[ccp4bb] Nonlinearities and Thresholds

2022-03-10 Thread Jacob Keller
Dear Crystallographers,

this is a sort of abstract question, but I think this listserve might have
some good answers.

In crystallography, we have a lot of nonlinearities or thresholds in
dealing with data. For example, the "atomic resolution" threshold, wherever
it be, defines whether we can use direct methods. There's a sort of
mathematical edge or non-linearity there.

Or, data sets with completeness below some threshold become useless. I
think back to a number of James Holton's movies which vary certain
parameters in data quality, and there are often certain non-linearities,
like holes in rings, which unpredictably leap out of nowhere. And, most if
not all programs appear to have non-linear edges between working and not
working, say with MR and sequence similarity, etc.

For a totally non-crystallographic example which I think is actually
parallel: if one were to make a movie of a hummingbird with slow
temporal resolution, but then improve the temporal resolution
incrementally, at some point there would be a eureka moment in which the
blurry things which mysteriously keep the bird afloat are discovered to be
actual wings, which would be highly "non-linear."

So, the question: does anyone know what these sorts of thresholds are
called, or how to think of them philosophically or mathematically? Or even
better, but much harder: can these thresholds be predicted, and if so, how?

I think it's just a general science question, but with some clarity, would
be really helpful for deciding which techniques to emphasize or advocate
for, i.e., which ones are at the "bleeding edge" of discovery.

All the best,

Jacob

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

+



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Re: [ccp4bb] am I doing this right?

2021-10-26 Thread Jacob Keller
Hi All,

haven't been following CCP4BB for a while, then I come back to this juicy
Holtonian thread!

Sorry for being more practical, but could you use a windowed approach:
integrate values of the same pixel/relp combo (roxel?) over time (however
that works with frame slicing) to estimate the error over many frames, then
shrink the window incrementally, see whether the  successive
plotted shrinking-window values show a trend? Most likely flat for
background? This could be used for the spots as well as background. Would
this lose the precious temporal info?

Jacob

On Fri, Oct 22, 2021 at 3:25 AM Gergely Katona  wrote:

> Hi,
>
>
>
> I have more estimates to the same problem using a multinomial data
> distribution. I should have realized that for prediction, I do not have to
> deal with infinite likelihood of 0 trials when observing only 0s on an
> image. Whenever 0 photons generated by the latent process, the image is
> automatically empty. With this simplification, I still have to hide behind
> mathematical convenience and use Gamma prior for the latent Poisson
> process, but observing 0 counts just increments the beta parameter by 1
> compared to the prior belief. With equal photon capture probabilities, the
> mean counts are about 0.01 and the std is about 0.1 with
> rate≈Gamma(alpha=1, beta=0.1) prior . With a symmetric Dirichlet prior to
> the capture probabilities, the means appear unchanged, but the predicted
> stds starts high at very low concentration parameter and level off at high
> concentration parameter. This becomes more apparent at high photon counts
> (high alpha of Gamma distribution). The answer is different if we look at
> the std across the detector plane or across time of a single pixel.
>
> Details of the calculation below:
>
>
>
>
> https://colab.research.google.com/drive/1NK43_3r1rH5lBTDS2rzIFDFNWqFfekrZ?usp=sharing
>
>
>
> Best wishes,
>
>
>
> Gergely
>
>
>
> Gergely Katona, Professor, Chairman of the Chemistry Program Council
>
> Department of Chemistry and Molecular Biology, University of Gothenburg
>
> Box 462, 40530 Göteborg, Sweden
>
> Tel: +46-31-786-3959 / M: +46-70-912-3309 / Fax: +46-31-786-3910
>
> Web: http://katonalab.eu, Email: gergely.kat...@gu.se
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Nave,
> Colin (DLSLtd,RAL,LSCI)
> *Sent:* 21 October, 2021 19:21
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] am I doing this right?
>
>
>
> Congratulations to James for starting this interesting discussion.
>
>
>
> For those who are like me, nowhere near a black belt in statistics, the
> thread has included a number of distributions.  I have had to look up where
> these apply and investigate their properties.
>
> As an example,
>
> “The Poisson distribution is used to model the # of events in the future,
> Exponential distribution is used to predict the wait time until the very
> first event, and Gamma distribution is used to predict the wait time until
> the k-th event.”
>
> A useful calculator for distributions can be found at
>
> https://keisan.casio.com/menu/system/0540
>
> a specific example is at
>
> https://keisan.casio.com/exec/system/1180573179
>
> where cumulative probabilities for a Poisson distribution can be found
> given values for x and lambda.
>
>
>
> The most appropriate prior is another issue which has come up e.g. is a
> flat prior appropriate? I can see that a different prior would be
> appropriate for different areas of the detector (e.g. 1 pixel instead of
> 100 pixels) but the most appropriate prior seems a bit arbitrary to me. One
> of James’ examples was 10^5 background photons distributed among  10^6
> pixels – what is the most appropriate prior for this case? I presume it is
> OK to update the prior after each observation but I understand that it can
> create difficulties if not done properly.
>
>
>
> Being able to select the prior is sometimes seen as a strength of Bayesian
> methods. However, as a strong advocate of Bayesian methods once put it,
> this is a bit like Achilles boasting about his heel!
>
>
>
> I hope for some agreement among the black belts. It would be good to end
> up with some clarity about the most appropriate probability distributions
> and priors. Also, have we got clarity about the question being asked?
>
>
>
> Thanks to all for the interesting points.
>
>
>
> Colin
>
> *From:* CCP4 bulletin board  *On Behalf Of *Randy
> John Read
> *Sent:* 21 October 2021 13:23
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] am I doing this right?
>
>
>
> Hi Kay,
>
>
>
> No, I still think the answer should come out the same if you have good
> reason to believe that all the 100 pixels are equally likely to receive a
> photon (for instance because your knowledge of the geometry of the source
> and the detector says the difference in their positions is insignificant,
> i.e. part of your prior expectation). Unless the exact position of the spot
> where you detect the photon is relevant, detecting 1 photon on a big 

Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-21 Thread Jacob Keller
Can you kill the lights? Kill the engine? Kill a buzz? And did you know
kill means river in Dutch, I think it is? Lots of kills in upstate NY,
where I was young and easy under the apple boughs.

I would suggest, generally, some small molecule dependent protein to add to
the genome (recombinase maybe?) Maybe it could be light dependent, then we
would show the former US president's genius and foresight.

