[ccp4bb] SAXS models and data

[Gloria Borgstahl]

Do we deposit these anywhere?



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[ccp4bb] TEV vs HRV3C

[Gloria Borgstahl]

Hello my fellow structural biologists,  I am contemplating why some choose
the HRV3C protease site over TEV for their fusion proteins.  Does anyone
know?  Can HRV3C be made easily in homelab?  Does anyone have a plasmid?
Thank you, G



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[ccp4bb] MBP Ab for native protein

[Gloria Borgstahl]

Hello friends,  Can anyone recommend an antibody that works well
forbbonding native MBP tag?  Many thanks, G



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[ccp4bb] Contouring Patterson map?

[Gloria Borgstahl]

What is the best way to display Harker sections... these days?



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[ccp4bb] Contouring Patterson map?

[Gloria Borgstahl]

What is the best way to display Harker sections these days?



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Re: [ccp4bb] dimeric tag to induce the homodimerization of protein

[Gloria Borgstahl]

I was thinking the form of GFP that dimerizes.  This would also make it
easy to track where the protein is.

On Tue, Sep 22, 2020 at 1:28 PM Diana Tomchick <
diana.tomch...@utsouthwestern.edu> wrote:

> Any dimeric tag should work if you add a long enough linker to satisfy
> your distance criterion.
>
> GST, for example. Download the coordinates and get a rough idea how long
> the linker would have to be for your protein.
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj <
> dhiraj-srivast...@uiowa.edu> wrote:
>
> EXTERNAL MAIL
>
> Hi
> I want to make my protein dimeric to increase its affinity for its
> interaction partner which is a dimer. does anyone know a suitable
> tag/fusion protein which can be used as C terminal fusion for this purpose?
> I can not use any of the leucine zipper as I am looking for the distance
> between the c terminus to be around 30-40 A.
>
>
> Thank you
> Dhiraj
>
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[ccp4bb] AcTEV protease

[Gloria Borgstahl]

Another protein purification question:  Does anyone know what is AcTEV
protease that is sold by Thermofisher Scientific?  Is this the same as
SuperTEV?
It works well but is so expensive.
Thanks for any advice, Gloria



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Re: [ccp4bb] disinfecting keyboards

[Gloria Borgstahl]

We were looking at these, they look like fun.

https://www.wetkeys.com/Soft-touch-Comfort-Hygienic-Washable-Keyboard-USB-p/kbstfc106-w.htm



On Tue, May 5, 2020, 7:20 PM James Holton  wrote:

> All joking aside, there has been a furor of attention on UV-based
> disinfection of late.  Some of it is not entirely crazy.  I.E. Columbia
> University’s Center for Radiological Research has put forward the idea of
> illuminating occupied public areas with ultra-narrow-band UV-C (222 nm).
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552051/
>
> Mind you, UV-C normally covers 100 - 280 nm, and the PPE requirements for
> that (at LBNL at least) are extensive: polycarbonate safety glasses and
> face shield with a mark U6 (UV protection), long-sleeved clothing, and
> gloves.  Basically: do not expose skin!
>
> The idea behind using monochromatic 222 nm radiation is that it is at the
> edge of a very steep increase in the absorption of water, protein, and
> other biologicals.  Penetration depths are hard to estimate because of the
> steep slope, but they are on the order of 1 micron.  So, smaller than a
> typical mamalian cell, but bigger than a bacterium or virus.  The paper
> above did not have any human subjects, nor did it discuss how to deal with
> all the ozone, but the results are intruiging. Needs further study.
>
> Personally, I think this would probably fog your corneas and perhaps burn
> the thin skin on lips and other exposed mucosa. Hair I'd expect to
> embrittle and fall apart eventually. Yes, hair is 40 microns thick and the
> penetration depth is 1 micron, but photon's don't "stop" at the penetration
> depth.  36% of them go deeper. Plastic in keyboards too would probably
> bleach and flake with prolonged exposure.  Ever seen a keyboard left out in
> the sun for a few weeks?  I'd worry a bit about this micro-damage creating
> crevices where bugs could hide.
>
> I encourage you to bring this up with your Health and Safety people, but
> make sure they are sitting down first.
>
> -James Holton
> MAD Scientist
>
> On 4/29/2020 12:41 PM, Andrea Thorn wrote:
>
> Hi Tim!
>
>
> 100% alcohol is less effective than 80%, and in order to completely be
> sure, the keyboard needs not only to be wiped. One can buy keyboards that
> can be disinfected because they are waterproof, such as the Cherry
> JK-1068DE-2 for about 50 €.
>
>
> We clean the keyboards in our lab occasionally anyway, and have used 70%
> alcohol on them without problem. Disinfectant wipes, a detergent cleaner
> (such as Viss Glass & Flächen) and cotton swabs also offer some help. We
> wipe our mobile phones with a disinfectant wipe after washing our hands
> when arriving home/at work.
>
> I would also be really interested in what could be done with a UV light,
> if someone knows?
>
> If the computer is used by one person during the shift, individual
> keyboards for each person could be a solution. If people sit down, the desk
> surface, which may be touched, should likely also be wiped at the beginning
> and end of the shift I would say.
>
> Stay save and best wishes,
>
>
>
> Andrea.
>
>
>
> Am 29/04/2020 um 21:04 schrieb Diana Tomchick:
>
> ​100% ethanol or isopropanol work really well on the microscopes, I soak a
> Kimwipe and then clean the eyepieces and the knobs for changing
> magnification and focus, as well as the door handles, bench tops, etc.
>
>
> Diana
>
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> --
> *From:* CCP4 bulletin board 
>  on behalf of Diana Tomchick
>  
> *Sent:* Wednesday, April 29, 2020 2:00 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] disinfecting keyboards
>
>
> EXTERNAL MAIL
>
> ​You could try doing what my technician does with her keyboard; she wraps
> it in a clear, thin food wrap that can be taped to the back of the
> keyboard. This is usually done to keep food and other things (liquids) from
> damaging the keyboard, but you could simply replace the wrap every time
> someone else uses it.
>
>
> Personally I like using a Kimwipe soaked with 100% isopropanol, I've never
> yet encountered a keyboard that suffered from having the writing removed
> with that or 100% ethanol. Both work and as long as they are 100% (no
> water), the keyboard and mouse have no issues.
>
>
> Diana
>
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> --
> *From:* CCP4 bulletin board 
>  on behalf of Tim Gruene 
> 
> *Sent:* Wednesday, April 29, 

[ccp4bb] insect secretion recommendations?

[Gloria Borgstahl]

Hi Friends,  We are secreting Spike Ecto domain into the media from insect
cells for purification.  As we scale up I am wondering what is recommended
for collecting the media from large volumes of culture.  Centrifugation?
Filtration of some kind?  I imagine we need to be gentle to not lyse the
insect cells.  Thank you, Gloria



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Re: [ccp4bb] Vote for cryoEM

[Gloria Borgstahl]

I personally don't tweet.


On Tue, Mar 31, 2020 at 12:21 PM Sweet, Robert <
27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk> wrote:

> Real Men (and possibly Women too) Don't Tweet.
>
> Bob
>
> 
> From: CCP4 bulletin board  on behalf of James
> Holton 
> Sent: Tuesday, March 31, 2020 12:18 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Vote for cryoEM
>
> Allessandro,
>
> The link you provide directs to a website hosted at someting called "
> twitter.com".  My spam filter flagged it as junk.
>
> -James Holton
> MAD Scientist
>
> On 3/31/2020 8:41 AM, Alessandro Vannini wrote:
> We are head to head with mass-spectrometry in the #JBCMethodsMadness
> CHAMPIONSHIP. This can’t happen!
>
> Get out and VOTE! 15 min to go!
>
> https://twitter.com/jbiolchem/status/1244655631316987905?s=20<
> https://urldefense.com/v3/__https://twitter.com/jbiolchem/status/1244655631316987905?s=20__;!!P4SdNyxKAPE!QXCVmZuWG8FEmwM9FbPP-f3LEbH6bMcORBgYwRGHCpxZF7cWEHW9OsXmS5uQ_Q$
> >
>
> [cid:part2.4691618D.6AA0935A@lbl.gov]
>
> Sent from my iPhone
>
> 
>
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Re: [ccp4bb] Shipping samples for neutron diffraction

[Gloria Borgstahl]

Actually we don't declare anything to TSA, Jahaun just carries them in his
carryon and it is fine.  Easy peasy?


On Wed, Feb 19, 2020 at 3:48 PM <
0c2488af9525-dmarc-requ...@jiscmail.ac.uk> wrote:

>
> ... and don't say you're travelling with heavy water!
>
> Jon Cooper
>
> On 19 Feb 2020 17:12, "Azadmanesh, Jahaun" 
> wrote:
>
> Hello,
>
> I have traveled to a neutron beamline ~6 times over the past several years.
>
> I have had awful luck shipping crystals, so I decided to hand-carry and I
> found this best.
>
> I'm not sure what conditions you plan to shoot your crystals.  I mount my
> crystals in a sturdy quartz capillary (from vitrocom), seal the ends with a
> layer of wax, then two coats of nail polish (choose your favorite prettiest
> color!)
>
> I would then wrap the capillary in cotton from a cottonball, then "stuff"
> the capillary-cotton combination in a 15 mL falcon tube for a snug fit. For
> extra assurance, the falcon tubes were placed inside a generous amount of
> bubble-wrap that was then taped inside a small cardboard box.
>
> I have sought anaerobic conditions for several of my samples to acquire a
> desired redox state of my metallo-protein. This was tough, so instead I
> used a high-ish concentration of my redox agent dissolved in my reservoir
> solution "slugs" in the capillary. These would be replaced in intervals
> with fresh slugs prior to the beamtime, and for sure right before the
> beamtime. The redox state held for my ~10 days of beamtime and several
> months after.
>
> For cryo-conditions, I just placed a bunch of my crystals in a capillary
> filled with reservoir solution and sealed as above. I would do my chemical
> manipulations and freezing at the beamline.
>
> Much more details are found in our publication:
> https://www.ncbi.nlm.nih.gov/pubmed/30279321
>
> Hope this helps!
> --
> *From:* CCP4 bulletin board  on behalf of
> stephen.c...@rc-harwell.ac.uk 
> *Sent:* Wednesday, February 19, 2020 4:22 AM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Shipping samples for neutron diffraction
>
> Non-UNMC email
>
> Dear CCP4 community,
>
>
> I have an impending trip to a neutron source and was wondering how people
> tend to ship their samples prior to beam time?  Is sending something frozen
> best or is sealed in a capillary more sensible or is there another better
> way?  One caveat is that ideally my sample should remain anaerobic which
> has me leaning towards frozen, but I guess in pcr type tubes sealed in gas
> tight vessels is also a viable option?
>
>
> Any thoughts or suggestions are very much appreciated.
>
>
> 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
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[ccp4bb] protein expression in human cells

[Gloria Borgstahl]

Hello CCP4-ers,
I was wondering what people have found to be the best human cell line
expression system for making a large quantity of purified recombinant
protein.
Any information and protocols would be greatly appreciated.
Happy 2020, Gloria



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[ccp4bb] invitation for in-person or remote participation in ORNL STS workshop

[Gloria Borgstahl]

Dear Friends in Crystallography,

Below is a blurb about the workshop we are organizing for December 9-10.
It will be a brainstorming session to define the needs of the structural
biology community that will influence the design of the next generation of
neutron beamlines.  Please let me know if you have any questions.  G



This is an exciting time for neutron scattering in the U.S. as the future
Second Target Station (STS) at the Spallation Neutron Source (SNS) at Oak
Ridge National Laboratory (ORNL) will provide transformative new
capabilities that both complement and greatly extend our current resources
for neutron scattering. Scientists of all disciplines, from beginners to
experts, interested in the STS project and in the new capabilities it will
enable are invited to ORNL on December 9-10. *You can attend in person or
by remote access. The details for registering can be found here:
https://conference.sns.gov/event/193/
<https://conference.sns.gov/event/193/>*.

This workshop is a critical step in the STS project. It will lead to the
identification of the essential performance parameters for a prospective
suite of instruments and to the formation of teams that will continue to
refine the science case and participate in defining the requirements for
individual instruments to be built at the STS. The workshop is organized
around four breakout sessions for each of seven main science topical areas.
The focus of these breakouts is to facilitate discussion of the
opportunities enabled by the STS across current and prospective future
scientific fields and to identify the capabilities and performance
parameters that need to be enabled by STS neutron instruments. The biology
breakout sessions are:



(1) Time-Resolved Proton Transfer in Biology, Biochemistry, and Medicine

(2) Cell Signaling at Membranes

(3) Macromolecular Complex Assembly and Disassembly

(4) Bioinspired Materials and Technology



We are looking forward to your participation!



