> Visit www.embl.org/events  for a complete list of 
> all EMBL events.
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 
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> 

[ccp4bb] AW: [EXTERNAL] chirality with electron diffraction

[Schreuder, Herman /DE]

Hi Tim,

thank you for your reply. The 1998 Schenk paper is "new" to me, I had seen this 
one: https://science.sciencemag.org/content/sci/364/6441/667.full.pdf
The background of my question was about the status in practice: Is it possible 
to routinely determine the absolute configuration of small molecules by 
electron diffraction, or is it something that in theory can be done, but only 
difficult in practice?

Best,
Herman



-Ursprüngliche Nachricht-
Von: Tim Gruene  
Gesendet: Montag, 20. Juli 2020 11:03
An: CCP4 bulletin board ; Schreuder, Herman /DE 

Betreff: [EXTERNAL] chirality with electron diffraction

EXTERNAL : Real sender is  tim.gru...@univie.ac.at   



Dear Herman,

The absolute configuration can be determined, although very differently from 
X-ray ccrystallography.

So far, two different experimental approaches have been published: 
Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890

and Brazda et al (2019), 10.1126/science.aaw2560 

The former is based on imaging, the latter is based on dynamical
refinement: Jansen, Tang, Zandbergen, Schenk (1998),
https://doi.org/10.1107/S0108767397010489

There is a very interesting paper by Burmester and  Schroeder Scanning 
Microscopy Vol. 11, 1997 (Pages 323-334). According to this paper, the 
anomalous signal in ED is actually stronger than in X-ray crystallography. As 
far as I know, this has not been pursued further.

Best wishes,
Tim

P.S.: This is a response to your email to Jessica Bruhns in the thread 'quote 
source inquiry'. This thread has reached an overflow, so I took the liberty to 
adjust the subject.

--
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University 
of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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[ccp4bb] chirality with electron diffraction

[Tim Gruene]

Dear Herman,

The absolute configuration can be determined, although very differently
from X-ray ccrystallography.

So far, two different experimental approaches have been published: 
Ma, Oleynikov, Terasaki (2017), https://doi.org/10.1038/nmat4890

and Brazda et al (2019), 10.1126/science.aaw2560 

The former is based on imaging, the latter is based on dynamical
refinement: Jansen, Tang, Zandbergen, Schenk (1998),
https://doi.org/10.1107/S0108767397010489

There is a very interesting paper by Burmester and  Schroeder
Scanning Microscopy Vol. 11, 1997 (Pages 323-334). According to this
paper, the anomalous signal in ED is actually stronger than in X-ray
crystallography. As far as I know, this has not been pursued further.

Best wishes,
Tim

P.S.: This is a response to your email to Jessica Bruhns in the thread
'quote source inquiry'. This thread has reached an overflow, so I took
the liberty to adjust the subject.

-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

GPG Key ID = A46BEE1A



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pgpb5PLDIIARa.pgp
Description: OpenPGP digital signature

Re: [ccp4bb] protein oligomer

[mesters]

Hi,

low expression levels could indicate (just a few thoughts):

- the protein is not folded properly and already degraded during 
production which in turn may indicate problems with the construct 
(insert + plasmid) itself or problems with the speed of expression (i.e. 
no time to fold). Lower the expression temperature or induce at a much 
higher OD600 (near log phase) and grow for 2 hours only after induction


- it is not always advisable to keep and purify the protein of interest 
at 4ºCelsius (keyword "cold denaturation")



Things to try:

- implement the method described in the paper: Optimum solubility (OS) 
screening: an efficient method to optimize buffer conditions for 
homogeneity and crystallization of proteins, ACTA D D60, 1670-1673 
(Jancarik et al.)


-At pH 7.5 and pI 8.6, your protein will be positively charged. Trying 
to "stabilize" the protein according to the Hofmeister series of salts 
is not helpful. Remember, the Hofmeister series of salts will inverse 
with positively charged proteins! Meaning, NaCl is not a good salt to 
use in order to solubilize the protein. Try to add 50-200 mM 
ammoniumsulfate to help solubilize the protein.



Good luck,

Jeroen



Am 19.07.20 um 11:07 schrieb 张士军:


Dear all:

Any ideas to decrease protein polymer formation? my protein was easy 
to form oligomers and precipitation when do purification,I have tried 
add glycerol and DTT,both didn't work. Does anyone has experience to 
avoid it happening. Thanks!