On Fri, Feb 19, 2021 at 2:17 PM Tim Gruene  wrote:

> Dear Jacob,
>
> you cannot kill a virus. It is not alive, but a complex chemical
> compound that interferes with the chemistry of the host. So why don't
> you work on the part of your conept over the week-end and present the
> concept?
>
> Cheers,
> Tim
>
>
> On Fri, 19 Feb 2021 12:55:32 -0500 Jacob Keller
>  wrote:
>
> > I don't think seeing the big picture resolves, or even addresses, the
> > question of possibly using a live vaccine. Some big-picture
> > considerations favor each side.
> >
> > The concern of mutation is a grave one, and an unknown. I would point
> > out, however, that the same considerations apply to the wild virus
> > currently scourging the planet (well, and every other virus currently
> > slinking around the biome). The distinction would be, I guess, that
> > we would be actively contributing in some way. On the other hand,
> > maybe being passive is like not throwing a rope to a drowning man?
> >
> > Maybe having a "v-day" would address this: introduce the
> > virus-vaccine at well-chosen locations, aka super-spreader events,
> > which, like well-placed demolition dynamite, would cause a "flash
> > pan-infection." Funnily, this would require all of the pandemic rules
> > to be turned on their heads! Presumably this generates herd immunity
> > within a couple of weeks, as well as its fair share of adverse
> > reactions and deaths. As a safety measure, have two orthogonal
> > chemical kill switches based on plentiful inexpensive well-tolerated
> > compounds, say a vitamin or pesticide (yes, pesticide, that stuff
> > that's always sprayed all over your food). Use those to quench the
> > vaccine before mutation, say 6-8 wks. Then, back to normal life, and
> > start honing similar tools for coming pandemics, a "holohomoimmune
> > system."
> >
> > Here's a question to the informed-consent hawks: would exposing
> > everybody to the virus while providing two compounds to block
> > completely the effects be considered a valid opt-in/out? Or what if
> > the default was switched, such that both compounds were required for
> > infection, and therefore one had to actively opt in?
> >
> > I don't know--it seems that many of the objections to the idea are
> > based on the bioethical concept "first do no harm," but that
> > principle is not necessarily adopted in all cases.
> >
> > One of the best things I learned in med school:
> >
> > Medicine is, fundamentally, "uncertainty management:"
> >
> > there are almost never any certainties in medicine, and one has to
> > use a Bayesian framework and a few bioethical principles to figure
> > out what to do next.
> >
> > Anyway, rest assured, I have not yet ordered the primers for creating
> > such a virus-vaccine...
> >
> > All the best, and have a good weekend everyone,
> >
> > Jacob
> >
> >
> >
> > On Fri, Feb 19, 2021 at 5:54 AM Robbie Joosten
> >  wrote:
> >
> > > Hi Tim,
> > >
> > > Very good points. The big picture is hard to grasp and we end up
> > > taking political choices rather than anything else. I'm very glad
> > > that we can outsource these choices to others every four year here.
> > >
> > > Lockdowns may save lives in the here and now, but the global
> > > economic damage makes life for others much harder to a point that
> > > it may actually kill them. Economic decline in the First World may
> > > be something with which that we can deal but, like viruses, it
> > > blows over to other parts of the world where economic growth is the
> > > real life saver. Does the prolonging of a reasonably measurable
> > > number well-lived lives in the West outweigh the extinguishing of a
> > > hard-to-assess number of much younger lives in the rest of the
> > > world? I'm glad I don't have to make that call.
> > >
> > > Cheers,
> > > Robbie
> > >
> > > > -Original Message-
> > > > From: CCP4 bulletin board  On Behalf Of Tim
> > > > Gruene
> > > > Sent: Friday, Fe

Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-19 Thread Jacob Keller
us to
> > > Australia, which is 90-99% fatal for European rabbits, but less lethal
> > > for the native rabbits. They intentionally released this virus and in
> > > the first year the mortality rate was 99.8% for the European rabbits.
> > > Yay, right??? Unfortunately, in the subsequent year the mortality rate
> > > fell to 25% and steadily continued to fall until it was lower than the
> > > reproductive rate of the European rabbits. The host-virus interaction
> > > played itself out: less-virulent viruses arose and resistant rabbits
> > > were selected for.
> > >
> > > To me it seems unwise to assume a replication competent virus
> > > (engineered or not) would refrain from mutating and adapting upon
> > > release, especially over the time course that would be required to
> > > infect all 7 billion+ humans on this planet. To me, I feel our options
> > > are (1) reach herd immunity through natural infection and accept the
> > > preventable deaths of many millions of people or (2) continue with
> > > non-pharmaceutical interventions (mask wearing, distancing, etc) until
> > > we can vaccinate enough people to reach herd immunity and hopefully by
> > > that time we have robust testing and treatment options available for
> > > those who continue to fall ill after we reach herd immunity. We as
> > > humans did something amazing by producing multiple safe and effective
> > > vaccines in less than one year, and I would like us to continue trying
> > > to save as many lives as possible by employing these vaccines as
> > > widely as possible.
> > >
> > > Anyways, take care. I know the pandemic is hard on all of us.
> > >
> > > Best regards,
> > > Jessica
> > >
> > > On Thu, Feb 18, 2021 at 6:15 AM Patrick Shaw Stewart
> > >  wrote:
> > >
> > > > I agree with those who say that A and B are usually incompatible.
> > > >
> > > > If we're like
> > > > chickens-in-a-barn-that-have-been-infected-with-bird-flu, the virus
> > > > very rapidly becomes more virulent (hospital and care-home
> > > > infections?).  It's hard for a virus to infect your nose and throat
> > > > quickly, and then stop.
> > > >
> > > > In the medium term the herd will build up some immunity and then
> > > > we'll become more like wandering albatrosses: the virus has to keep
> > > > us on the move if it's going to get itself near another susceptible
> > > > host.
> > > >
> > > > IMO the way a *respiratory *virus tries to "have its cake and eat
> > > > it" - that is, get as much of both A and B as possible - is to
> > > > develop thermal sensitivity.  I.e. infect nose and throat but keep
> > > > out of lungs and brain :
> > > >
> > > > https://www.preprints.org/manuscript/202101.0389/v1
> > > >
> > > >
> > > >
> > > > Thanks, Patrick
> > > >
> > > >
> > > > On Wed, Feb 17, 2021 at 9:46 PM Edwin Pozharski
> > > >  wrote:
> > > >
> > > >> I guess for such vehicle to be "extremely contagious" (or
> > > >> contagious at all for that matter) it should be capable of rapidly
> > > >> multiplying inside the host, so that it outruns immune system
> > > >> mediated destruction for at least some time in order to be present
> > > >> in high enough concentration to effectively spread via aerosols.
> > > >> Given the range of immunodeficiencies present in any population,
> > > >> you are essentially guaranteed to kill at least some people whose
> > > >> immune system will not be able to cope with rapidly multiplying
> > > >> virus.  You can theoretically fine tune the lethality of such virus
> > > >> to make sure that number of people you thus murder will be less
> > > >> than those that die either in no vaccine or traditional vaccination
> > > >> scenario, but that would be ethical equivalent of that modern
> > > >> crypto fascist suggestion that we just have to take it easy until
> > > >> herd immunity is established, even though few million grandparents
> > > >> will die in the process while the rest of us enjoy indoor dining.
> > > >>
> > > >>
> > > >>
> > > >> On Wed, Feb 17, 2021 at 12:33 PM Jacob Keller
> &g

Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
I was under the impression that A,B,C are not strongly or intrinsically
coupled, for one, since there are so many varieties of viruses with so much
range of infectiousness and clinical severity. False impression?