On behalf of Biology Session Chairs:

 Dr. Gloria Borgstahl

University of Nebraska Medical Center

Dr. Yun Liu

National Institute of Standards and Technology / University of Delaware



PLEASE REGISTER AT THE CONFERENCE WEBSITE :
https://conference.sns.gov/event/193/



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[ccp4bb] E. coli BirA biotin ligase expression/purification

[Gloria Borgstahl]

Dear friends in crystallography,
I know this seems unrelated, but it really isn't ... please forgive me.
We are trying to use the Avitag/BirA system to specifically biotinylate
target proteins in an economical manner.  Are any of you purifying the BirA
enzyme in your lab for biotinylation and if so can you help me figure out
the right plasmid/purification protocol?   Please let me know.  Thank you.
Gloria



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Re: [ccp4bb] challenges in structural biology

[Gloria Borgstahl]

Sorry to be late chiming in on this post (survived RAGBRAI).  I think the
challenges (crystallization, perdeuteration) and benefits of neutron
crystallography (where are those protons) could be included.  We are now in
an era of using cryotrapping with neutrons which I think is really cutting
edge for time-resolved structural information.  My two cents, G

On Mon, Jul 22, 2019 at 1:55 AM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Dear Artem, Tom, Janet,
>
> for me and probably others the usage of words like 'magic bullet' (which
> you defend, or try to redefine) implies a belief-based esoteric approach
> that has little to do with science. I suggest that to obtain funding,
> 'magic bullets' should not be promised, because these cannot be delivered
> (I gave the lo-gravity hi-funding example).
>
> That this discussion (including messages by Janet and Tom) happens at all
> suggests that crystallization is currently not a science - it lacks a
> consistent nomenclature and way of documentation, and suffers from strong
> publication bias (many unpublished negative results).
>
> On the other hand, what you (Janet, Tom) write about the research that
> should/could be performed - this sounds a lot like a scientific approach,
> and is not different from what has been realized in other areas of
> crystallography. Yes, existing tools for predicting crystallization success
> are not consulted because the rate of false positives and false negatives
> is high. If those rates could be reproducibly reduced, I bet the usage
> would go up - that could start a feedback loop leading to even better
> predictions. Is work in this direction sexy? No. Is it useful? Yes. Is it
> hard work? Yes. Does it contribute to make crystallization a science? Yes.
>
> What about 'deep learning' applied to crystallization outcomes? Can it
> guide individual trials better than intuition? Can it find previously
> unknown promising combinations on a larger scale?
>
> Can this be funded? Yes of course. Your statement that crystallization
> gets no funding may be true in some countries (but aren't CCP4BB readers
> from the U.S. also reviewers?), but it's untrue in others - think of groups
> in France that obviously got long-term funding. And for space (low-gravity)
> - that amount of funding could have been used for a lot of meaningful
> earth-bound research.
>
> Kay
>
>
> Am 21.07.19 um 23:04 schrieb Artem Evdokimov:
> > Dear Kay
> >
> >
> > I disagree that 'magic bullet' is impossible. I think the definition is
> wrong here - magic bullet to me is a rational set of methods that (when
> executed with precision and care) enable crystallization to the maximum
> possible benefit. This includes everything - constructs, crystallization
> design, etc. Part of the magic bullet is also a precise knowledge when
> crystallization is unlikely (i.e. an actual proven predictor that
> consistently discriminates between "you're going to succeed if you work
> hard" and "it's doomed to fail, don't bother" scenarios in crystallization.
> >
> > The above is not sexy. It does not present itself as a lovely subject on
> which to have international cocktail parties with politicians delivering
> fancy speeches. But that is what is needed, and no one is funding that to
> the best of my knowledge.
> >
> > What needs to be done is a significant amount of testing,
> standardization, and methods development from the perspective of holistic
> outcome (i.e. crystals that work) - and none of the previously advertised
> 'magic bullets' work the way I just described.
> >
> > Having written this, I think you're right - this is a bit of a
> distraction from James' original point. However it's a valid opportunity
> for a lively discussion on its own :)
> >
> > Artem
> >
> > - Cosmic Cats approve of this message
> >
> >
> > On Sun, Jul 21, 2019 at 4:52 PM Kay Diederichs <
> kay.diederi...@uni-konstanz.de >
> wrote:
> >
> > Dear Artem,
> >
> > black or white is not my way of thinking, which is why I don't
> believe in Hannibal's approach when it comes to crystallization.
> >
> > None of the magic bullets that were advertised over the past decades
> have proven generally applicable.  I believe more in incremental
> improvement which in this case includes a few biophysical characterization
> methods, possibly improved microfluidics or other apparatus, and expanded
> screens. And a lot of hard work, perseverance, intuition, frustration
> >  tolerance. Nothing that really needs huge funding - of course it
> does need money, but just a  share of what is anyway needed for the usual
> lab work including expression, purification, functional characterization,
> binding studies and the like.
> >
> > One area where a huge amount of money was burnt is crystallization
> in space, on board of e.g. the spacelab and ISS. This is for me an example
> of a mis-led approach to throw money at a difficult problem, with the
> 

Re: [ccp4bb] how many crystallographers are there?

[Gloria Borgstahl]

You might want to quote how many researchers use the PDB instead.  Maybe an
inquiry to the PDB could give you that statistic.
That would give you a big number

On Wed, May 29, 2019 at 1:54 PM Scott Horowitz  wrote:

> Hi all, I was recently asked how many biological
> crystallographers plus cryo-EM users there are in the world (in relation to
> how many people could therefore be theoretically impacted by Foldit
> electron density tools, for the purposes of grant funding). I'm a bit at a
> loss as to even what order of magnitude to provide. Any thoughts about how
> to estimate a number?
>
>
> Thanks,
>
> Scott
>
>
> Scott Horowitz, Ph.D.
>
> Assistant Professor
>
> Department of Chemistry & Biochemistry
>
> Knoebel Institute for Healthy Aging
>
> University of Denver
>
>
>
> ECS Building
>
> 2155 E. Wesley Ave
>
> Denver, CO 80208
>
> Phone: 303-871-4326
>
> Fax: 303-871-7915
>
> Email: scott.horow...@du.edu
>
> Office: Room 561   Lab: Room 505
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Gloria Borgstahl]

Hi Matthew,  I am also a bit late in responding, I have a few
incommensurately modulated protein crystal datasets that you would be
welcome to use in your course.  It would be neat for students to at
least know that this type of diffraction exists.  As far as I know,
they can only be processed with Eval software.  Please let me know if
you are interested and we can transfer images to you.  Thanks, Gloria


On Thu, Sep 27, 2018 at 4:42 AM Andrew Leslie  wrote:
>
> Dear Matthew,
>
>I am also late in responding to this, but as part of a 
> Nature Protocols paper on iMosflm (Supplementary Information for Nature 
> Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem 
> datasets”. Some of these are just two images, to show issues in indexing, 
> others are complete datasets showing a variety of pathologies.
>
> All the images and a tutorial on how best to process them (with iMosflm) are 
> available at the following URL:
>
> www.mrc-lmb.cam.ac.uk/harry/imosflm/examples
>
>
> Best wishes,
>
> Andrew
>
>
> On 26 Sep 2018, at 03:15, Whitley, Matthew J <> wrote:
>
> For some reason, the September 19th ccp4bb digest got caught in my spam 
> filter and didn't come through until a few minutes ago, so I didn't see 
> several responses concerning interesting datasets for processing until just 
> now.
>
> Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David Waterman 
> for responding (and also to everyone else who responded if I am still 
> overlooking anyone.)
>
> As I mentioned before, I will be happy to compile a list of suggested 
> datasets and make it available via this list.
>
> Matthew
>
> ---
> Matthew J. Whitley, Ph.D.
> Research Instructor
> Department of Pharmacology & Chemical Biology
> University of Pittsburgh School of Medicine
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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[ccp4bb] nonenzymatic removal of His tag?

[Gloria Borgstahl]

Hello, friends in crystallography,
A colleague just asked me this question.  He is worried about trace
protease interfering with the receptors he is studying in cell-based
experiments using a 110 amino acid protein we made for him.  He has
been unable to make the peptide synthetically.  The company is having
trouble getting that to happen.  Any ideas?  Happy Thursday, G



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Re: [ccp4bb] protein quasicrystals?

[Gloria Borgstahl]

I am very interested in this topic, and have found incommensurately
modulated crystals and few examples of possible protein quasicrystals.
Do you have any images you could share?

On Tue, Feb 13, 2018 at 9:09 AM, Yu Qiu  wrote:
> Hi,
>
>
>
> I have been trying to crystallize a protein complex and keep getting sphere
> shape crystals. The diffraction is around 3 angstrom, but looks like
> multiple lattices. I am wondering if it could be a quasi crystal? Is there
> anyone has such experience?
>
>
>
> Thanks,
>
> Yu

Re: [ccp4bb] "Atomic resolution"

[Gloria Borgstahl]

My answer is not precise, I just try to give students something they
can remember, so they don't go and model water for example at an
inappropriate resolution.  Of course phasing matters as well.

On Thu, Jan 11, 2018 at 2:31 PM, Keller, Jacob  wrote:
>>I tell people it is when your resolution is less than the bond length that 
>>connects the two atoms.
>
> I thought this was sort of a pitfall, since the Bragg spacings don't 
> necessarily map on to conventional resolution. But it would fit the 1.5 Ang 
> estimate.
>
> Also, resolution would depend a lot on phase accuracy/precision, no?
>
> JPK
>
>
> On Thu, Jan 11, 2018 at 1:59 PM, Thomas Edwards  
> wrote:
>> Dear Jacob,
>>
>> Ah... this old chestnut!
>>
>> Current EM people say that they are at atomic resolution because they
>> are building atomic models (naive??).
>>
>> I have been criticised in the past for using the term with say 2.2A
>> diffraction data. By co-authors and reviewers alike. When I was young
>> and naive.
>>
>> My (current) definition would be yours - visible with data.
>> I think 1.5A is about right for X-ray. Maybe higher res?
>>
>> I’m sure there are lots of rigorous ways to think. I probably haven’t
>> taken that route. However, I think it is a semantic problem that might
>> benefit from some disambiguation rather than rigour.
>>
>> It depends why you are asking the question...
>>
>> Sorry..!
>>
>> Ed is: Out and about...
>> Sent from iPhone6sPlus.
>>
>> On 11 Jan 2018, at 19:31, Keller, Jacob  wrote:
>>
>> Dear Crystallographers,
>>
>>
>>
>> Has there been a consensus as to what is meant by “atomic resolution?”
>> Seems like the term is taken by various practitioners to mean different 
>> things.
>>
>>
>>
>> A related question: at what resolution are atoms “visible” using only
>> the data? I have an empirical feeling that this would be around 1.5
>> Ang Bragg spacings, but on the other hand, one can contour up most
>> maps and see individual atom peaks. I would be interested to hear a
>> more rigorous way to think about this.
>>
>>
>>
>> All the best,
>>
>>
>>
>> Jacob Keller
>>
>>
>>
>> +
>>
>> Jacob Pearson Keller
>>
>> Research Scientist / Looger Lab
>>
>> HHMI Janelia Research Campus
>>
>> 19700 Helix Dr, Ashburn, VA 20147
>>
>> (571)209-4000 x3159
>>
>> +
>>
>>
>>
>> The content of this email is confidential and intended for the
>> recipient specified in message only. It is strictly forbidden to share
>> any part of this message with any third party, without a written consent of 
>> the sender.
>> If you received this message by mistake, please reply to this message
>> and follow with its deletion, so that we can ensure such a mistake
>> does not occur in the future.
>>
>>

[ccp4bb] phosphates

[Gloria Borgstahl]

Doe any one know what resolution is needed to distinguish phosphate
from carbon in a difference map?

Re: [ccp4bb] "Atomic resolution"

[Gloria Borgstahl]

I tell people it is when your resolution is less than the bond length
that connects the two atoms.

On Thu, Jan 11, 2018 at 1:59 PM, Thomas Edwards  wrote:
> Dear Jacob,
>
> Ah... this old chestnut!
>
> Current EM people say that they are at atomic resolution because they are
> building atomic models (naive??).
>
> I have been criticised in the past for using the term with say 2.2A
> diffraction data. By co-authors and reviewers alike. When I was young and
> naive.
>
> My (current) definition would be yours - visible with data.
> I think 1.5A is about right for X-ray. Maybe higher res?
>
> I’m sure there are lots of rigorous ways to think. I probably haven’t taken
> that route. However, I think it is a semantic problem that might benefit
> from some disambiguation rather than rigour.
>
> It depends why you are asking the question...
>
> Sorry..!
>
> Ed is: Out and about...
> Sent from iPhone6sPlus.
>
> On 11 Jan 2018, at 19:31, Keller, Jacob  wrote:
>
> Dear Crystallographers,
>
>
>
> Has there been a consensus as to what is meant by “atomic resolution?” Seems
> like the term is taken by various practitioners to mean different things.
>
>
>
> A related question: at what resolution are atoms “visible” using only the
> data? I have an empirical feeling that this would be around 1.5 Ang Bragg
> spacings, but on the other hand, one can contour up most maps and see
> individual atom peaks. I would be interested to hear a more rigorous way to
> think about this.
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> (571)209-4000 x3159
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>

Re: [ccp4bb] AW: Differences in a homodimer protein

[Gloria Borgstahl]

I have seen this in MnSOD.  We had a tetramer in the ASU and each
active site had different ligands in our peroxide soak. I assumed the
crystal lattice can influence these things or it is inappropriate
metal incorporation.  I would highly recommend doing ICP-MS on a
dissolved crystal and on your purified metalloprotein to make sure
your metal incorporation is 100% the metal you want.