Best Regards




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--
*Dr.math. et dis. nat.Jeroen R. Mesters*
Deputy, Lecturer, Program Coordinator /Infection Biology
/ 
Visiting 
Professorship (South Bohemian University) in Biophysics

Logo Uni Lübeck
*University of Lübeck*
Center for Structural and Cell Biology in Medicine
*Institute of Biochemistry*

Tel +49 451 3101 3105 (secretariate 3101)
Fax +49 451 3101 3104
jeroen.mest...@uni-luebeck.de 
www.biochem.uni-luebeck.de 

*Ratzeburger Allee 160
23538 Lübeck, Schleswig-Holstein
Germany*




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Re: [ccp4bb] protein oligomer

[Barone, Matthias]

You could try SERp? Gave some good suggestions for our non-cristallizing 
paralog. However, some of the mutants I created did not express well/were 
rather insoluble. So that might be a bad idea in your case.. anyway here's the 
link:
https://services.mbi.ucla.edu/SER/

Best, Matthias

From: CCP4 bulletin board  on behalf of 张士军 
<21620150150...@stu.xmu.edu.cn>
Sent: Monday, July 20, 2020 8:38:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein oligomer


Dear:

The protein was purified in 4 degree, and the expression level is low, so the 
aggregation is not by high concentration; the buffer pH is 7.5 which is not 
colse to the PI 8.6. It should be a dimer when function, but it was aggregated 
when negative staining. Maybe I could try to add arginine when purification, or 
do mutantions. anyone has website for prediction the mutation sites of protein?

Thanks!

Best,

shijun


-原始邮件-
发件人:"Nikolay Dobrev" 
发送时间:2020-07-19 20:29:49 (星期日)
收件人: CCP4BB@JISCMAIL.AC.UK
抄送:
主题: Re: [ccp4bb] protein oligomer


It really depends from the nature of the protein and if is 
oligomerizing/agregating/forming polymers if any of this is reversible.
On the other side if you are working with one of the fibril forming protein it 
will require optimizaiton on its on as they will form naturally polymers.

Do you observed different specises when you analyze your protein by SEC or if 
you are able to perfom DLS?
Additional information regarding your protein will be really helpful for more 
detalied suggestions how to overcome your protein.

Best,
Nikolay

Nikolay Dobrev
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of 
all EMBL events.


On 19/07/2020 14:15, S. Mohanty wrote:
Keep the protein concentration low during purification steps along with using 
other anti-aggregation agent/s. Make sure that the pH at which you are 
purifying is not close to the pI of the protein. Until completely purified, all 
purification steps should be performed in a cold room if it is a soluble 
protein.

Smita


On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga 
 wrote:


I am not sure what you mean by polymer formation. Presuming that you have 
optimized your protein concentration, pH and salt concentration, you could try 
arginine as an anti-aggregation agent in your purification (I presume you do 
FPLC). Have a look at chaotropic agents used in protein purification, The 
answer is generally dependent on the protein/proteins you are trying to purify 
and is not necessarily straightforward.

Kinds regards
[X]
On Sun, Jul 19, 2020 at 12:08 PM 张士军 
<21620150150...@stu.xmu.edu.cn> wrote:

Dear all:

Any ideas to decrease protein polymer formation? my protein was easy to form 
oligomers and precipitation when do purification,I have tried add glycerol and 
DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks!

Best Regards



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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--
Nikolay Dobrev
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of 
all EMBL events.



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[ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Quote source inquiry

[Schreuder, Herman /DE]

Dear Jessica,

Thank you for this positive news on electron diffraction on small molecules. A 
point which is still not clear to me: is it possible to determine the absolute 
configuration with electron diffraction? Some claim that it cannot be done, 
others claim that it can be done using multiple diffraction events.

What is your experience?
Best, Herman

Von: CCP4 bulletin board  Im Auftrag von Jessica Bruhn
Gesendet: Samstag, 18. Juli 2020 02:16
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Quote source inquiry


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Hi Garib, Tim and James,

Thank you for the helpful information. I look forward to testing this out on 
some of our data. Hopefully it helps!

To Garib, I think that electron diffraction/microED of small molecules is 
actually in a pretty good position for this technique to really take off. To 
your concerns:
1. Our group is personally very happy with the data coming from our detector 
(CETA-D). The thicker scintillator really seems to have helped. And there are 
other good detectors out there. I have some data posted in zenodo in case you 
are interested (10.5281/zenodo.3905397 and 10.5281/zenodo.3937740).
2. Crystal handling is thankfully very straightforward for dry, small molecule 
crystals. Just dab a TEM grid on some (crystalline) powder and in most cases 
you should be ready to collect. Protein crystals are unfortunately 
significantly more difficult to work with. We'll see how work in that area 
progresses...
3. As for rotation, I am curious to hear what concerns you have about rotation? 
Are you concerned about completeness? Or the lower data quality in the high 
tilt angle frames? Or the accuracy of the goniometer with regard to position 
and constant speed maintenance? If your concerns are about completeness, I 
would say that we have been able to get fairly decent completeness by combining 
data from multiple crystals. In our hands (18 small molecule ED structures 
solved in house), about half of these reached >95% completeness, another 
quarter were >90% and the rest were in the 81-90% range.