B + C = vaccine

A + B + C = immunosysadmin pandemic updater virus. Patient 0 honors given
for each (yearly?) edition to curry political favor.

JPK


On Wed, Feb 17, 2021 at 3:03 PM Tim Gruene  wrote:

> Hi Jacob,
>
> how do you think this should be possible? In order to infect others,
> the virus particle needs to proliferate (that's the only thing it
> does). It profilerates by hijacking the machinery of one cell of your
> body and turn it into a virus factory. Your body does not like this
> abuse and kills the cell, and also tries to kill the virus particles.
> The virus does not make you sick, it only captures one cell. The
> reaction of your body to kick out the virus, and the cell that does not
> do it's job anymore, make you sick. A and B are mutually exclusive. B +
> C is named vaccine.
>
> Best,
> Tim
>
>
> On Wed, 17 Feb 2021 12:33:09 -0500 Jacob Keller
>  wrote:
>
> > It would seem to me that it should be possible to generate versions
> > of the Covid virus that would:
> >
> > A. be extremely contagious and yet
> > B. be clinically benign, and
> > C. confer immunity to the original covid virus.
> >
> > If, then, this virus could be released, with appropriate "kill switch"
> > safeguards built in, would this not solve the world's pandemic
> > problems? Is there any reason, practically, why this approach would
> > not be feasible?
> >
> > Maybe we don't really know enough to manipulate A, B, C yet?
> >
> > Or maybe it's too scary for primetime...nightmare bio-warfare
> > apocalypse?
> >
> > Has this sort of thing been done, or does it have a name?
> >
> > Jacob
>
>
>
> --
> --
> Tim Gruene
> Head of the Centre for X-ray Structure Analysis
> Faculty of Chemistry
> University of Vienna
>
> Phone: +43-1-4277-70202
>
> GPG Key ID = A46BEE1A
>


-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

+



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Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
You were right: I did go to medical school, but you were wrong that I was
not instructed about informed consent; I was, and came out thinking it is
not as simple as you seem to portray it. We discussed the topic quite a
bit, and there were all sorts of borderline cases, weird twists, and so on.
That was one thing I loved about medical school: real
clinical/epidemiological life is much more vivid, complex, and nuanced than
the rules we try to impose. So...

Under some conditions the rules have to change, like during wars or
pandemics. I am very sympathetic to the idea of informed consent and not
forcing people to do things, especially medically, but don't you agree that
my personal freedom from things like vaccination sometimes begins to impose
on others' freedoms from pandemics? Or mandatory mask-wearing...?

And by the way, we are still at the sci-fi stage here, so let's not get too
exercised!

JPK

On Wed, Feb 17, 2021 at 2:29 PM David Schuller  wrote:

> It was my understanding that you attended medical school. I am surprised
> that there was no instruction pertaining to the ethics of obtaining consent
> for human subject trials.
>
>
>
> On 2/17/21 2:09 PM, Jacob Keller wrote:
>
> But does it end better than the current best-seller "Smoldering Pandemic
> Paralyzes Human Race, Fuels Contention, Kills Millions, Drives the Rest
> Mad?"
>
> JPK
>
>
>
> On Wed, Feb 17, 2021 at 12:40 PM David Schuller 
> wrote:
>
>> I read that book. It does not end well.
>>
>>
>> On 2/17/21 12:33 PM, Jacob Keller wrote:
>>
>> It would seem to me that it should be possible to generate versions of
>> the Covid virus that would:
>>
>> A. be extremely contagious and yet
>> B. be clinically benign, and
>> C. confer immunity to the original covid virus.
>>
>> If, then, this virus could be released, with appropriate "kill switch"
>> safeguards built in, would this not solve the world's pandemic problems? Is
>> there any reason, practically, why this approach would not be feasible?
>>
>> Maybe we don't really know enough to manipulate A, B, C yet?
>>
>> Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?
>>
>> Has this sort of thing been done, or does it have a name?
>>
>> Jacob
>> --
>>
>> +
>>
>> Jacob Pearson Keller
>>
>> Assistant Professor
>>
>> Department of Pharmacology and Molecular Therapeutics
>>
>> Uniformed Services University
>>
>> 4301 Jones Bridge Road
>>
>> Bethesda MD 20814
>>
>> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>>
>> Cell: (301)592-7004
>>
>> +
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>>
>> --
>> ===
>> All Things Serve the Beam
>> ===
>>David J. Schuller
>>modern man in a post-modern world
>>MacCHESS, Cornell University
>>schul...@cornell.edu
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>
>
> --
>
> +
>
> Jacob Pearson Keller
>
> Assistant Professor
>
> Department of Pharmacology and Molecular Therapeutics
>
> Uniformed Services University
>
> 4301 Jones Bridge Road
>
> Bethesda MD 20814
>
> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>
> Cell: (301)592-7004
>
> +
>
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 

++

Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
Had to research on Wiki for your arcane pop culture reference, but okay,
you can call it the Keller Virus; but I want a credited performance, and I
want someone else to make the thing, and for it to cure cancer as well and
not wipe out the human race. Weird GFP-related artifacts would be okay
though.


   - Emma Thompson <https://en.wikipedia.org/wiki/Emma_Thompson> as Dr.
   Alice Krippin: The doctor who creates the cancer cure, she inadvertently
   brings mankind to the brink of extinction. Neville dubs the virus "the
   Krippin virus". Her performance is uncredited.