On Wed, Nov 29, 2017 at 1:58 AM,   wrote:
> Dear Denis,
>
>
>
> I would first superimpose both monomers to see if you can find a reason why
> one subunit has a bound water and the other not, which would in general be
> flanking side chains in (slightly) different positions. Next I would look
> for some global differences between the subunits that could be linked to
> crystal contacts explaining the non-random distribution of the subunits.
> However, these differences might be quite subtle and difficult to detect.
>
>
>
> As Emily suggested, you could also do some functional assay’s to see if
> there is any positive (or negative) cooperativity between the subunits
> providing independent support for functional differences between the
> subunits.
>
>
>
> My 2 cts,
>
> Herman
>
>
>
>
>
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Denis
> Rousseau
> Gesendet: Dienstag, 28. November 2017 20:04
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein
>
>
>
> Hi BB members
>
>
>
> I have a homodimeric protein, which contains metal centers. In several
> different derivatives I find a water molecule on a metal center in one
> subunit which is absent on the other.  It is always the same subunit that
> contains the water molecule. The resulution is ~2.4 A. Is this an artifact
> or a functional difference? If it is truly homodimeric I would expect
> differences to be random. The space group is P212121.
>
>
>
> Thanks for the advice.
>
>
>
> Denis Rousseau
>
> 
>
>

Re: [ccp4bb] AW: Re: [ccp4bb] another unknown density problem

[Gloria Borgstahl]

If you mean you truncated the low resolution data to 5 angstrom, I
wouldn't recommend that.

On Fri, Nov 3, 2017 at 12:24 PM, Eleanor Dodson
 wrote:
> That map does not look like a 5A map?
> I guess you mean something else..
> Eleanor
>
> On 3 November 2017 at 11:00,  wrote:
>>
>> You are right. In this case, I would put some waters in it, refine and see
>> if the density gets any clearer. However, since from this perspective the
>> density is quite far away from the protein, it could be a very disordered
>> PEG, which, even at high resolution, might be impossible to fit.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> Von: Abhishek Anan [mailto:rendezvous.a...@gmail.com]
>> Gesendet: Freitag, 3. November 2017 11:10
>> An: Schreuder, Herman /DE
>> Cc: CCP4BB@jiscmail.ac.uk
>> Betreff: [EXTERNAL] Re: [ccp4bb] another unknown density problem
>>
>>
>>
>> Dear Prof Schreuder
>>
>> Here are another couple of perspectives from coot. The density is too far
>> and isolated from the peptide chain to be an alternate conformation or
>> conformational change. The density of the peptide chain does not look good
>> because it was truncated at 5A for clarity.
>>
>> Best regards
>>
>> Abhishek
>>
>>
>>
>> On Fri, Nov 3, 2017 at 9:33 AM,  wrote:
>>
>> Dear Abhishek,
>>
>>
>>
>> To me, it looks like an alternative conformation of the peptide chain or
>> maybe even a conformational change with respect to the starting model. The
>> peptide chain does not look too well defined, despite high resolution
>> electron density.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
>> Abhishek Anan
>> Gesendet: Freitag, 3. November 2017 09:26
>> An: CCP4BB@JISCMAIL.AC.UK
>> Betreff: [EXTERNAL] [ccp4bb] another unknown density problem
>>
>>
>>
>> Hi all,
>>
>> I have an "unknown" density in the map. I have tried to fit it to PEG but
>> it doesn't fit very well. I was wondering if there are other PEG-related or
>> other molecules I could try.
>>
>> The crystal grew in TRIS-HCl and PEG MME 2K.
>>
>> Thank you
>>
>> Abhishek
>>
>>
>
>

Re: [ccp4bb] PDB search help

[Gloria Borgstahl]

Our RPA14/32 crystals from 2007 included full length protein for both
subunits (RPA14 and RPA32), but only the central OB fold of RPA32
could be modelled.  The crystals included RPA32(1-270) but only 42-176
could be modelled.  I remember being very frustrated by not being able
to visualize the wHLH domain at the CT or RPA32.  There is disordered
density for the rest due to the flexible linkers.  All of RPA14 could
be modelled.  This happened for all 3 crystal forms we solved...  see
2PI2, 2PQA, and 2Z6K

On Mon, Oct 2, 2017 at 9:22 AM, zaigham mahmood khan
 wrote:
> Hey Rajesh
>
> You may try the following link:
>
> Please enter the protein sequence, and scroll down the "Select standard
> database" tab, and choose "pdb_nr". Once you will "submit" the job, you will
> most likely get what you want to see!
>
> You may pick any pdb ID from the result section, and from pdb.org, you may
> find the residues of that protein that were observed in the electron density
> map as well as whole expression casstte that was attempted to crystallize
> for that particular pdb entry.
>
> Best wishes
>
> -Z
>
>
> Zaigham Mahmood Khan, PhD
>
> Icahn School of Medicine at Mount Sinai
> Department of Oncological Sciences
> 1470 Madison Avenue
> New York
>
> On Wed, Sep 27, 2017 at 6:47 PM, Rajesh
> <1642be9504b8-dmarc-requ...@jiscmail.ac.uk> wrote:
>>
>> Dear BB,
>>
>> Sorry for the off topic.
>>
>> Does anyone know how to search the PDB for the entries that have the
>> density only for part of the protein molecule rather than for the entire
>> length of the protein attempted to crystallize?
>>
>>
>>
>> Thanks,
>> Rajesh..
>
>

Re: [ccp4bb] Aperiodic protein crystals

[Gloria Borgstahl]

Thanks James, Yes Charles I have an active NSF grant on this topic.
We should talk, Gloria

On Mon, Sep 18, 2017 at 11:05 AM, James Holton
 wrote:
>
> I believe Gloria Borgstahl's lab at UNMC has done a little bit of work on
> this.
>
>
> -James Holton
>
> MAD Scientist
>
>
> On 9/16/2017 9:18 PM, Stewart, Charles E [BIOTC] wrote:
>
> Dear all,
>
>
> I am looking for suggestions on research groups that study or have
> experience with aperiodic crystals in macromolecular crystallography. I am
> part of the local organizing committee for the Aperiodic 2018 international
> conference. Although aperiodic crystals are normally not desired and largely
> avoided in protein crystallography I am interested in exploring recent work
> in this area.
>
>
> Thank you for any suggestions.
>
>
> Best regards,
>
>
> Charles Stewart Jr, Ph.D.
> Iowa State University
> Associate Scientist, Office of Biotechnology
> Manager, Macromolecular X-ray Crystallography Facility
> http://www.biotech.iastate.edu/X-ray/
>
> 0202 Molecular Biology Building
> 2437 Pammel Drive
> Ames, IA 50011
> Phone: 515-294-2846
> Email: cstew...@iastate.edu
>
>

[ccp4bb] Fwd: Granada Crystallization Boxes?

[Gloria Borgstahl]

Dear friends, I am resending this post just in case any of you have
Granada Crystallation Boxes (GCBs) in your lab supplies that you do
not need.  The NASA protein crystal growth community could really use
them.  Please see my message below.  Thank you, Gloria


-- Forwarded message --
From: Gloria Borgstahl <gborgst...@gmail.com>
Date: Tue, Jul 11, 2017 at 1:04 PM
Subject: Granada Crystallization Boxes?
To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@jiscmail.ac.uk>


I have recently found out that these are no longer being manufactured
or sold commercially.  But, as fortune has it, we have just been
funded to fly some large quartz capillaries crystallization
experimente up to the International Space Station for neutron
crystallography.  Our experimental design is to fly the experiments in
the Granada Crystallation Boxes!  NASA has already approved the
3x10x0.5 cm plastic Granada boxes for flights.   Does anyone have any
in their lab supplies that they do not plan to use?  We would be
willing to buy them from you!  Thanks, Gloria

Re: [ccp4bb] Granada Crystallization Boxes?

[Gloria Borgstahl]

We probably would be interested in used ones.

On Thu, Jul 13, 2017 at 4:18 AM, Patrick Shaw Stewart <patr...@douglas.co.uk
> wrote:

>
> Gloria, would you be interested in used ones?  I don't actually have any -
> we threw them out a few months ago, I've just checked - but someone might
> have some.
>
> Best wishes, Patrick
>
>
> On 11 July 2017 at 19:04, Gloria Borgstahl <gborgst...@gmail.com> wrote:
>
>> I have recently found out that these are no longer being manufactured or
>> sold commercially.  But, as fortune has it, we have just been funded to fly
>> some large quartz capillaries crystallization experimente up to the
>> International Space Station for neutron crystallography.  Our experimental
>> design is to fly the experiments in the Granada Crystallation Boxes!  NASA
>> has already approved the 3x10x0.5 cm plastic Granada boxes for flights.
>> Does anyone have any in their lab supplies that they do not plan to use?
>> We would be willing to buy them from you!  Thanks, Gloria
>>
>
>
>
> --
>  patr...@douglas.co.ukDouglas Instruments Ltd.
>  Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
>  Directors: Peter Baldock, Patrick Shaw Stewart
>
>  http://www.douglas.co.uk
>  Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
> <(877)%20225-2034>
>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>

Re: [ccp4bb] Granada Crystallization Boxes?

[Gloria Borgstahl]

Triana is no longer making or selling them

On Tue, Jul 11, 2017 at 1:39 PM, Diana Tomchick <
diana.tomch...@utsouthwestern.edu> wrote:

> You can continue to buy new ones from Tirana Science & Technology, which
> is a Spanish company.
>
> http://www.trianatech.com/index.php?option=com_content;
> view=article=65=88=en
>
> Diana
>
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> University of Texas Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
>
> On Jul 11, 2017, at 1:04 PM, Gloria Borgstahl <gborgst...@gmail.com>
> wrote:
>
> I have recently found out that these are no longer being manufactured or
> sold commercially.  But, as fortune has it, we have just been funded to fly
> some large quartz capillaries crystallization experimente up to the
> International Space Station for neutron crystallography.  Our experimental
> design is to fly the experiments in the Granada Crystallation Boxes!  NASA
> has already approved the 3x10x0.5 cm plastic Granada boxes for flights.
> Does anyone have any in their lab supplies that they do not plan to use?
> We would be willing to buy them from you!  Thanks, Gloria
>
>
> --
>
> UT Southwestern
>
> Medical Center
>
> The future of medicine, today.
>

[ccp4bb] Granada Crystallization Boxes?

[Gloria Borgstahl]

I have recently found out that these are no longer being manufactured or
sold commercially.  But, as fortune has it, we have just been funded to fly
some large quartz capillaries crystallization experimente up to the
International Space Station for neutron crystallography.  Our experimental
design is to fly the experiments in the Granada Crystallation Boxes!  NASA
has already approved the 3x10x0.5 cm plastic Granada boxes for flights.
Does anyone have any in their lab supplies that they do not plan to use?
We would be willing to buy them from you!  Thanks, Gloria

[ccp4bb] Fwd: Please register - 7th Annual SBMB Workshop - July 13 - Omaha, NE

[Gloria Borgstahl]

You are invited to attend this year's Structural Biology and Molecular
Biophysics workshop and below is a link where you can register to attend.
The meeting is free with breakfast, lunch and afternoon snacks provided.
We need you to register to know how many are attending and to make name
tags, etc.  If you can, please register to present a research poster.  The
associated journal club is included in the website and will be broadcasted
remotely if you wish to participate from your office.
Thank you for your interest, Gloria Borgstahl  and Luis Marky

Please share this email.

To register, please cut and paste the following into your browser
sbmb.unmc.edu/workshop2017

Re: [ccp4bb] Shipping crystals for RT data collection

[Gloria Borgstahl]

I would recommend just mounting them in capillaries without loops, as this
will travel better.
Put slugs of mother liquor on either side, seal with wax and then coat wax
and onto the end of capillary glass lightly with fingernail polish.

We have had terrible luck with FedEx delivering damaged crystals, if you
try this make sure you have cushioned your capillaries and have plenty of
room temperature thermal packs in your styrofoam container.

The best way to carry them in your carryon.  Weput them individually in 15
mL falcon tubes packed with cotton
and put them in our briefcase or backpack and let TSA x-ray the bag.
We have done this, TSA asks no questions.
Yet Omaha is a small airport.
Good luck!  Gloria

On Thu, Dec 22, 2016 at 1:42 PM, Ricardo Padua  wrote:

> Hi all,
>
> I'm looking for a way to ship crystals for room temperature data
> collection.
>
> I want to ship them already mounted on loops with capillaries, not in
> drops like the In situ trays from Mitegen.
>
> Any experiences on that?
>
> Thanks
>
>
> --
> Ricardo Padua
> HHMI Postdoctoral fellow
> Kern Lab
> Brandeis University
> Waltham, MA
>
>

Re: [ccp4bb] Crystal with ZERO diffraction

[Gloria Borgstahl]

I have learned to refrain from catapulting my crystals.

Happy holidays, God bless us ... everyone (with diffracting crystals)

On Wed, Dec 21, 2016 at 2:35 PM, Keller, Jacob 
wrote:

> >In a second case we were working on a beamline on the west coast of the
> US. Every crystal we put on vanished before we could even collect data –
> the loops appeared empty as soon as we looked through the microscope (this
> sets the decade). The beamline had an extra strong magnet on the goniometer
> and on closer inspection we saw that the hutch wall next to the goniometer
> was peppered with many crystals - they were being catapulted out of the
> loop when the base ‘flipped’ onto the magnet. There were so many that
> others will know this beamline.
>
>
>
> Yes, I also catapulted some crystals—but I actually saw one of mine fly
> off the loop….
>
>
>
> JPK
>
>
>

Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle curiosity ...