You may also be interested to know that of these 18 small molecule structures, 
three had to be refined in REFMAC5 because the resolution was a little low 
(1.2-1.7A) or the data to parameter ratio was too poor for SHELXL. I understand 
your time is limited, but I do think that electron diffraction for small 
molecules is really gaining momentum. We have collected data from almost fifty 
different samples from our clients all across pharma since installing our new 
camera in September. Many of these probably won't end up in public databases, 
but they have been hugely impactful for these chemists.

Have a wonderful weekend.

Best wishes,
Jessica



On Fri, Jul 17, 2020 at 2:11 AM Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:
Dear Tim,


I understand the problem. If the problem is the distance only then only one 
parameter is needed for refinement of lattice parameters.

I do think that microED has good potential. However engineering problems need 
to be sorted out (detector, crystal handling, rotation etc).

When the problem becomes urgent then I can coniblue working on this problem. I 
have already implemented using all data (chemistry and crystal data) for 
lattice refinement. They need to be tested properly.
There are several issues that need to be sorted out.

Regards
Garib



On 17 Jul 2020, at 08:29, Tim Gruene 
mailto:tim.gru...@univie.ac.at>> wrote:

Dear Garib,

thank you very much for the details! If everything goes to plan, we are
going to use the Dectris QUADRO in September(ish), ideally also with
some protein crystals. In ED, distance calibration is more difficult
than with X-rays because of instabilities in the lens system (at least
with the older instruments), and because I do not work with a parallel
beam, but focus the beam onto the detector surface. This is not a very
reproducible process. In those cases where I got high resolution data,
the cell is often quite stable, and the distance can vary by about 5%...

Best regards,
Tim

On Thu, 16 Jul 2020 23:21:54 +0100
Garib Murshudov mailto:ga...@mrc-lmb.cam.ac.uk>> wrote:


One correction: Model should after molecular replacement and few
cycles of refinement (perhaps with a little bir relaxed geometry, but
not too much).

There is an option to use a model after molecular replacement but it
is being migrated to another program that will have proper tests.

Regards
Garib



On 16 Jul 2020, at 22:34, Garib Murshudov 
mailto:ga...@mrc-lmb.cam.ac.uk>>
wrote:

Hi Tim,

There is an option to do unit cell parameter refinement (for all
six parameters in general which can only happen in P1). It is
undocumented.

Celrefine/lattice refine all # if you give scale instead of all
then only one parameter is refined.

Cellrefine select .  # use only atomic B value < Bmedian +
alpha * Binterquartile_range


The last command was 

Re: [ccp4bb] protein oligomer

[张士军]

Dear:

The protein was purified in 4 degree, and the expression level is low, so the 
aggregation is not by high concentration; the buffer pH is 7.5 which is not 
colse to the PI 8.6. It should be a dimer when function, but it was aggregated 
when negative staining. Maybe I could try to add arginine when purification, or 
do mutantions. anyone has website for prediction the mutation sites of protein?

Thanks!

Best,

shijun



-原始邮件-
发件人:"Nikolay Dobrev" 
发送时间:2020-07-19 20:29:49 (星期日)
收件人: CCP4BB@JISCMAIL.AC.UK
抄送:
主题: Re: [ccp4bb] protein oligomer



It really depends from the nature of the protein and if is 
oligomerizing/agregating/forming polymers if any of this is reversible.
On the other side if you are working with one of the fibril forming protein it 
will require optimizaiton on its on as they will form naturally polymers.

Do you observed different specises when you analyze your protein by SEC or if 
you are able to perfom DLS?
Additional information regarding your protein will be really helpful for more 
detalied suggestions how to overcome your protein.

Best,
Nikolay

Nikolay Dobrev 
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of all EMBL events.




On 19/07/2020 14:15, S. Mohanty wrote:

Keep the protein concentration low during purification steps along with using 
other anti-aggregation agent/s. Make sure that the pH at which you are 
purifying is not close to the pI of the protein. Until completely purified, all 
purification steps should be performed in a cold room if it is a soluble 
protein.


Smita 




On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga  
wrote:




I am not sure what you mean by polymer formation. Presuming that you have 
optimized your protein concentration, pH and salt concentration, you could try 
arginine as an anti-aggregation agent in your purification (I presume you do 
FPLC). Have a look at chaotropic agents used in protein purification, The 
answer is generally dependent on the protein/proteins you are trying to purify 
and is not necessarily straightforward.


Kinds regards


On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn> wrote:


Dear all:

Any ideas to decrease protein polymer formation? my protein was easy to form 
oligomers and precipitation when do purification,I have tried add glycerol and 
DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks!

Best Regards




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-- 
Nikolay Dobrev 
Scientific Officer, Protein Expression and Purification Core Facility
EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany
T +49 6221 387 8633 | M +49 173 684 0532
twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of all EMBL events.



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