On Wed, Feb 17, 2021 at 2:15 PM Philip D. Jeffrey 
wrote:

> You're casting yourself in the Emma Thompson role for the remake of "I Am
> Legend" ?
>
> Phil
>
> --
> *From:* CCP4 bulletin board  on behalf of Jacob
> Keller 
> *Sent:* Wednesday, February 17, 2021 2:09 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"
>
> But does it end better than the current best-seller "Smoldering Pandemic
> Paralyzes Human Race, Fuels Contention, Kills Millions, Drives the Rest
> Mad?"
>
> JPK
>
>
>
> On Wed, Feb 17, 2021 at 12:40 PM David Schuller 
> wrote:
>
> I read that book. It does not end well.
>
>
> On 2/17/21 12:33 PM, Jacob Keller wrote:
>
> It would seem to me that it should be possible to generate versions of the
> Covid virus that would:
>
> A. be extremely contagious and yet
> B. be clinically benign, and
> C. confer immunity to the original covid virus.
>
> If, then, this virus could be released, with appropriate "kill switch"
> safeguards built in, would this not solve the world's pandemic problems? Is
> there any reason, practically, why this approach would not be feasible?
>
> Maybe we don't really know enough to manipulate A, B, C yet?
>
> Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?
>
> Has this sort of thing been done, or does it have a name?
>
> Jacob
> --
>
> +
>
> Jacob Pearson Keller
>
> Assistant Professor
>
> Department of Pharmacology and Molecular Therapeutics
>
> Uniformed Services University
>
> 4301 Jones Bridge Road
>
> Bethesda MD 20814
>
> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>
> Cell: (301)592-7004
>
> +
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>
>
> --
>
> +
>
> Jacob Pearson Keller
>
> Assistant Professor
>
> Department of Pharmacology and Molecular Therapeutics
>
> Uniformed Services University
>
> 4301 Jones Bridge Road
>
> Bethesda MD 20814
>
> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>
> Cell: (301)592-7004
>
> +
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

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Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
I was also thinking about vaccine distribution, how difficult it is.

The infectious vaccine is like lighting back-fires in forest fires.

One can certainly see why this would be instantly shot down by any pharma
CEO: all you need, theoretically, is enough for one infection, so negative
profit margin. (Unless  you charge $billions for that one infection)

JPK

On Wed, Feb 17, 2021 at 2:09 PM Emilia Arturo  wrote:

> I hoped to see a vaccine available that can be taken as a pill or a mist,
> which would avoid some issues that plague both distribution and vaccine
> hesitancy. A faster and broader distribution, obviously, would increase
> population immunity more rapidly, consequently reducing transmission
> dramatically.
>
> Emilia ("Emily") C Arturo
> Postdoctoral associate
> Laboratory of Dr. Erica Ollmann Saphire
> Division of Structural Biology & Infectious Diseases
> La Jolla Institute for Immunology, California USA
> Twitter @moonlighterturo @ljiresearch @EOSpahire
>
>
>
>
> On Wed, Feb 17, 2021 at 10:07 AM Quyen Hoang  wrote:
>
>>  I guess that one might need a way to avoid natural mutations that could
>> turn B into B’ or B".
>>
>> Quyen
>>
>> Quyen Hoang, PhD
>> Associate Professor of Biochemistry and Molecular Biology
>> Adjunct Associate Professor of Neurology
>> Primary Investigator of the Stark Neuroscience Research Institute
>> Indiana University School of Medicine
>> 635 Barnhill Drive, MS0013C
>> Indianapolis, IN, 46202
>> (317)274-4371
>> https://qqhoang.pages.iu.edu
>>
>> It would seem to me that it should be possible to generate versions of
>> the Covid virus that would:
>>
>> A. be extremely contagious and yet
>> B. be clinically benign, and
>> C. confer immunity to the original covid virus.
>>
>> If, then, this virus could be released, with appropriate "kill switch"
>> safeguards built in, would this not solve the world's pandemic problems? Is
>> there any reason, practically, why this approach would not be feasible?
>>
>> Maybe we don't really know enough to manipulate A, B, C yet?
>>
>> Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?
>>
>> Has this sort of thing been done, or does it have a name?
>>
>> Jacob
>> --
>>
>> +
>>
>> Jacob Pearson Keller
>>
>> Assistant Professor
>>
>> Department of Pharmacology and Molecular Therapeutics
>>
>> Uniformed Services University
>>
>> 4301 Jones Bridge Road
>>
>> Bethesda MD 20814
>>
>> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>>
>> Cell: (301)592-7004
>>
>> +
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>>
>> --
>> ===
>> All Things Serve the Beam
>> ===
>>David J. Schuller
>>modern man in a post-modern world
>>MacCHESS, Cornell University
>>schul...@cornell.edu
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

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Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
But does it end better than the current best-seller "Smoldering Pandemic
Paralyzes Human Race, Fuels Contention, Kills Millions, Drives the Rest
Mad?"

JPK



On Wed, Feb 17, 2021 at 12:40 PM David Schuller 
wrote:

> I read that book. It does not end well.
>
>
> On 2/17/21 12:33 PM, Jacob Keller wrote:
>
> It would seem to me that it should be possible to generate versions of the
> Covid virus that would:
>
> A. be extremely contagious and yet
> B. be clinically benign, and
> C. confer immunity to the original covid virus.
>
> If, then, this virus could be released, with appropriate "kill switch"
> safeguards built in, would this not solve the world's pandemic problems? Is
> there any reason, practically, why this approach would not be feasible?
>
> Maybe we don't really know enough to manipulate A, B, C yet?
>
> Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?
>
> Has this sort of thing been done, or does it have a name?
>
> Jacob
> --
>
> +
>
> Jacob Pearson Keller
>
> Assistant Professor
>
> Department of Pharmacology and Molecular Therapeutics
>
> Uniformed Services University
>
> 4301 Jones Bridge Road
>
> Bethesda MD 20814
>
> jacob.kel...@usuhs.edu; jacobpkel...@gmail.com
>
> Cell: (301)592-7004
>
> +
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

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[ccp4bb] Contagious, Self-Distributing "Vaccines?"

2021-02-17 Thread Jacob Keller
It would seem to me that it should be possible to generate versions of the
Covid virus that would:

A. be extremely contagious and yet
B. be clinically benign, and
C. confer immunity to the original covid virus.

If, then, this virus could be released, with appropriate "kill switch"
safeguards built in, would this not solve the world's pandemic problems? Is
there any reason, practically, why this approach would not be feasible?

Maybe we don't really know enough to manipulate A, B, C yet?

Or maybe it's too scary for primetime...nightmare bio-warfare apocalypse?

Has this sort of thing been done, or does it have a name?

Jacob
-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

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Re: [ccp4bb] 3D Printer Model of the Coronavirus

2021-01-29 Thread Jacob Keller
Is it infectious?

JPK

On Fri, Jan 29, 2021 at 2:14 AM Dale Tronrud  wrote:

>
> Sometimes when explaining something, or even in private
> contemplation, it is nice to have a physical object as a point of focus.
>   We, at the Corovavirus Structure Task Force, have created the design
> files that allow you to 3D print a model of the Coronavirus.  Our goal
> was to create a visual aid which accurately reflects the structure of
> the virus while still being easy to print and assemble.  I've attached
> photos of three models painted with differing color schemes.
>
> The model is comprised of two halves for the body (separated to
> allow printing w/o support) and 100 spikes which can be printed in
> batches of 25. The assembled model has a 15 cm diameter which results
> from a magnification of 1 million fold.  (1 mm:1 nm).
>
> You can read a description of the building process at
>
> https://insidecorona.net/3d-print-corona/
>
> and download the files at
>
> https://www.thingiverse.com/thing:4543692/files
>
> Give it a try.  We'd love to hear your stories of the build and how
> you've used the models!
>
> Dale Tronrud
> Coronavius Structure Task Force
> https://insidecorona.net
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
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>


-- 

+

Jacob Pearson Keller

Assistant Professor

Department of Pharmacology and Molecular Therapeutics

Uniformed Services University

4301 Jones Bridge Road

Bethesda MD 20814

jacob.kel...@usuhs.edu; jacobpkel...@gmail.com

Cell: (301)592-7004

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Re: [ccp4bb] MAX IV Life Science Director position

2013-06-03 Thread Jacob Keller
Brighter than XFEL? Or is it going to be an XFEL?