[Gloria Borgstahl]

Eddie Snell for President

On Wed, Nov 9, 2016 at 11:02 AM, Edward Snell 
wrote:

> As a Brexit and Trumpet affected person having a foot in both countries
> ,this topic is too far off the normal discussion on CCP4 and probably
> better taken up privately.  CCP4 is not a political discussion site. With
> CCP4 the signal is unusually high and the noise low when compared to any
> discussion board. I for one would like to keep it there. Political views
> aside, we’re all trying to achieve the same scientific goals. Let’s
> remember that and keep that the focus.
>
>
>
> Edward Snell Ph.D.
>
> President and CEO Hauptman-Woodward Medical Research Institute
>
> Assistant Prof. Department of Structural Biology, University at Buffalo
>
> 700 Ellicott Street, Buffalo, NY 14203-1102
>
> Phone: (716) 898 8631 Fax: (716) 898 8660
>
> Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
>
> Heisenberg was probably here!
>
>
>

Re: [ccp4bb] [RANT] Reject Papers describing non-open source software

[Gloria Borgstahl]

Did they stop supporting it due to lack of renewed funding and having to
cut staff that had the knowledge?
I'm pretty sure you only know part of the story.


On Tue, May 12, 2015 at 11:48 AM, James Stroud xtald...@gmail.com wrote:

 I hereby call on the broadest community of academics and researchers,
 including scientists, historians, economists, sociologists, psychologists,
 and whoever else has ever published a paper or read from the literature
 thereof, to reject any and all papers that describe new software that
 itself is not released under an open source model.

 I further declare that this post is designed to ruffle feathers and incite
 incendiary conversation, to provoke all-caps and evoke multiple exclamation
 marks with interposed “1”s where anger prevents one from properly holding
 the shift key.

 My rationale for this post: I have just spent a week installing software
 for structural biology (not crystallography) only to find that some of the
 key utilities needed were described in a recent publication but were not
 OSS. The authors have decided to stop supporting the software but have not
 retracted their paper, which is completely irrelevant without the
 availability of the software package they describe.

 Let’s hammer this one out and come to the rational conclusion that non-OSS
 software should not be awarded publications.

 James

Re: [ccp4bb] cryo condition

[Gloria Borgstahl]

High concentration of ammonium formate is a cryosolvent

On Mon, May 4, 2015 at 5:20 PM, Tristan Croll tristan.cr...@qut.edu.au
wrote:

 What about nature's favourite cryoprotectant, trehalose?

 
 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Roger
 Rowlett rrowl...@colgate.edu
 Sent: Tuesday, 5 May 2015 7:22 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] cryo condition

 We rarely use glycerol anymore, because it seems to fail so often for
 many of our current proteins. Try glucose, 25-30%. This is most
 conveniently done by weighing 125-150 mg of glucose in a microcentrifuge
 tube, then addding well solution to the 0.5 mL mark and mixing until
 completely dissolved. Then you can try dunking crystals in the cryo
 solution, or, you can try the no-fail method (which does fail on
 occasion) of cryoprotecting directly in the crystallization drop by slow
 addition of the cryoprotectant. See

 http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals
 .
 We have often found the slow addition of a glucose cryoprotectant works
 for fragile, high solvent content crystals that are prone to cracking
 under osmotic stress.

 Other alternatives include high concentrations of sodium malonate
 (1-2M), or high concentrations of sodium formate (I think around 4 M?).
 These could also be introduced gradually if required.

 Good luck!

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

 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu

 On 5/4/2015 2:43 PM, Faisal Tarique wrote:
  Hello everyone
 
  Can anybody suggest me a cryo condition for a crystal obtained in
  MIDAS screen of Molecular Dimension:
 
  G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0
 
  G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0
 
  Crystals are in beautiful cuboid shaped but all sorts of PEG
  combinations and Glycerol formulation failed to prevent it from
  cracking and dissolving.
 
  Has anybody faced a similar situation as mentioned above and what
  precaution was taken to prevent it from cracking or dissolving.
 
  Your suggestions will be of immense help
 
  Thanks in advance

Re: [ccp4bb] Strange Ancient Diffraction Pattern...

[Gloria Borgstahl]

Ah Ha!

drum roll

A quasicrystal   !

On Wed, Apr 1, 2015 at 7:00 AM, Julia Griese gri...@dbb.su.se wrote:

  This one appears to be of a similar age. It has a most puzzling, but
 pretty pentagonal pattern (and a backstop). Unfortunately Mosflm doesn't
 appear to support the image format.

 /Julia



 On 01/04/15 13:08, Harry Powell wrote:

 Hi Jacob

  I noticed that there's no backstop shadow that might give a clue as to
 the direct beam position.

  Do you know what wavelength radiation was used to bake this?

  On 1 Apr 2015, at 12:03, Keller, Jacob wrote:

  Can anyone index this? It's got mostly split spots and a strange diffuse
 scattering background

 JPK

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


 roundmatzah.jpg


Harry
 --
Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis
 Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH
 Chairman of International Union of Crystallography Commission on
 Crystallographic Computing
 Chairman of European Crystallographic Association SIG9 (Crystallographic
 Computing)











 --
 Dr. Julia Griese
 Postdoctoral Researcher
 Stockholm Center for Biomembrane Research
 Department of Biochemistry and Biophysics
 Stockholm University
 106 91 Stockholm
 Sweden

 phone: +46-(0)8-162 778
 email: gri...@dbb.su.se

Re: [ccp4bb] Demonstration for 2nd graders?

[Gloria Borgstahl]

I have had young ones grow lysozyme crystals in just a few minutes, using
eye droppers and petri dishes.  The crystals grow very fast, you can watch
them grow in the microscope.  Also they grow large enough you can see them
by eye.  Some izit dye would be fun to add (never did that).   Then I let
them take the setups home with them (nothing toxic in it).  They all wanted
to take them home.

Stock solutions:

100 mg/ml sigma lysozyme in 50 mM sodium Acetate pH 4.5

4 M stock NaCl

50% w/v MPEG 5,000

1 M stock sodium acetate pH 4.5



 Reservoir MasterMix for 60 reactions

9 mL water

22.5 mL NaCl

4.5 mL sodium acetate

54 mL MPEG


 with transfer pipette into small 35X10 mm Petri dish

1 drop protein + 1 drop reservoir


Mix equal amount of lysozyme with reagent. Dilute protein and/or MPEG
and/or Sodium Chloride for less nucleation, larger, and better shaped
crystals.

On Thu, Jan 8, 2015 at 1:02 PM, David Schuller schul...@cornell.edu wrote:

  This one is probably above second grade, but the equipment setup is
 pretty easy

 http://ipl.physics.harvard.edu/wp-uploads/2013/03/15c_s07_5.pdf
 Measuring the wavelength of light with a ruler




 On 01/08/15 13:35, John Lee wrote:

 Hi everyone,

  Slightly off topic here but I got myself volunteered by my 2nd grade son
 to do a show and tell at his class. I have the rock candy experiment ready
 with some background info on what I do.

  Can anyone direct me to some resources or your personal demo's that you
 have done?

  Thanks a bunch

  -John




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

[ccp4bb] Nova F- (topic off in left field)

[Gloria Borgstahl]

Apparently these are not for sale anymore by EMD Millipore
yet it is what is recommended for plasmid preps with pETcoco-2.
Does anyone know of a source of these cells
or a substitute cell line we could use?
Thanks, Gloria

Re: [ccp4bb] Strategies to bring out over-expressed protein from inclusion bodies to soluble fraction!!!

[Gloria Borgstahl]

Thanks Mark, this is a good tip
what would you use for Rosetta cells though?
Tetracyclin?

On Fri, Oct 17, 2014 at 8:31 AM, Mark Wilson mwilso...@unl.edu wrote:

 Hi Ivan,
 We've had good luck with the addition of chloramphenicol ~1 hour prior to
 harvest, as described in:
 Carrió, M. M., and Villaverde, A. (2001) Protein aggregation as bacterial
 inclusion bodies is reversible. FEBS Lett. 489, 29­33.
 We usually combine this with lower temperature growth (20 C).
 Best regards,
 Mark

 Mark A. Wilson
 Associate Professor
 Department of Biochemistry/Redox Biology Center
 University of Nebraska
 N118 Beadle Center
 1901 Vine Street
 Lincoln, NE 68588
 (402) 472-3626
 mwilso...@unl.edu





 On 10/17/14 7:51 AM, xaravich ivan xaravich.i...@gmail.com wrote:

 Dear cc4bb enthusiasts,
 
 This is slightly off topic but many protein crystallographers might be
 familiar with this problem.
 
 
 I have been trying to over-express a bacterial (non-E.Coli) protein  in
 E.Coli and more than 80% goest to inclusion bodies.
 
 
 
 I tried the following
 
 
 Lowering the IPTG concentration to 0.1mM (range test from 0.075 mM - 0.5
 mM)
 
 
 Cold shock for 30 minutes in ice before induction
 
 
 Slow rotation speed at 18 degrees O/N after induction
 
 
 While these steps helped a bit I still get about 50-60% of my protein in
 inclusion bodies.
 
 
 I would like to know what other steps do you suggest to enhance the yield
 in the soluble fraction (without changing the host strain or manipulating
 the DNA)
 
 
 Thanks in advance
 
 Ivan
 
 

[ccp4bb]

[Gloria Borgstahl]

Be aware that some buffers are temperature sensitive and change pH, if this
pH change heads toward the pI of the protein it can crash out.


On Tue, Aug 19, 2014 at 6:37 PM, Prince, D Bryan 
dbryan.pri...@astrazeneca.com wrote:

  Dear Prashant,


  I have been working with a protein-protein complex expressed in
 mammalian cells, and that complex in very poorly soluble. Even with 500mM
 NaCl in the buffer, I cannot concentrate the complex to above 3 mg/mL. I
 tried an old school technique and precipitated my protein complex with
 ammonium sulphate (~80% saturation) on ice. When I recovered my complex, I
 was able to get almost 9mg/mL without any precipitation at all. The sample
 still crystallizes, but I believe now that the sulphate ion is
 shielding/masking part of the protein better than the NaCl did. Perhaps
 this would be worth a try for you as well?


  Another thing you can try with a weakly soluble protein is to set up
 various ratios between the protein and the reservoir solution in your
 crystallization drop.


  One point I have not yet seen mentioned is that some proteins are hyper
 sensitive to changes in temperature. In my opinion, any protein sample
 should be kept cold, (on ice at the bench) UNLESS you have good reason and
 biophysical data to show otherwise. If you are concentrating at room
 temperature, try concentrating at 4C if you can, you might be pleasantly
 surprised.


  Good Luck!


  Bryan Prince

  --

 *Confidentiality Notice: *This message is private and may contain
 confidential and proprietary information. If you have received this message
 in error, please notify us and remove it from your system and note that you
 must not copy, distribute or take any action in reliance on it. Any
 unauthorized use or disclosure of the contents of this message is not
 permitted and may be unlawful.



--
 *From:* CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Roger
 Rowlett rrowl...@colgate.edu
 *Sent:* Tuesday, August 19, 2014 5:50 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb]


 Some things to try to increase solubility:

 1. Move the buffer pH away from the expected pI. Proteins have minimum
 solubility near their pI values.
 2. Add solubilizing agents to the solution, e.g., 20-50% glycerol. (This
 may alter you crystallization screening strategy)
 3. Include some inert salt in the solution to minimize electrostatic
 interactions, e.g. 100-500 mM NaCl.

 Ultimately, your protein just may not be very soluble. That is potentially
 OK...it will ppt and maybe xtallize well at low concentration.

 Roger Rowlett
 On Aug 19, 2014 1:52 PM, Prashant Deshmukh prashantbiophys...@gmail.com
 wrote:

 Hi,
 i am concentrating my protein using centricon filter, but it is
 precipitated soon. Please help me solving this problem.
 Thanks.
  Prashant Deshmukh
 Dept. of Biophysics,
 NIMHANS,
 Bangalore 560 029,
 E-mail:prashantbiophys...@gmail.com
 Mob.No.: +919620986525

[ccp4bb] dynapro DLS cuvettes

[Gloria Borgstahl]

Does any one know of a source of these cuvettes?
Protein Solution doesn't exist anymore
and Wyatt no longer has these.

Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?

[Gloria Borgstahl]

You can prevent them from falling off by also
removing 5 microliter or so of mother liquor from the drop and
repositioning it back over the reservoir


On Wed, Jul 2, 2014 at 3:38 PM, Patrick Loll pat.l...@drexel.edu wrote:

 You can cut a small piece of sponge and put that into the reservoir; this
 prevents the reservoir buffer from splashing up into the drop.

 The sitting drops should be reasonably safe, but the 10 uL hanging drops
 are big; they'll be vulnerable to falling off if the tray is jarred.

 Good luck!

 Pat

 On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote:

  Dear CCP4ers
 
  I need to transfer some crystals mainly in sitting drops to the site of
 data collection without freezing them.
 
  I do not know what is the best solution to secure the boxes in their
 place to minimize the disturbance.
 
  I am using the 24 well VDX plates with 10-80 microliter sitting drops. I
 have one or two hanging drop boxes as well with 10 microliter size drops.
 
  If you have any experience about this matter, I greatly appreciate, if
 you share it with me.
 
  Thanks
 
  Meisam Nosrati




 ---
 Patrick J. Loll, Ph. D.
 Professor of Biochemistry  Molecular Biology
 Director, Biochemistry Graduate Program
 Drexel University College of Medicine
 Room 10-102 New College Building
 245 N. 15th St., Mailstop 497
 Philadelphia, PA  19102-1192  USA

 (215) 762-7706
 pat.l...@drexelmed.edu

Re: [ccp4bb] PDB passes 100,000 structure milestone

[Gloria Borgstahl]

I vote for Z's idea


On Wed, May 14, 2014 at 12:32 PM, Zachary Wood z...@bmb.uga.edu wrote:

 Hello All,

 Instead of placing the additional burden of policing on the good people at
 the PDB, perhaps the entry page for each structure could contain a comments
 section. Then the community could point out serious concerns for the less
 informed users. At least that will give users some warning in the case of
 particularly worrisome structures. The authors of course could still reply
 to defend their structure, and it may encourage some people to even correct
 their errors.

 Best regards,

 Z


 ***
 Zachary A. Wood, Ph.D.
 Associate Professor
 Department of Biochemistry  Molecular Biology
 University of Georgia
 Life Sciences Building, Rm A426B
 120 Green Street
 Athens, GA  30602-7229
 Office: 706-583-0304
 Lab:706-583-0303
 FAX: 706-542-1738
 ***







 On May 14, 2014, at 12:47 PM, Mark Wilson mwilso...@unl.edu wrote:

 Hi Tim,
 I agree with everything you've said about the importance of validation,
 but aren't we really talking about something different here?  Users of
 structural information should of course be keeping a careful eye on
 validation reports. On the other hand, what possible reason is there for
 the PDB to continue to archive and offer for public use models whose
 fundamental integrity (rather than quality or reliability) are highly
 suspect?  I hope that I'm not the only one who is frustrated that the page
 for 2HR0 is still available and unblemished by warnings.
 Best regards,
 Mark

 Mark A. Wilson
 Associate Professor
 Department of Biochemistry/Redox Biology Center
 University of Nebraska
 N118 Beadle Center
 1901 Vine Street
 Lincoln, NE 68588
 (402) 472-3626
 mwilso...@unl.edu






 On 5/14/14 11:35 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 Dear Eric,

 On 05/14/2014 06:05 PM, Eric Williams wrote:

 [...]
 We seem to be at an impasse. The PDB won't evict highly suspect
 structure
 models unless journals retract them, and the journals in question have
 shown no indication of desiring to retract them. Is there anything that
 can
 be done? [...]

 What's the appropriate course of action for conscientious consumers of
 PDB
 data? Is there a way to petition journals to issue retractions? I wonder
 what the gents at Retraction Watch (http://retractionwatch.com) would
 recommend.

 Eric


 you can teach the consumers how to help themselves - you are welcome to
 join my session MS-84 at the IUCr 2014 :-) because I believe that one of
 the New Paradigms in Crystallography is the requirement to how to
 correctly interpret crystallographic models, and validation is becoming
 more and more important as subject.

 Best,
 Tim


 On Wed, May 14, 2014 at 10:04 AM, Bernhard Rupp

 hofkristall...@gmail.comhttps://mail.google.com/mail/?view=cmfs=1tf=1
 to=hofkristall...@gmail.com

 wrote:


 which structure ended up as number 100.000?

 I guess that depends if we still count the Murthy corpses like 2a01
 This
 3-armed Swastika for example still does not come with a single warning
 short of a poor quality report
 http://www.ebi.ac.uk/pdbe-srv/view/entry/2a01/summary_details.html So,
 sorry, 0 (or lessŠ.) valid entries only at the time of
 announcement.

 Cheers, BR



 Supplemental material:



 ³The PDB says it will remove the other ten structures only when
 editors at
 the journals in which they were originally published or the authors
 themselves retract them²

 *http://www.nature.com/news/2009/091222/full/462970a.html
 http://www.nature.com/news/2009/091222/full/462970a.html*





 ³With the support of the structural-biology community, the mission of
 the
 wwPDB is to safeguard the integrity and improve the quality of the PDB
 archive.²

 http://www.nature.com/nature/journal/v463/n7280/full/463425c.html



 Not to be overly cynical, but



 http://tinyurl.com/pmupalt





 *From:* CCP4 bulletin board
 [mailto:CCP4BB@JISCMAIL.AC.UKhttps://mail.google.com/mail/?view=cmfs=1
 tf=1to=CCP4BB@JISCMAIL.AC.UK]
 *On Behalf Of *mesters
 *Sent:* Mittwoch, 14. Mai 2014 14:42
 *To:*
 CCP4BB@JISCMAIL.AC.UKhttps://mail.google.com/mail/?view=cmfs=1tf=1to
 =CCP4BB@JISCMAIL.AC.UK

 *Subject:* Re: [ccp4bb] PDB passes 100,000 structure milestone



 Amazing, great!

 And, which structure ended up as number 100.000?

 - J. -


 Am 14.05.14 10:42, schrieb battle:

 The Worldwide Protein Data Bank (wwPDB) organization is proud to
 announce
 that the Protein Data Bank archive now contains more than 100,000
 entries.

 Established in 1971, this central, public archive of
 experimentally-determined protein and nucleic acid structures has
 reached a
 critical milestone thanks to the efforts of structural biologists
 throughout the world.

 Read the full story at:
 http://www.wwpdb.org/news/news_2014.html#13-May-2014

 --
 Gary Battle
 on behalf on the wwPDB



 --
 Dr. Jeroen R. Mesters
 Deputy, Senior Researcher  

Re: [ccp4bb] Google Gets it Right

[Gloria Borgstahl]

Made my day
Her biography was inspirational to me!


On Mon, May 12, 2014 at 9:52 AM, lbetts0508 . laurie.betts0...@gmail.comwrote:

 I should have said, ALL crystallographers rejoice!!   Laurie


 On Mon, May 12, 2014 at 10:01 AM, lbetts0508 . laurie.betts0...@gmail.com
  wrote:

 Oh Hallelujah! Woman crystallographers rejoice.
 On May 12, 2014 9:39 AM, Robert Sweet sw...@bnl.gov wrote:

 Check out google.com.  They're announcing what would have been Dorothy
 Hodgkin's 104th b-day.  I saw the molecule, and said to myself, My
 goodness, that's penicillin G, and the mouseover announced the big day.

 She was a great scientist and a friend to thousands of us.

 Bob

 
 =
 Robert M. Sweet E-Dress: sw...@bnl.gov
 Group Leader, PXRR: Macromolecular   ^ (that's L
   Crystallography Research Resource at NSLSnot 1)
   http://px.nsls.bnl.gov/
 Photon Sciences and Biosciences Dept
 Office and mail, Bldg 745, a.k.a. LOB-5
 Brookhaven Nat'l Lab.   Phones:
 Upton, NY  11973631 344 3401  (Office)
 U.S.A.  631 344 2741  (Facsimile)
 
 =

Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate

[Gloria Borgstahl]

You can increase the formate concentration and it will be a cryoprotectant
on its own.  Test increasing amounts until you find the concentrationthat
freezed clear as glass and the transfer your crystal to this.  We did this
with sodium formate at 7 M quite easily.


On Tue, Dec 17, 2013 at 2:59 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 Dear Junyu,

 You can test cryo conditions conveniently in the absence of your
 crystals if you have access to e.g. an inhouse machine. Prepare your
 solutions with the usual suspects (PEG400; PEG400+glycerol;
 butanediol; Na Malonate etc. pp.) at various concentrations, pick up a
 large as possible drop with a nylon loop and take a shot to see if you
 have ice rings.
 Try to be on the safe side with respect to the decision about the
 minimal concentration because the presence of the crystal might give a
 slightly different result.

 Best,
 Tim

 On 12/16/2013 10:36 PM, Xiao, Junyu wrote:
  Dear all, sorry if this topic does not interest you. I wonder
  whether anyone has experience with freezing crystals grown in ~0.2
  M Magnesium Formate. Garman and Mitchell suggested that A major
  anomaly is solution 44, 0.2 M magnesium formate, which requires 50%
  glycerol for cryoprotection in their 1996 paper (J Appl. Cryst.
  29, 584-587).  Since 50% glycerol is kind of harsh, I wonder
  whether anyone has tried alternative cryo protectant. Your kind
  help will be highly appreciated.
 
  Best regards, Junyu
 
  --- Junyu
  Xiao, Ph.D. University of California, San Diego Leichtag Room 283
  9500 Gilman Drive, 0721 La Jolla, CA 92093-0721 Lab phone:
  858-822-0684
  
 
 

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

 GPG Key ID = A46BEE1A

 -BEGIN PGP SIGNATURE-
 Version: GnuPG v1.4.12 (GNU/Linux)
 Comment: Using GnuPG with Icedove - http://www.enigmail.net/

 iD8DBQFSsBJ/UxlJ7aRr7hoRApCiAJ0b3LyawgscAsIzwFUUHNDqRSplPwCeOZPv
 pF9iloWrCgMjIwo4hKlAgN8=
 =l4tU
 -END PGP SIGNATURE-

Re: [ccp4bb] in vitro Tyr phosphorylation

[Gloria Borgstahl]

Thank you all for your suggestions, I copied them below
I also found a paper for making Src and Abl catalytic domains Protein
Science Dec 2005
---

Before, I found a paper of Bhandari R (2000) where they describe the
co-expression of tyrosine kinase EphB1 (attached file). I haven’t checked
how generic the kinase is.

Kind regards



Seppe Leysen

---
Guillermo Montoya gmont...@cnio.es



I do not have the full reference here
But there is a febs lett paper from crhistoph muller group where they co
express stat transcription factor with a kinase to obtain the dimer

Dimerisation depends on tyr phosphorylation

Hope it helps

G.
-
Hi,

 This is in response to your post on the CCP4 bulletin board.
We either express Src independently and mix it with the target protein to
be phosphorylated in the presence of ATP at 37°C (works well) or co-express
the target protein with a non-specific PTK.

 Best

 Nicolas Nassar, PhD
On Tue, Dec 17, 2013 at 2:13 AM, Edward A. Berry ber...@upstate.edu wrote:

 The kinase domain of FAK can be made constitutively active by a mutation
 and expressed in E. coli. It's called Super FAK Kinase. But i am afraid
 it is rather specific. If you don't get a better lead I can send you the
 sequence and a reference.


 Gloria Borgstahl wrote:

 Does anyone know of a good way to phosphorylate the Tyr on a protein for
 structural
 studies?  Is there a generic kinase that can be coexpressed or purified
 for phosphorylation?

 The pCMF Amber codon system is very expensive
 and Glu really doesn't mimic pTyr all that well.

 Any ideas/help would be appreciated, G

[ccp4bb] in vitro Tyr phosphorylation

[Gloria Borgstahl]

Does anyone know of a good way to phosphorylate the Tyr on a protein for
structural studies?  Is there a generic kinase that can be coexpressed or
purified for phosphorylation?

The pCMF Amber codon system is very expensive
and Glu really doesn't mimic pTyr all that well.

Any ideas/help would be appreciated, G

Re: [ccp4bb] A question on protein microheterogenity for crystalization

[Gloria Borgstahl]

We had this happen with RPA14/32 heterodimer, we kept the two peaks
separate, and then grew crystals from each but in different space groups
and diffraction resolution.   See Habel, J. E., Ohren, J. F. and Borgstahl,
G. E. O. Dynamic light scattering analysis of full-length, human RPA14/32
dimer: purification crystallization and self-association *Acta
Cryst.*D57, 254-259 (2001).


On Sun, Dec 15, 2013 at 2:33 PM, Phoebe A. Rice pr...@uchicago.edu wrote:

  It might be that the protein is innocent and the FPLC is guilty. If
  FPLC pump needs maintenance and is stuttering a bit when running at high
 pressure, the salt gradient won't be as smooth as you'd like.  If you have
 an ionic strength detector, that trace will tell you.  Otherwise, see if
 other people's proteins are also looking a bit iffy under similar
 circumstances.
   Phoebe
  --
 *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
 Katherine Sippel [katherine.sip...@gmail.com]
 *Sent:* Sunday, December 15, 2013 11:53 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] A question on protein microheterogenity for
 crystalization

   Hi Acoot,

 There are a lot of great suggestions here already. I've also run into this
 phenomenon in couple of cases. The first was a binding protein in mixed
 bound and unbound forms. The second was a case of heterogeneous
 post-translational modification.  In both cases I could only get crystals
 from purified peaks and not pooled protein. If protein is precious I'd
 second Mark's suggestion to try screening pooled protein while you scale up
 your prep.

  Cheers,
  Katherine


 On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett acootbr...@yahoo.com wrote:

  Dear All,

  When I purified my protein by ion exchange chromatography for
 crystallization, there were several peaks containing the target protein as
 analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel
 filtration coupled MALLS.

  For crystallization purpose, can I merge the corresponding ion exchange
 chromatography peaks together? Otherwise the protein yield will be too low.
 And how to explain the heterogeneity by ion exchange chromatography in this
 situation?

  I am looking forward  to getting a reply from you.

  Acoot




 --
 Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?

[Gloria Borgstahl]

Are you talking about the Fig 3.1 in their book, Laboratory-build
oscillation camera (Arndt, Champness, Phizackerley and Wonacott, 1973)


On Thu, Oct 31, 2013 at 2:28 AM, Eleanor Dodson
eleanor.dod...@york.ac.ukwrote:

 one in their book, i am sure.
 eleanor


 On 30 Oct 2013, at 16:05, Gerard Bricogne wrote:

  Dear all,
 
  Apologies for such a retro and non-biological question, but would
  anyone have a photograph of an Arndt-Wonacott rotation camera that he/she
  would be willing to share? I collected data on the first two prototypes
 in
  the early seventies, then on one of the first commercial models, but I
  cannot find any images of this ground-breaking piece of equipement on the
  Web. I found images for the Enraf-Nonius precession camera and the CAD-4
  diffractometer, but not for the A-W rotation camera.
 
  This would be for use as visual material in presentations, and I
 would
  gratefully acknowledge the source of it. Thank you in advance!
 
 
  With fingers crossed ... .
 