JPK


On Mon, Jun 3, 2013 at 10:36 AM, Derek Logan derek.lo...@biochemistry.lu.se
 wrote:

  Hi,

  If anyone fancies becoming Life Science Director at the world's brightest
 light source, look no further than here:

  http://www.lunduniversity.lu.se/o.o.i.s?id=24914Dnr=547454Type=E

  /Derek
   
 Derek Logan tel: +46 46 222 1443
 Associate Professor mob: +46 76 8585 707
 Dept. of Biochemistry and Structural Biology  www.cmps.lu.se
 Centre for Molecular Protein Science   www.maxlab.lu.se/node/307
 Lund University, Box 124, 221 00 Lund, Sweden








-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Strand distorsion and residue disconnectivity in pymol

2013-05-30 Thread Jacob Keller
Can't you just show both cartoon (smoothed) and sticks (not smoothed) for
the given area?

JPK

On Thu, May 30, 2013 at 11:06 AM, Jason Vertrees 
jason.vertr...@schrodinger.com wrote:

 Hi Donghui,

 Bernhard is correct: PyMOL flattens out secondary structure to produce
 more aesthetically appealing images. You can disable this for beta sheets
 and loops by typing:

 # disable smoothing of sheets

 set cartoon_flat_sheets, 0


 # disable smoothing of loops

 set cartoon_smooth_loops, 0


 The cartoon_sidechain_helper setting automatically modulates these
 settings. If for some reason you need to disable cartoon_sidechain_helper
 you can imitate the look with:

 hide
 show cartoon
 show sticks, not (n. C+CA+O+N) extend 1
 set cartoon_smooth_loops, 0
 set cartoon_flat_sheets, 0

 Again as Bernhard noted, smoothing representations adjusts the
 representations' positions in space; therefore, you have the option of
 being positionally correct or aesthetically more pleasing.

 Cheers,

 -- Jason




 On Wed, May 29, 2013 at 10:29 PM, wu donghui wdh0...@gmail.com wrote:

 Dear all,

 I found a problem when I use pymol to prepare structure interface. Strand
 is distorted when residue from the strand is connected to the strand by
 turning on side_chain_helper on. However when side_chain_helper is off,
 the strand turns to normal shape but the residue from it is disconnected to
 the strand. I attached the picture for your help. I know there must be some
 tricks for this. Welcome for any input. Thanks a lot.

 Best,

 Donghui




 --
 Jason Vertrees, PhD
 Director of Core Modeling Products
 Schrödinger, Inc.

 (e) jason.vertr...@schrodinger.com
 (o) +1 (603) 374-7120




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] a new version of XDS

2013-05-29 Thread Jacob Keller
Any thoughts of making a Windows executable? Might help a lot of users

JPK


On Wed, May 29, 2013 at 4:20 PM, Kay Diederichs 
kay.diederi...@uni-konstanz.de wrote:

 ... is available for academic users at http://homes.mpimf-heidelberg.**
 mpg.de/~kabsch/xds/ http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds/
 Please note that there are some incompatibilities; most notably, the new
 format of XPARM.XDS is different so that the new INTEGRATE does not work
 with an old XPARM.XDS.

 best,

 Kay




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] a new version of XDS

2013-05-29 Thread Jacob Keller
I use Cygwin normally, which seems to work fine for most things...

Jacob


On Wed, May 29, 2013 at 5:08 PM, Jim Fairman fairman@gmail.com wrote:

 You can always use VMWare player to run a virtual machine of a Linux
 distribution inside Windows.  It's free and it works fairly well.


 On Wed, May 29, 2013 at 1:29 PM, Jacob Keller 
 j-kell...@fsm.northwestern.edu wrote:

 Any thoughts of making a Windows executable? Might help a lot of users

 JPK


 On Wed, May 29, 2013 at 4:20 PM, Kay Diederichs 
 kay.diederi...@uni-konstanz.de wrote:

 ... is available for academic users at http://homes.mpimf-heidelberg.**
 mpg.de/~kabsch/xds/ http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds/
 Please note that there are some incompatibilities; most notably, the new
 format of XPARM.XDS is different so that the new INTEGRATE does not work
 with an old XPARM.XDS.

 best,

 Kay




 --
 ***

 Jacob Pearson Keller, PhD

 Looger Lab/HHMI Janelia Farms Research Campus

 19700 Helix Dr, Ashburn, VA 20147

 email: kell...@janelia.hhmi.org

 ***




 --
 Jim Fairman, Ph D.
 Crystal Core Leader I
 Emerald BioStructures http://www.emeraldbiostructures.com/
 Tel: 206-780-8914
 Cell: 240-479-6575
 E-mail: fairman@gmail.com jfair...@embios.com




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Call for Proposals: IMAGINE, a New Neutron Crystallography Diffractometer

2013-05-23 Thread Jacob Keller
Is this Laue diffraction in the sense that the neutrons are a spectrum of
energies, or does Laue mean something else here?

Jacob


On Thu, May 23, 2013 at 3:53 PM, Meilleur, Flora meille...@ornl.gov wrote:

 IMAGINE, Neutron Crystallography Diffractometer

 High Flux Isotope Reactor (HFIR), Oak Ridge National Laboratory

 *Call for proposals for experiments anticipated to run from August
 through December 2013*

 ** **

 You are invited to apply for beam time on the neutron quasi-Laue
 diffractometer IMAGINE, at the High Flux Isotope Reactor. Proposal will be
 accepted via the web-based proposal system until NOON Wednesday, July 31,
 2013.

 This call is for experiments anticipated to run from August through
 December 2013.

 ** **

 Please see the attached flyer for additional information. For technical
 information about the capabilities of IMAGINE go to
 neutrons.ornl.gov/imagine/ or contact Flora Meilleur, meille...@ornl.gov,
 or Andrey Kovalevsky, kovalevsk...@ornl.gov.