   Gerard.
 
  --
 
  ===
  * *
  * Gerard Bricogne g...@globalphasing.com  *
  * *
  * Global Phasing Ltd. *
  * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
  * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
  * *
  ===

[ccp4bb] limited proteolysis / ms

[Gloria Borgstahl]

We are trying to apply this approach for the first time to define a
crystallizable domain of our protein of interest.  Can anyone send a
protocol that includes exactly how to do the mass spectrometry
measurement?  Our core lab here doesn't know and I'm just not that gifted.
Happy October, G

[ccp4bb] Structural transmission of signal across a membrane

[Gloria Borgstahl]

Hello ccp4ers,
I am helping a colleague develop a grant and have a vague recollection of
structures of transmembrane protein receptors that signal across the
membrane.  Can anyone send me specific examples?
Many thanks, G

[ccp4bb] structural search for homologs in pdb?

[Gloria Borgstahl]

We have a protein sequence that probably contains OB folds.  What is the
best way to search for the top structural homologs to this sequence in the
pdb?  G

[ccp4bb] 96 well DLS plates

[Gloria Borgstahl]

We have found funding for a 96 well DLS from Wyatt.  I was wondering what
peoples experiences with this instrument was.   Is there a better
instrument out there?  Does it matter where you buy the 96 well plates from?
Many thanks, Gloria

Re: [ccp4bb] off topic: good peak on gel filtration

[Gloria Borgstahl]

In our lab we do DLS on the SEC fractions and only set up those that are
monodisperse  :-)


On Mon, Jul 1, 2013 at 7:37 PM, El Arnaout, Toufic 
elarnao...@biochem.wustl.edu wrote:

 Hello Peter,
 In addition to the great comments/details, please check the following
 points I have now in mind.. since you want to relate the size exclusion
 peak/profile to the crystallization:
 - occasionnaly, some perfect peaks that you might think are homogeneous
 actually correspond to a sample of hetergeneous protein (maybe the target
 protein will still crystallize, but problems happen during crystal
 optimization, or/and observing missing electronic density of the N- or
 C-terminus for example). It might also be reflected on the specific
 activity (btw if the protein you have is easy to assay, you could check the
 activity from different fractions if you think broad peak = problem). In
 some occasions, analyzing fractions from a perfect peak shows on SDS-PAGE
 a double band or sometimes far bands (I won't comment on oligomerization in
 this case).
 - not all proteins from good SE chromatograms crystallize...
 - some people only collect fractions from the centre of the peak (or for
 example they measure the A280 max, divide it by two, draw an horizontal
 line at that value, and collect the fractions/projection between where the
 line crosses the peak from both sides.. If you add one fraction before or
 after, the protein might not crystallize anymore.
 - from the literature, I have seen many shapes of chromatograms: perfect,
 bleeding, skewed (tail) to the right or left, little broad, etc.. which
 resulted in diffraction quality crystals.
 - re-running a protein sample (that crystallizes after the first SEC) for
 a second SEC might cause the protein not to crystallize anymore (for
 example membrane proteins might lose lipids etc).
 - for proteins that are subjected to SEC straight after Ni-NTA, and which
 have a perfect peak shape: a 4-16 hours delay before injection might show
 the aggregation effect. A peak shoulder will form, which you might not have
 seen if the sample was directly injected. The point is: maybe what you
 incubate for crystallization or work on after SEC might not be anymore that
 protein with the beautiful peak you had. Using different
 additives/chemicals/mutations may help in making the protein more
 stable/thermostable. You can check previous publications of Dr Tate CG,
 GPCRs for example. This is also a nice paper:
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809.
 Regards



 toufic el arnaout
 School of Medicine - 660 S Euclid Ave
 Washington University in St. Louis
 St Louis, MO 63110, USA

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A.
 Berry [ber...@upstate.edu]
 Sent: Saturday, June 29, 2013 9:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] off topic: good peak on gel filtration

 Thanks- guess I'm old-fashioned, using low-pressure columns.
 So apparently theoretical plates are still calculated, and have
 improved a lot- 25000/m is HETP .04 mm, way better than the
 figure I mentioned. (TP per dollar not so much.)
 No more sour grapes from me-
 eab

 Zhijie Li wrote:
  Hi Ed,
 
  I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which
  most of us should be quite familiar with. According to GE healthcare, a
 new
  Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a
 new
  Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP
  difference of the two should be mainly caused by the different resin
 sizes.
  Of course in reality columns change over time and in cases like Peter's,
 it
  might be a good idea to test the performance of the column before
 drawing a
  conclusion. When we are concerned about resolution of a column, we load a
  standard sample and calculate the TP based on the peak shape. As I
 remember,
  GE healthcare's SEC manuals has recommended procedures on TP
 determination.
 
  Zhijie
 
 
  -Original Message-
  From: Edward A. Berry
  Sent: Saturday, June 29, 2013 7:43 PM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] off topic: good peak on gel filtration
 
  Peter Hsu wrote:
  Hi all,
 
  I've generally always thought as long as the peak was symmetrical and
 not
  too broad would suggest a good sample. However, looking at my previous
  runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200,
 or
  slightly broader peaks with about 3mL (all symmetrical peaks, roughly
  similar amounts loaded on the columns). I'm curious to see what people's
  views are as far as what constitutes a broad peak and how much that can
  end up affecting crystallization of the sample.
 
  Thanks for any responses.
 
  Peter
 
  The width itself may not be a good indicator unless its always the same
  protein- in general a molecule that elutes later
  will have a broader peak.
  Supposing that each time a molecule diffuses into the 

[ccp4bb] Artic Express: problem with chaperone copurification

[Gloria Borgstahl]

We have had good luck with making a protein soluble using the Artic Express
bacterial cell line BUT we can't get rid of the chaperone that copurifies.
We have tried adding ATP, MgCl2 and potassium to lysate and extensive
washes and this releases a bit of the chaperone.  Has anyone solved this
problem?  We have heard of adding denatured proteins to the lysate to
confuse el chaperono.  Does this work?
Many thanks in advance for a advice, Gloria

Re: [ccp4bb] suitable buffer for CD studies

[Gloria Borgstahl]

I have found it is best to test the absorption of your buffer in the
wavelength range you are interested in.
If you are going to do a temperature study with CD perhaps at 222 nm, then
test your buffer there with your UV spec.
You want to have little or no absorption.  Or do the range 200-270 nm and
choose a buffer that does not absorb in that range.

For example we thought Bicine pH 8.5 with BME would be ok for CD studies.
Bicine pH 7 did not absorb at 222 nm, but 8.5 gave a high background. bME
really absorbs
We ended up using PBS with a smidgeon of BME.

Also you should use a protein concentration that gives you an absorption of
1.0 at the wavelength of interest
This is crucial to keep noise out of your data.

Also the CD dynod voltage should be monitored and keep it below 400 for
protein studies

Here is an excellent reference:  Kelly, Ness and Price (2005) How to study
proteins using circular dichroism Biochemica et Biophysica Acta *1751*,
119-139

Good Luck, Gloria

On Wed, Mar 20, 2013 at 1:59 AM, Harsh Bansia spideysp...@gmail.com wrote:

 Sorry for a simple and non-CCP4 question.

  I have determined the structures of three different mutants of a
 thermostable protein by X-ray crystallography method. I feel that Mg2+ has
 a role in protein stability.

  So I want to perform a thermal denaturation study by CD spectroscopy
 both in presence and absence of Mg2+ ion.

 In this regards, what should be the suitable buffer for CD studies? May I
 use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is
 it advisable to use PBS buffer. If so, what is maximum concentration of
 Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and
 lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4.


  Thanking you in advances,

 harsh

Re: [ccp4bb] Nobel Prizes for 3D Molecular Structure

[Gloria Borgstahl]

Daniel Schectman's 2012 nobel prize for the discovery of quasicrystals is
missing.

On Sat, Oct 13, 2012 at 5:01 PM, Joel Sussman
joel.suss...@weizmann.ac.ilwrote:

  Just want to make sure anyone interested in Nobel Prizes knows about this
 existing page:

 http://proteopedia.org/wiki/index.php/Nobel_Prizes_for_3D_Molecular_Structure
 best regards,
 Joel Sussman

Re: [ccp4bb] Professor Dame Louise Johnson

[Gloria Borgstahl]

This indeed is sad news for today.
I just wanted to note that Professor Johnson's early papers on
time-resolved crystallography truly inspired me to continue in
crystallography, influenced my decision for my first postdoctoral position
and to push the limits.  I still have the carefully highlighted photocopies
(yes used a photocopier and a real bound journal in gradual school) in my
filing cabinet next to my office.

My condolences to those close to her and her family.  Gloria

On Tue, Oct 2, 2012 at 6:40 AM, elizabeth.d...@diamond.ac.uk wrote:

  It is with great sadness that I would like to inform the
 crystallographic community of the death of one of the great pioneers of the
 field, Professor Dame Louise Johnson.

 ** **

 Those of us who had the privilege to work alongside her benefitted greatly
 from her vision for extending technique and instrumentation such that
 increasingly complex problems could be successfully solved and found her
 quiet determination to succeed inspirational. 

 ** **

 Dr. Liz Duke

 Diamond Light Source

 Harwell Science and Innovation Campus

 Chilton, Didcot

 Oxon OX11 0DE

 UK

 ** **

 Tel. +44 (0) 1235 778057

 Mob. +44 (0)7920 138148

 ** **



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[ccp4bb] CONH2 not COOH at TER

[Gloria Borgstahl]

How would I tell REFMAC to replace the
C-terminal Pro in my synthetic peptide
with a CONH2
not
the natural CT.

[ccp4bb] retract Fwd: CONH2 not COOH at TER

[Gloria Borgstahl]

I figured it out

ATOM 48  C   PRO A  11  59.322 -42.497 -19.375  1.00
39.11   C
ATOM 49  O   PRO A  11  58.425 -42.192 -20.159  1.00
36.42   O
HETATM   50  N   NH2 A  11  60.520 -42.416 -19.709  1.00
46.31   O
TER


-- Forwarded message --
From: Gloria Borgstahl gborgst...@gmail.com
Date: Thu, Aug 23, 2012 at 12:25 PM
Subject: CONH2 not COOH at TER
To: ccp4bb@jiscmail.ac.uk


How would I tell REFMAC to replace the
C-terminal Pro in my synthetic peptide
with a CONH2
not
the natural CT.

Re: [ccp4bb] Calculation of volume/size of cavity

[Gloria Borgstahl]

We used VOIDOO recently and it worked well

Kleywegt, G. J.  Jones, T. A. (1994). Detection, delineation,
measurement and display of cavities in macromolecular structures. Acta
Crystallogr. D Biol. Crystallogr. 50, 178-185.


On Thu, May 31, 2012 at 12:41 PM, Zhou, Tongqing (NIH/VRC) [E]
tz...@mail.nih.gov wrote:
 Dear All,



 I am looking for a way to calculate the size of a protein cavity which is
 occupied by a loop from a ligand protein. The goal is to see what’s the
 maximum length of peptide allowed in this cavity.



 Thanks,



 Tongqing



 Tongqing Zhou, Ph.D.

 Staff Scientist

 Structural Biology Section

 Vaccine Research Center, NIAID/NIH

 Building 40, Room 4609B

 40 Convent Drive, MSC3027

 Bethesda, MD 20892

 (301) 594-8710 (Tel)

 (301) 793-0794 (Cell)

 (301) 480-2658 (Fax)

 **

 The information in this e-mail and any of its attachments is confidential
 and may contain sensitive information. It should not be used by anyone who
 is not the original intended recipient. If you have received this e-mail in
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 storage devices. National Institute of Allergy and Infectious Diseases shall
 not accept liability for any statements made that are sender's own and not
 expressly made on behalf of the NIAID by one of its representatives.

 **

[ccp4bb] SUMO(ULP-1) protease

[Gloria Borgstahl]

My fellow crystallographers,
We are thinking the SUMO/His vectors would be nice to have in the lab
aresenal... but.  The stumbling block is that the protease needed for
cleavage is very expensive at crystallography scale.  SUMO(ULP-1)
protease costs ~$700/mg fusion protein.   It would not be a problem
for labs using micrograms of protein, but is prohibitive at our level
of protein purification.
Has anyone found a way around this?  Your pal, Gloria

Re: [ccp4bb] Disorder or poor phases?

[Gloria Borgstahl]

a recent experience in our lab with molecular replacement (wt and
disordered point mutant; same space group and unit cell)
was solved with a combination of two methods.

1.  We made omit maps in the disordered region at several lower
resolutions.  The region became interpretable after suffereing through
these maps, building residue by residue and refinement.
2.  Then we had the bright idea to make Fwt-Fmutant maps to confirm
our interpretation.  Happily this map did confirm the unexpected large
structural changed caused by a point mutant.

On Fri, Apr 13, 2012 at 1:31 PM, James Holton jmhol...@lbl.gov wrote:
 Francis,

 I think in the cases you describe the region in question is disordered.
  Time and time again I have users coming to my beamline wanting to clean up
 a questionable region by getting experimental phases.  Ahh!  If only I had
 a nickle for each one.  Oh wait, I suppose I kind of do?  I take that back!
  Go MAD everyone!

 Much as I hate to discourage people from using my favorite technique, Tim is
 right: phases are not region-specific in electron density maps.  Dale does
 make a good point that there is such a thing as model bias and one can
 argue that experimental phases don't have it.  But, this is only true if you
 have not yet applied solvent flattening.  How long has it been since you
 looked at a raw experimentally-phased map (before solvent flattening)?
  I'm willing to bet a while.  With very few exceptions, raw experimental
 phases are lousy.  We have actually become quite dependent on density
 modification to clean them up.  In fact, solvent flattening is the only
 reason why SAD works at all.

 However, you CAN use anomalous differences to clear up disordered regions in
 a different way.  Something I started calling SeMet scanning a number of
 years ago.  A few of my users have done this, and a good example of it is
 Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826).  Basically, you mutate
 residues in the disordered region one at a time to SeMet, and look at phased
 anomalous difference Fourier (PADF) maps.  These maps are surprisingly
 clear, even when the anomalous difference signal is so weak as to make
 experimental phasing hopeless.  Yes, the best phases to use for PADF maps
 are model phases, but, as always, it is prudent to refine the model after
 omitting the thing you are looking for before calculating such phases.

 Another way to get residue-specific labeling for low-resolution chain
 tracing is radiation damage.  If you expose for the right amount of time,
 Asp and Glu side chains will be specifically burnt off, but not Asn and
 Gln.  You will also see Met loosing its head, etc.  So, as long as you have
 read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to
 guide sequence assignment, even at 4.5 A and worse.

 Anyway, when it comes to the question of is it disordered or is it model
 bias?, I think it is usually the former.  It is very difficult to make
 model bias suppress a region that is actually well-ordered.  Try it!
  After all, this is the whole reason why we bother looking at fo-fc maps.
  Then again, it is always possible to have a model so bad that the phase
 error is enough to squash anything.  An excellent example of this can be
 found in the Book of Fourier.  Taking amplitudes from the image of a cat,
 you can see what happens when you use the phases of a duck:
 http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif
 as opposed to what happens if you use the phases of a manx:
 http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif
 A manx is a species of cat that doesn't have a tail, so no animals were
 harmed in obtaining these phases.  My point here is that the cat's tail can
 be seen quite readily in the 2fo-fc map if most of the structure is already
 right, but if your model is completely unrelated to the true structure
 (fitting a duck into a cat-shaped hole), then everything is in the noise.

 Real structures are usually somewhere between these two extremes, and I
 think an important shortcoming in modern crystallography is that we don't
 have a good quantitative description of this middle-ground.  We all like to
 think we know what model bias is, but we don't exactly have units for
 it.  Should we be using a scale of 0 to 1?  Or perhaps duck to cat?
  Yes, I know we have figure of merit, but FOM is not region-specific.

  In my experience, as long as you have ~50% of the electrons in the right
 place (and none of them in the wrong place), then you can generally trust
 that the biggest difference feature in the fo-fc map is real, and build
 from there.  As the model becomes more complete, the phases should continue
 to get better, not worse.  Eventually, this does break down, although I'm
 not really sure why.  With small molecules, the maximum fo-fc peak keeps
 getting bigger (on a sigma scale) as you add more and more atoms, and the
 biggest one you will ever see is the last one.  For macromolecules, the
 

[ccp4bb] molrep question - how get our ducks in a row?

[Gloria Borgstahl]

Hello all,
We are solving a superstructure of a protein complex with 2 parts.
Built 6 of the first part and they are all sensibly stacked next to each other.
Then we read this into molrep as the fixed model and solved for the
second part.
The solution was found but the 6 for the second model are in different
ASU's and unit cells.
What is the easiest way to get everyone together in one asu?

We can think of hard ways to do it, but any advice?
Thanks, Gloria

Re: [ccp4bb] Crystallizing protein sitting in PBS

[Gloria Borgstahl]

A thing we frequently forget is that phosphate can be a precipitating agent
try a phosphate grid screen, just like you would with ammonium sulfate.
If your protein likes PBS, it may want to crystallize with phosphate

See Enrico Stura's footprint screen for example

On Tue, Nov 15, 2011 at 5:25 PM, Jayakrishnan Nandakumar
sscna...@gmail.com wrote:
 Hi All,
 I have an RNA-binding protein that I can purify out of bacteria in PBS
 (Phosphate buffered saline;137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
 KH2PO4), but which is insoluble in Tris/NaCl-based buffers. My guess would
 be that the inorganic phosphates (by mimicking RNA) are binding the protein
 to keep it in solution. My question is whether I can leave the protein in a
 phosphate-based buffer (at lower salt maybe) to set up crystallization
 trials or are PBS-based buffers not suitable for crystallization in general.
 I have always used Tris/NaCl based buffers in the past.

 Thanks in advance for your suggestions.
 Regards,
 JK

Re: [ccp4bb] Crystalization in low PH

[Gloria Borgstahl]

Glutaraldehyde works best at low pH

On Mon, Nov 7, 2011 at 8:40 AM, Ed Pozharski epozh...@umaryland.edu wrote:
 On Mon, 2011-11-07 at 05:19 +, Sam Arnosti wrote:
 Hi everyone

 I have a protein that is extraordinarily stable at PH=3.0 or even 2.0.

 I want to crystallize it in the  low PH and compare the differences between 
 the crystals in regular PH and low PH.

 I was wondering how people set up the boxes in low PH, as usual buffers are 
 mostly less acidic.

 Regards

 Sam

 Not clear if you already have crystals at regular pH, but if you do,
 you may consider direct transfer to lower pH.  Of course, crystals may
 dissolve, which you could possibly prevent by cross-linking with
 glutaraldehyde.  Three caveats:
 a) If lattice is incompatible with lower pH, even with cross-linking the
 resolution may sink to essentially useless levels
 b) I have no idea if the cross-linking will not be disrupted at really
 low pH, perhaps someone else can comment on that
 c) the 3rd reviewer can always say that lattice forces could have
 prevented a conformational change.  But same goes for direct
 crystallization at low pH (but caries less weight).

 --
 I'd jump in myself, if I weren't so good at whistling.
                               Julian, King of Lemurs

Re: [ccp4bb] IUCr committees, depositing images

[Gloria Borgstahl]

I just want to jump in to state that I am ALL FOR the notion of
depositing the images that go with the structure factors and the
refined structure.

Through the years, I have been interviewing folks about the strange
satellite diffraction they saw, but ignored,
used the mains that they could integrate and deposited that structure,
does not help me to
justify the existance of modulated protein crystals to reviewers.

But if I could go and retrieve those images, and reanalyze with new methods.
Dream come true.  Reviewers convinced.

On Wed, Oct 26, 2011 at 10:59 AM, Patrick Shaw Stewart
patr...@douglas.co.uk wrote:

 Could you perhaps use the principle of capture storage that is used by
 wild-life photographers with high-speed cameras?
 The principle is that the movie is written to the same area of memory,
 jumping back to the beginning when it is full (this part is not essential,
 but it makes the principle clear).  Then, when the photographer takes his
 finger off the trigger, the last x seconds is permanently stored.  So you
 keep your wits about you, and press the metaphorical store button just
 after you have got the movie in the can so to speak

 Just a thought
 Patrick

 On Wed, Oct 26, 2011 at 2:18 PM, John R Helliwell jrhelliw...@gmail.com
 wrote:

 Dear Frank,
 re 'who will write the grant?'.

 This is not as easy as it sounds, would that it were!

 There are two possible business plans:-
 Option 1. Specifically for MX is the PDB as the first and foremost
 candidate to seek such additional funds for full diffraction data
 deposition for each future PDB deposiition entry. This business plan
 possibility is best answered by PDB/EBI (eg Gerard Kleywegt has
 answered this in the negative thus far at the CCP4 January 2010).

 Option 2 The Journals that host the publications could add the cost to
 the subscriber and/or the author according to their funding model. As
 an example and as a start a draft business plan has been written by
 one of us [JRH] for IUCr Acta Cryst E; this seemed attractive because
 of its simpler 'author pays' financing. This proposed business plan is
 now with IUCr Journals to digest and hopefully refine. Initial
 indications are that Acta Cryst C would be perceived by IUCr Journals
 as a better place to start considering this in detail, as it involves
 fewer crystal structures than Acta E and would thus be more
 manageable. The overall advantage of the responsibility being with
 Journals as we see it is that it encourages such 'archiving of data
 with literature' across all crystallography related techniques (single
 crystal, SAXS, SANS, Electron crystallography etc) and fields
 (Biology, Chemistry, Materials, Condensed Matter Physics etc) ie not
 just one technique and field, although obviously biology is dear to
 our hearts here in the CCP4bb.

 Yours sincerely,
 John and Tom
 John Helliwell  and Tom Terwilliger

 On Wed, Oct 26, 2011 at 9:21 AM, Frank von Delft
 frank.vonde...@sgc.ox.ac.uk wrote:
  Since when has the cost of any project been limited by the cost of
  hardware?  Someone has to implement this -- and make a career out of it;
  thunderingly absent from this thread has been the chorus of volunteers
  who
  will write the grant.
  phx
 
 
  On 25/10/2011 21:10, Herbert J. Bernstein wrote:
 
  To be fair to those concerned about cost, a more conservative estimate
  from the NSF RDLM workshop last summer in Princeton is $1,000 to $3,000
  per terabyte per year for long term storage allowing for overhead in
  moderate-sized institutions such as the PDB.  Larger entities, such
  as Google are able to do it for much lower annual costs in the range of
  $100 to $300 per terabyte per year.  Indeed, if this becomes a serious
  effort, one might wish to consider involving the large storage farm
  businesses such as Google and Amazon.  They might be willing to help
  support science partially in exchange for eyeballs going to their sites.
 
  Regards,
     H. J. Bernstein
 
  At 1:56 PM -0600 10/25/11, James Stroud wrote:
 
  On Oct 24, 2011, at 3:56 PM, James Holton wrote:
 
  The PDB only gets about 8000 depositions per year
 
  Just to put this into dollars. If each dataset is about 17 GB in
  size, then that's about 14 TB of storage that needs to come online
  every year to store the raw data for every structure. A two second
  search reveals that Newegg has a 3GB hitachi for $200. So that's
  about $1000 / year of storage for the raw data behind PDB deposits.
 
  James
 
 



 --
 Professor John R Helliwell DSc



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Re: [ccp4bb] Another paper structure retracted

[Gloria Borgstahl]

Dale, This is exactly the conversation  I just had with my student Jason,
right on!
The paper we are writing just now, this is figure 1.
But I always get rejected by Nature, so go figure.
On Thu, Aug 11, 2011 at 1:25 PM, Dale Tronrud det...@uoxray.uoregon.eduwrote:

   I agree with Prof. Tomchick: if the point of your paper is your crystal
 structure of
 the binding of a ligand to a protein you should include a figure with the
 omit map
 (displayed without a cover radius) that convinced you that binding took
 place.  I
 prefer that map over some simulated, after-the-fact, omit map calculated
 just for
 publication.

   This is not simply a matter for reviewers to be gatekeepers, it is
 important for the
 readers to know what level of confidence to place in this result, and it is
 instructional
 for everyone to see what ligand binding density looks like.  Apparently
 some people don't
 know what features to look for to distinguish between signal and noise.

 Dale Tronrud

 On 08/11/11 09:40, Diana Tomchick wrote:
  A quick glance at the header of the PDB file shows that there is one
 glaring discrepancy between it and the table in the paper that hasn't been
 mentioned yet in this forum. The data completeness (for data collection)
 reported in the paper is 95.7%, but in the header of the PDB file (actually,
 in both the 2QNS and the 3KJ5 depositions) the data completeness (for data
 collection) is reported as only 59.4%. The PDB header also contains an
 inconsistency, with the data completeness (for refinement) reported as
 95.7%. Since the numbers of reflections reported for refinement versus data
 collection in the PDB header differ by less than 1%, it appears that there's
 been a bit of magical thinking that took place somewhere along the process
 from data processing to final model refinement. Small wonder that the
 refined geometry is so poor. Perhaps if these scientists had actually
 collected a complete dataset, we would not be having this conversation.
 
  Diana
 
  P.S. I have, on occasion, provided the coordinates and a map file to
 reviewers when they requested it. The last time it was requested was many
 years ago; I decided it was safer and easier if I provided as much
 information as possible in the manuscript (including better quality electron
 density figures than appear in this paper) to allow the reader to determine
 whether the work is valid or not.
 
  * * * * * * * * * * * * * * * * * * * * * * * * * * * *
  Diana R. Tomchick
  Associate Professor
  University of Texas Southwestern Medical Center
  Department of Biochemistry
  5323 Harry Hines Blvd.
  Rm. ND10.214B
  Dallas, TX 75390-8816, U.S.A.
  Email: diana.tomch...@utsouthwestern.edu
  214-645-6383 (phone)
  214-645-6353 (fax)
 
 
 
  On Aug 10, 2011, at 5:45 PM, Dale Tronrud wrote:
 
I've made a quick look at the model and the paper - and it doesn't
  need more than a quick look.  The description of the model in
  the paper sounds great.  The problems in the model are clear.  My
  favorite is the quote Trp-477 of PTH1R makes several van der Waals
  contacts with Trp-339 and Lys-337 of G-beta-1   They are contacts
  all right.  The distances between the 477:CH2 and 337:CE is 2.75 A
  and between 477:NE1 and 339:CH2 is 2.26 A.  There are many more.
 
In general the geometry of this entire model is terrible.  In
  Table 1 the bond length rmsd is listed at 1.64 A and the bond angles
  are 0.0078 deg!  Perhaps one is to presume the numbers should be
  swapped.  In any case, the values I calculate for the model are
  0.160 A and 4.46 deg!  Absolutely dreadful.  The PDB header lists
  the (swapped) values from the paper and then reports hundreds of
  outliers.
 