 ** **

 Flora Meilleur, Ph. D

 Assistant Professor, Molecular and Structural Biochemistry

 North Carolina State University

 IMAGINE lead scientist, Neutron Sciences Directorate

 Oak Ridge National Laboratory

 Phone: 865-242-5747 

 ** **




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Why the name aimless

2013-05-02 Thread Jacob Keller
Moreorless?

Dolores?

JPK


On Thu, May 2, 2013 at 12:22 PM, Eric Williams ericwilli...@pobox.comwrote:

 I've always thought gormless is a woefully underused word.

 Eric


 On Thu, May 2, 2013 at 11:53 AM, Mark van Raaij mjvanra...@cnb.csic.eswrote:

 i.e. the next program will be Graceless or Feckless?

 On 2 May 2013, at 12:10, Phil Evans wrote:

  Reference:
  Gibbons, S. (1932) Cold Comfort Farm, Longmans, London
 
  On 2 May 2013, at 11:07, Roberto Battistutta 
 roberto.battistu...@unipd.it wrote:
 
  Hi everyone,
  just a curiosity, why the name aimless for the recent data reduction
 and analysis program in CCP4? You know, my students are curious ...
  Thank you,
  Roberto.
 
 
  Roberto Battistutta
  Associate Professor
  Department of Chemistry
  University of Padua
  via Marzolo 1, 35131 Padova - ITALY
  tel. +39.049.827.5262
  fax. +39.049.827.5829
  roberto.battistu...@unipd.it
  www.chimica.unipd.it/roberto.battistutta/
  VIMM (Venetian Institute of Molecular Medicine)
  via Orus 2, 35129 Padova - ITALY
  tel. +39.049.7923.236
  fax +39.049.7923.250
  www.vimm.it





-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Why the name aimless

2013-05-02 Thread Jacob Keller
Nevertheless?

JPK


On Thu, May 2, 2013 at 2:18 PM, Roger Rowlett rrowl...@colgate.edu wrote:

  Some of my undergraduate students need the CCP4 program feckless.

 :)
 ___
 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 5/2/2013 12:22 PM, Eric Williams wrote:

 I've always thought gormless is a woefully underused word.

  Eric

 On Thu, May 2, 2013 at 11:53 AM, Mark van Raaij mjvanra...@cnb.csic.eswrote:

 i.e. the next program will be Graceless or Feckless?

 On 2 May 2013, at 12:10, Phil Evans wrote:

  Reference:
  Gibbons, S. (1932) Cold Comfort Farm, Longmans, London
 
  On 2 May 2013, at 11:07, Roberto Battistutta 
 roberto.battistu...@unipd.it wrote:
 
  Hi everyone,
  just a curiosity, why the name aimless for the recent data reduction
 and analysis program in CCP4? You know, my students are curious ...
  Thank you,
  Roberto.
 
 
  Roberto Battistutta
  Associate Professor
  Department of Chemistry
  University of Padua
  via Marzolo 1, 35131 Padova - ITALY
  tel. +39.049.827.5262
  fax. +39.049.827.5829
  roberto.battistu...@unipd.it
  www.chimica.unipd.it/roberto.battistutta/
  VIMM (Venetian Institute of Molecular Medicine)
  via Orus 2, 35129 Padova - ITALY
  tel. +39.049.7923.236
  fax +39.049.7923.250
  www.vimm.it






-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Philosophical question

2013-03-19 Thread Jacob Keller
Never one to shrink from philosophizing, I wonder generally why the codon
conventions are the way they are? Is it like the QWERTY keyboard--basically
an historical accident--or is there some more beautiful reason? One might
argue that since basically all organisms share the convention (are there
exceptions, even?), that it must be the best of all possible conventions.
I have often wondered whether maybe this particular convention allows for
the most effective pathways between proteins of significant function, e.g.,
through the fewest mutations perhaps? One certainly cannot maintain that
every possible protein sequence has been made at some time or another in
the history of the biological world (go quantitate!) so there must be a way
to ensure that mostly the best ones got made. On the other hand, since
many organisms share DNA, maybe they had to agree on a system (I think
this is the dogma?). Was there a United Organisms convention at some
point, reminiscent of Les Immortels of the French language or POSIX or
something, to ensure compliance? What was the penalty for non-compliance?

Anyway, I like the question about the methionines,

Jacob

On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry ber...@upstate.edu wrote:

 Opher Gileadi wrote:

 Hi Theresa,

 To add to Anat's comments: Although the AUG codon for the first
 methionine and all other methionines in a protein coding sequence look the
 same, they are read in a very different way by the ribosomal machinery. The
 first AUG is recognized by the initiation complex, which includes the
 separate small ribosomal subunit (40s), a special tRNA-methionine, and
 initiation factors (proteins) including eIF2. This leads to assembly of a
 complete ribosome and initiation of protein synthesis. Subsequently, in the
 process of elongation, AUG codons are read by a different tRNA, which is
 brought to the 80s ribosome bound to a protein called elongation factor 1a.
 This is an oversimplification, of course, but the point is that the
 initiation codon (=the first amino acid to be incorporated to the protein)
 is read by a special tRNA, hence the universal use of methionine.

 Opher

  Yes, but why methionine? Half the time it has to be removed by
 N-terminal peptidase to give a small first residue, or by leader sequence
 processing. Why use a big expensive amino acid instead of choosing one of
 the glycine codons? Is there an obvious reason, or just it had to be
 something, and Met happened to get selected?

 And why sometimes alternate start codons can be used? and why doesn't
 initiation occur also at methionines in the middle of proteins? I'm
 guessing it has to do with 5' untranslated region and ribosome binding
 sites. So could the start codon actually be anything you want, provided
 there is a strong ribosome binding site there?

 Just being philosophical, and not afraid to display my ignorance,
 eab




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Philosophical question

2013-03-19 Thread Jacob Keller
I never said QWERTY just happened-- I said it was an accident of
history, based on the belief that some people nowadays have stopped using
manual typewriters, and they nevertheless still use the QWERTY keyboard.
I.e., because of the way history unfolded, we are now locked into using a
non-ideal keyboard configuration. I am dubious whether this model, however,
would apply to the codon conventions.