The tools proposed by the Validation Task Force should cause a
  model like this to pop out clearly.  Even the old tools show this
  model is quite unreliable.  We just have to use them.
 
  Dale Tronrud
 
  On 08/10/11 14:35, Jacob Keller wrote:
  On the surface it doesn't seem as bad as others, i.e., it does not
  seem to be a real fake--perhaps just a strong form of wishful thinking
  and creative density interpretation. I wonder what would be a good
  metric in which to establish a cutoff for present/not present in
  density. CC, maybe?
 
  Jacob
 
  On Wed, Aug 10, 2011 at 4:01 PM, David Schuller dj...@cornell.edu
 wrote:
  Time to fuel up the gossip engines for the approaching weekend:
 
 
  http://www.sciencedirect.com/science/article/pii/S096921260800186X
 
  RETRACTED: Structure of the Parathyroid Hormone Receptor C Terminus
 Bound to
  the G-Protein Dimer Gβ1γ2
  Structure, Volume 16, Issue 7, 9 July 2008, Pages 1086-1094
  Structure 2QNS withdrawn.
 
  --
 
 ===
  All Things Serve the Beam
 
 ===
David J. Schuller
modern man in a post-modern world
  

[ccp4bb] titering baculovirus ?

[Gloria Borgstahl]

Hi Guys,
we are learning to work with Sf9 cells and Carol in my lab wanted me to ask
you the following question.  Many thanks for any help, G

I need to titer a baculovirus stock in my suspension-adapted Sf9 cells.   I
know that these can be encouraged to attach better to tissue culture plastic
if they have added FBS (about 10%), but am not sure that they will not be
migrating and hiding plaques.  Does anyone have suggestions about how to
keep them more firmly anchored during the baculovirus titration, or about
another cell line that we could use instead?

Re: [ccp4bb] strange density

[Gloria Borgstahl]

I'm voting with Roger this time.
If I were you I would model a nickel in there (unless you have a better
candidate)
if it is right, the distances to the His should be like seen in a SOD active
site.
Then you can model the bonds and waters.  You may need partial occupancy on
the metal.

Reminds me of the time I found a 6th water in MnSOD as I was making the
token electron denisty
figure of the active site.  Found myself doing more refinement and a much
better paper.

On Thu, Feb 24, 2011 at 8:15 AM, Roger Rowlett rrowl...@colgate.edu wrote:

 This looks suspiciously like a metal ion with 3 or more resolved water
 ligands. It's hard to tell from the images provided, but it looks like the
 metal could be close to octahedral, with 2 His and 3 water ligands
 well-defined. The estimated metal-nitrogen bond distances might help narrow
 down the metal ion. For example, tetrahedral Zn(II) has a Zn-N bond distance
 of around 2.0 A. I suppose this could be a Mg(II) ion, since that is in your
 crystallization mix, but I think that Mg(II) typically prefers harder
 protein  ligands, e.g., carboxylates. Zn(II) is a very common contaminant in
 solution, however, and should not necessarily be discounted; indeed Zn(II)
 and other first row transition metal ions would be expected to have a
 reasonable affinity for medium hard/soft ligands such as vicinial His
 residues.

 Cheers.

 On 2/23/2011 7:34 PM, Alex Singer wrote:


 cocrystallized in 2.5mM Glycero-3Phosphocholine and cryoprotected by
 dipping in

 --
 --
 Roger S. Rowlett
 Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346

 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu

[ccp4bb] TEV protease

[Gloria Borgstahl]

We are using TEV protease to make a big batch of protein for
structural studies.  We usually use thrombin, so this is our first
time using this enzyme on a large scale.  Boy is it expensive!  Does
anyone know of a bulk source for this enzyme and what ratios of use do
you recommend.  Imidazole inhibits?
Many thanks. Gloria

[ccp4bb] Thank you! TEV protease

[Gloria Borgstahl]

I have learned alot about TEV protease
and thank you all for your help
Going to start making it ourselves!!!

[ccp4bb] Fwd: [ccp4bb] Wyckoff positions and protein atoms

[Gloria Borgstahl]

I've gotten some interesting responses, that I will summarize for the
group later, but I thought I should clarify why I asked.

I was worrying about this because I have been working out the steps in
how to determine the (3+1)D superspace group for a protein crystal.
The last step listed in IT vol C chapter 9.8, is to consider any atoms
that lie ON a Wyckoff position, and what restrictions this would apply
to the modulation function that is refined for each atom.

My first reaction, was Wyckoff positions?  I vaguely remember those,
my recollection from my experience was they were really cool, but were
usually in the solvent, so I can't imagine a protein crystallographer
would ever need to apply the modulation function to a protein atom
that happened to be on one.  But to a crystallographer working on a
modulated mineral, it would happen all the time, I'll bet.
So maybe this was one more thing that just didn't really apply to
protein structures and lucky us
we don't worry about this last step (just as I never did model that
solvent water that was on one, back in the 90s).

Then I thought, maybe I'm missing something, or there are special
cases out there (and so far I have heard of a disulfide bond on a
2-fold connecting two homodimers).

So I polled the collective knowledge of the great ccp4bb group.

On Wed, Dec 8, 2010 at 10:57 AM, Gloria Borgstahl gborgst...@gmail.com wrote:
 My fellow crystallographers,
 I wanted to take a poll.

 How many of you have ever had a protein atom on a Wyckoff position
 (AKA a special position).
 What kind of molecules have you found at special positions (it would
 have to contain the symmetry of the special position, right?)
 I'm thinking it is impossible to have a protein atom at a special position
 or am I exposing my ignorance yet again...

 my experience is that only once I found an atom in a special position,
 it was a strange solvent molecule,
 that blew my mind for a while until I learned about special positions
 in crystallography.

 Looking forward to your responses, Gloria

 
 Gloria Borgstahl
 Eppley Institute for Cancer Research and Allied Diseases
 987696 Nebraska Medical Center
 10732A Lied Transplant Center
 Omaha, NE 68198-7696

 http://sbl.unmc.edu
 Office (402) 559-8578
 FAX (402) 559-3739

 Professor
 Hobbies:  Protein Crystallography, Cancer, Biochemistry,
 DNA Metabolism, Modulated Crystals,  Crystal Perfection
 Interests:  Manga, Led Zepplin, Cold Play, piano, BRAN,
 RAGBRAI, golf and lately superspace groups
 

Re: [ccp4bb] small lines in diffraction pattern

[Gloria Borgstahl]

Gees, I go to Washington DC for a couple of days and then a superbowl 
party, come back to my stack of emails
and find out I missed out on all the fun... again!

At first glance I thought it looked like a problem with the CCD detector 
overloads, but that apparently has been ruled out.
Looks like you have already analyzed this, and from what I briefly read it 
sounds like a superstructure
that is commensurate and can be solved by molecular replacement or twinned 
plates (if the crystals look like plates).

Would love to see more of the images though at different distances and 
delta phi. 

**
Gloria Borgstahl 
Eppley Institute for Cancer Research and Allied Diseases
987696 Nebraska Medical Center 
10732A Lied Transplant Center 
Omaha, NE 68198-7696

http://sbl.unmc.edu
Office (402) 559-8578
FAX (402) 559-3739

Professor 
Hobbies:  Protein Crystallography, Cancer, Biochemistry, DNA Metabolism, 
Modulated Crystals,  Crystal Perfection, X-ray Topography,
Interests:  City of Ember, skateboarding, RAGBRAI, basketball, and 
rollerskating
***



James Holton jmhol...@lbl.gov 
Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK
01/28/2009 09:59 PM
Please respond to
James Holton jmhol...@lbl.gov


To
CCP4BB@JISCMAIL.AC.UK
cc

Subject
Re: [ccp4bb] small lines in diffraction pattern






I'm sure Gloria would be delighted if that were the case, but I don't 
think this is an incommensurate lattice.  These actually don't so much 
give you diffuse scattering as little satellite spots near the main 
spots at spacings that don't make any sense given the lattice repeat. 
My understanding is that these arise from something that is slowly 
varying from unit cell to unit cell (could be as simple as a side chain 
waving back and forth) in a repetitive pattern that just doesn't line up 
in any way with the repeat of the unit cells. 

Still, I'll ask her.

However, I think that the difference is that modulated lattices are 
gradual changes of structure across many unit cells and what I was 
talking about is a more simplistic case of two different kinds of unit 
cells with varying degrees of randomness in their arrangement.  That is, 
using the formalism I described below, a modulated lattice would have 
unit cells that go: ABCDEFGHGFEDCBABCDEFG... etc. where A is not that 
different from B, B similar to C, etc., but A and H are very different.

-James Holton
MAD Scientist

Jürgen Bosch wrote:
 Hi James,

 what your descriptions aims at is I think shown in this publication
 Borgstahl, G. E. O. Incommensurate Crystallography by Sander van 
 Smaalen Crystallography reviews 14 , 259-260 (2008).

 Or am I misunderstanding something here ?

 Jürgen


 On 28 Jan 2009, at 12:39, James Holton wrote:

 I recommend you have a look at a book from OUP called Diffuse X-Ray
 Scattering and Models of Disorder by T. R. Welberry.  The first 
chapter
 explains quite well (I think) where all these streaky things come from.
 It will also make you feel better about having it when you see all the
 small molecule structures that have horrible diffuse scattering! (such
 as urea).

 This looks to me like a fairly classic case of correlated static
 disorder.  Best way to think about it is this:

 Imagine you have two different kinds of unit cells: A an B.  Doesn't
 really matter what the difference between A and B is, could be a
 two-headed side chain in conformer A vs conformer B, or it could be as
 complicated as a domain motion.  But, for simplicity, lets assume it is
 two rotamers of a side chain and also assume that each unit cell in 
your
 crystal can only be one or the other (no in betweens).

 Now, if the arrangement of these unit cells is perfectly correlated and
 an A always occurs right next door to a B along the c-axis (say),
 then what you really have is a bigger unit cell than you think.  That
 is, you can draw a unit cell around each A-B pair and call it a
 supercell with the contents of B as a simple NCS mate of A (with one
 side chain in a different rotamer).  Some people might call this a
 pseudotranslation.  The effect on the diffraction pattern in this 
case
 would be the appearance of a very weak spot in between each old spot
 along your c axis.  That is, your supercell is twice as big along
 c so the reciprocal-space lattice has twice as many spots in it.  The
 new spots are weak because they only correspond to the differences
 between A and B, which in this case is only a few atoms.

 Now lets say A and B are not perfectly correlated, but only slightly.
 That is, in some parts of the crystal A and B are side-by-side, but in
 other parts you get AAB, ABBA, BABBA, etc.  In each of these cases the
 supercell you must draw is 3, 4 and 5x your original unit cell.  Each
 of these will produce new weak spots with progressively tighter
 spacings.  As the supercell becomes very long

[ccp4bb] Buried Surface Area - summary

[Gloria Borgstahl]

Here was the query.What is the easiest way, these days, to calculate the buried surface area between two subunits of a protein? Here are the software and links received (thank you all):1.4+ votes for PISA - This following website is great and does a very nice job of analyzing protein interfaces (including, but not limited to, buried surface area calculations):http://www.ebi.ac.uk/msd-srv/prot_int/pistart.htmlFYI. the next website can also yield useful infoto figure out if an interface is physiologically relevant or not (less detailed output though):http://www.ebi.ac.uk/thornton-srv/databases/pita/2. surface ( http://www.ccp4.ac.uk/dist/html/surface.html ). 3. Two or three votes for areaimol in CCP4 works well - but beware that for cases we have worked on the default value for point density needs to be increased to get accurate values. With the subunits A and B and their surfaces surf(A) and surf(B) the equation should hold: surf(A) + surf(B) = surf(A+B) +2x where x is the surface between them. So if you calculate the surfaces of each subunit separately and of the complex that yields x. 4. NACCESS http://wolf.bms.umist.ac.uk/naccess/5. CCP4mg will do this - and show you graphically..http://www.ysbl.york.ac.uk/~ccp4mg/ccp4mg_help/analysis.html#sas6. see the discussion and references in: http://xray.bmc.uu.se/cgi-bin/gerard/reprint_mailer.pl?pref=847. Chimera.http://www.cgl.ucsf.edu/pipermail/chimera-users/2006-December/001131.htmlThanks again.**GloriaBorgstahlEppleyInstituteforCancerResearchandAlliedDiseases987696NebraskaMedicalCenter10732ALiedTransplantCenterOmaha,NE68198-7696http://sbl.unmc.eduOffice(402)559-8578FAX(402)559-3739AssociateProfessorInterests:Cancer,Biochemistry,DNAMetabolism,ProteinCrystallography,ModulatedCrystals,CrystalPerfection,X-rayTopography,Varley,Rowling,Avatar,BRAN,RAGBRAI,swimmingsoccer**

[ccp4bb] buried surface area

[Gloria Borgstahl]

Hello all,
What is the easiest way, these days, to calculate the buried surface area
between two subunits of a protein?
Thanks, and Happy Friday, Gloria**GloriaBorgstahlEppleyInstituteforCancerResearchandAlliedDiseases987696NebraskaMedicalCenter10732ALiedTransplantCenterOmaha,NE68198-7696http://sbl.unmc.eduOffice(402)559-8578FAX(402)559-3739AssociateProfessorInterests:Cancer,Biochemistry,DNAMetabolism,ProteinCrystallography,ModulatedCrystals,CrystalPerfection,X-rayTopography,Varley,Rowling,Avatar,BRAN,RAGBRAI,swimmingsoccer**