Jacob

On Tue, Mar 19, 2013 at 10:44 AM, David Schuller dj...@cornell.edu wrote:

  On 03/19/13 10:34, Jacob Keller wrote:

 Never one to shrink from philosophizing, I wonder generally why the codon
 conventions are the way they are? Is it like the QWERTY keyboard--basically
 an historical accident-


 QWERTY didn't just happen. It was designed. Don't kids today know how to
 use Wikipedia or Google?

 http://en.wikipedia.org/wiki/QWERTY

 Still used to this day, the QWERTY layout was devised and created in the
 early 1870s by Christopher Latham 
 Sholeshttp://en.wikipedia.org/wiki/Christopher_Latham_Sholes,
 a newspaper http://en.wikipedia.org/wiki/Newspaper editor and printer
 who lived in Milwaukee http://en.wikipedia.org/wiki/Milwaukee...
 The solution was to place commonly used letter-pairs (like th or st)
 so that their typebars were not neighboring, avoiding jams. Contrary to
 popular belief, the QWERTY layout was not designed to slow the typist down,
 [5] http://en.wikipedia.org/wiki/QWERTY#cite_note-5, but rather to
 speed up typing by preventing 
 jams.http://en.wikipedia.org/wiki/QWERTY#cite_note-why-4
 

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




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Strange density in solvent channel and high Rfree

2013-03-19 Thread Jacob Keller
Isn't lowering the symmetry equivalent to using multiple
models/conformations for one map? I remember seeing this done with the
infamous MSBA structure from a few years ago, so caveat emptor I guess. And
further, wouldn't using strict NCS make things equivalent to the
higher-symmetry space group? And then violating the NCS in places would
then just be equivalent to modelling multiple conformations, no?

JPK

On Tue, Mar 19, 2013 at 11:34 AM, Phoebe A. Rice pr...@uchicago.edu wrote:

 Hi Zbyszek,
   If the issue is perfect twinning, I agree - good point!
   But you don't want to confuse people who simply have
 nearly-but-not-quite crystallographic symmetry (OK, I'm being a bit
 pedagogical here, but a lot of newbies read the BB).  We had a case of P31
 that was so close to P61 we actually solved the molecular replacement
 problem in P61, then expanded it back and re-rigid-bodied it.  We've played
 similar games with translational pseudo-symmetry (ignoring the weak spots
 at first).  In cases like that it is important to properly reprocess the
 data in the lower symmetry space group (or smaller unit cell) because there
 is real information in those small differences.  However, the point about
 Rfree holds for twinning or rotational pseudo-symmetry: the Rfree flags
 should be expanded by the xtal symmetry operators, not re-picked in the
 lower symmetry space group.
Phoebe

 ++

 Phoebe A. Rice
 Dept. of Biochemistry  Molecular Biology
 The University of Chicago
 773 834 1723; pr...@uchicago.edu
 http://bmb.bsd.uchicago.edu/Faculty_and_Research/
 http://www.rsc.org/shop/books/2008/9780854042722.asp

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek
 Otwinowski [zbys...@work.swmed.edu]
 Sent: Tuesday, March 19, 2013 9:37 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

 It is a clear-cut case of crystal packing disorder. The tell-tale sign is
 that data can be merged in the higher-symmetry lattice, while the number
 of molecules in the asymmetric unit (3 in P21) is not divisible by the
 higher symmetry factor (2, by going from P21 to P21212).
 From my experience, this is more likely a case of order-disorder than
 merohedral twinning. The difference between these two is that structure
 factors are added for the alternative conformations in the case of
 order-disorder, while intensities (structure factors squared) are added in
 the case of merohedral twinning.

 Now an important comment on how to proceed in the cases where data can be
 merged in a higher symmetry, but the structure needs to be solved in a
 lower symmetry due to a disorder.

 !Such data needs to be merged in the higher symmetry,assigned R-free flag,
 and THEN expanded to the lower symmetry. Reprocessing the data in a lower
 symmetry is an absolutely wrong procedure and it will artificially reduce
 R-free, as the new R-free flags will not follow data symmetry!

 Moreover, while this one is likely to be a case of order-disorder, and
 these are infrequent, reprocessing the data in a lower symmetry seems to
 be frequently abused, essentially in order to reduce R-free. Generally,
 when data CAN be merged in a higher symmetry, the only proper procedure in
 going to a lower-symmetry structure is by expanding these higher-symmetry
 data to a lower symmetry, and not by rescaling and merging the data in a
 lower symmetry.

 Zbyszek Otwinowski

  Dear all,
  We have solved the problem. Data processing in P1 looks better (six
  molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
  in
  ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
  of refinement (without put waters, ligands, etc.).
 
  Indeed, there were one more molecule in ASU, but the over-merged data in
  an orthorhombic lattice hid the correct solution.
 
  Thank you very much for all your suggestions, they were very important to
  solve this problem.
 
  Cheers,
 
  Andrey
 
  2013/3/15 Andrey Nascimento andreynascime...@gmail.com
 
  *Dear all,*
 
  *I have collected a good quality dataset of a protein with 64% of
  solvent
  in P 2 21 21 space group at 1.7A resolution with good statistical
  parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4;
  Complet.=93%
  Redun.=2.4, the overall values are better than last shell). The
  structure
  solution with molecular replacement goes well, the map quality at the
  protein chain is very good, but in the final of refinement, after
  addition
  of a lot of waters and other solvent molecules, TLS refinement, etc. ...
  the Rfree is a quite high yet, considering this resolution
  (1.77A).(Rfree=
  0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower
  symmetry space group (P21), but I got the same problem, and I tried all
  possible space groups for P222, but with other screw axis I can not even
  solve the structure.*
 
  *A 

Re: [ccp4bb] Philosophical question

2013-03-19 Thread Jacob Keller
I don't understand this argument, as it would apply equally to all features
of the theoretical LUCA (protein and DNA sequences, etc). To make it
logically sound, I think you have either to include some kind of super-high
boundary to getting to other possible conventions (you probably imply this)
or, as I have suggested, it may be a particularly good, if not the best,
solution (a global minimum, one might say). The first hypothesis is similar
to the QWERTY keyboard, which is cemented in place by many factors, whereas
the second is more survival of the fittest.

It should perhaps be noted parenthetically that prima facie the accident
of history QWERTY hypothesis is at variance with radical Darwinism.

JPK


On Tue, Mar 19, 2013 at 1:56 PM, David Waterman dgwater...@gmail.comwrote:

 I believe that the reason all organisms share the convention (more or
 less) is that it dates back to LUCA - the Last Universal Common Ancestor of
 all extant life. LUCA must have had the basic transcription and translation
 machinery that we now see somewhat divergently-evolved versions of in all
 cells. This does not answer why that particular convention was chosen,
 but it does count against the idea that it is the best possible system, or
 indeed should continue to be selected for (except that mutations to this
 machinery tend to be very much deleterious).

 -- David


 On 19 March 2013 14:34, Jacob Keller j-kell...@fsm.northwestern.eduwrote:

 Never one to shrink from philosophizing, I wonder generally why the codon
 conventions are the way they are? Is it like the QWERTY keyboard--basically
 an historical accident--or is there some more beautiful reason? One might
 argue that since basically all organisms share the convention (are there
 exceptions, even?), that it must be the best of all possible conventions.
 I have often wondered whether maybe this particular convention allows for
 the most effective pathways between proteins of significant function, e.g.,
 through the fewest mutations perhaps? One certainly cannot maintain that
 every possible protein sequence has been made at some time or another in
 the history of the biological world (go quantitate!) so there must be a way
 to ensure that mostly the best ones got made. On the other hand, since
 many organisms share DNA, maybe they had to agree on a system (I think
 this is the dogma?). Was there a United Organisms convention at some
 point, reminiscent of Les Immortels of the French language or POSIX or
 something, to ensure compliance? What was the penalty for non-compliance?

 Anyway, I like the question about the methionines,

 Jacob


 On Tue, Mar 19, 2013 at 9:46 AM, Edward A. Berry ber...@upstate.eduwrote:

 Opher Gileadi wrote:

 Hi Theresa,

 To add to Anat's comments: Although the AUG codon for the first
 methionine and all other methionines in a protein coding sequence look the
 same, they are read in a very different way by the ribosomal machinery. The
 first AUG is recognized by the initiation complex, which includes the
 separate small ribosomal subunit (40s), a special tRNA-methionine, and
 initiation factors (proteins) including eIF2. This leads to assembly of a
 complete ribosome and initiation of protein synthesis. Subsequently, in the
 process of elongation, AUG codons are read by a different tRNA, which is
 brought to the 80s ribosome bound to a protein called elongation factor 1a.
 This is an oversimplification, of course, but the point is that the
 initiation codon (=the first amino acid to be incorporated to the protein)
 is read by a special tRNA, hence the universal use of methionine.

 Opher

  Yes, but why methionine? Half the time it has to be removed by
 N-terminal peptidase to give a small first residue, or by leader sequence
 processing. Why use a big expensive amino acid instead of choosing one of
 the glycine codons? Is there an obvious reason, or just it had to be
 something, and Met happened to get selected?

 And why sometimes alternate start codons can be used? and why doesn't
 initiation occur also at methionines in the middle of proteins? I'm
 guessing it has to do with 5' untranslated region and ribosome binding
 sites. So could the start codon actually be anything you want, provided
 there is a strong ribosome binding site there?

 Just being philosophical, and not afraid to display my ignorance,
 eab




 --
 ***

 Jacob Pearson Keller, PhD

 Looger Lab/HHMI Janelia Farms Research Campus

 19700 Helix Dr, Ashburn, VA 20147

 email: kell...@janelia.hhmi.org

 ***





-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Philosophical question

2013-03-19 Thread Jacob Keller
I don't understand this argument, as it would apply equally to all features
of the theoretical LUCA

 No it won't.  Different features would have different tolerance levels to
 modifications.


Yes, this tolerance is the second (hidden or implicit) principle I
referred to. So you'd have to explain why the codon convention is so
intolerant/invariant relative to the other features--it seems to me that
either it is at an optimum or there is some big barrier holding it in
place. And you'd have to explain this without invoking interchange of DNA,
viruses, etc, as we're talking about a LUCA here, right? And you'll have to
make sure that whatever reason you invoke cannot be applied to other
features of this LUCA which are indeed seen to be variable.

JPK


***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


[ccp4bb] Most Abundant Eukaryotic Membrane Protein List?

2013-03-18 Thread Jacob Keller
Dear List,

Does anyone know of a source of quantification of membrane proteins in
garden-variety eukaryotic cell lines, e.g. HEK or HeLa cells (incidentally,
it just occurred to me that probably all HeLa cells are XX--seems right,
no?) I am looking for the highest-expressed, particularly, and definitely
want to include single-pass proteins as well.

Jacob

-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Most Abundant Eukaryotic Membrane Protein List?

2013-03-18 Thread Jacob Keller
I don't want to crystallize the protein--I have another reason

Jacob

On Mon, Mar 18, 2013 at 1:18 PM, Edward A. Berry ber...@upstate.edu wrote:

 In the mitocondrial inner membrane (which is very protein-dense) I believe
 the most abundant protein is the adenine nucleotide transporter. (e.g.
 pdb1OKC).  Single chain or homodimer, but apparently its not very easy to
 crystallize.


 Jacob Keller wrote:

 Dear List,

 Does anyone know of a source of quantification of membrane proteins in
 garden-variety eukaryotic cell lines, e.g. HEK or
 HeLa cells (incidentally, it just occurred to me that probably all HeLa
 cells are XX--seems right, no?) I am looking for
 the highest-expressed, particularly, and definitely want to include
 single-pass proteins as well.

 Jacob

 --
 *

 Jacob Pearson Keller, PhD

 Looger Lab/HHMI Janelia Farms Research Campus

 19700 Helix Dr, Ashburn, VA 20147

 email: kell...@janelia.hhmi.org 
 mailto:kell...@janelia.hhmi.**orgkell...@janelia.hhmi.org
 

 *





-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?

2013-03-15 Thread Jacob Keller
Well, wouldn't NCS be a parallel situation? I have heard, for example, that
the maps of viruses are considerably better at a given resolution than
monomeric proteins. So I would guess that someone has looked at this topic
in the case of NCS. Maybe high solvent content would be equivalent to
filling the solvent holes with equivalent proteins (assuming
(unrealistically) that the crystal diffract to the same resolution, since
the parameter ratio would be the same?

JPK

On Fri, Mar 15, 2013 at 9:27 AM, Guangyu Zhu g...@hwi.buffalo.edu wrote:

 Ian,

 Because it is same protein, the high thermal motion is likely caused by
 crystal packing, and should be corrected by TLS refinement. The B left over
 should be similar.

 Anyway, this is just a hypothetical question. I tried to make other things
 same and just compare resolution and d/p. But you can still find
 difference. So if 80% crystal diffract to 3.0A too, then d/p ratio is much
 higher than 3.0A 50% crystal, it must be a more accurate refinement. What
 if 80% crystal diffract to 3.1, 3.2A, or 3.3A? Or I change the question to:
 could d/p ratio compensate some resolution?

 Thanks!
 Guangyu

 From: Ian Tickle ianj...@gmail.com
 Date: Friday, March 15, 2013 6:33 AM
 To: System Administrator g...@hwi.buffalo.edu
 Cc: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk
 Subject: Re: [ccp4bb] Resolution and data/parameter ratio, which one is
 more important?


 Hi Guangyu,

 I think it's not as straightforward as comparing d/p ratios, that is only
 one of several factors that influences precision.  Another important factor
 would be the overall level of thermal motion  disorder which will most
 likely be significantly higher in the 3.6A 80% case; after all that's
 probably the reason that it only diffracts to 3.6A!

 All things considered I would go for the 3A form.

 Cheers

 -- Ian


 On 15 March 2013 00:27, Guangyu Zhu g...@hwi.buffalo.edu wrote:

 I have this question. For exmaple, a protein could be crystallized in two
 crystal forms. Two crystal form have same space group, and 1
 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the
 other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal
 must be 5/2=2.5 times larger because of higher solvent content. If both
 data collecte to same completeness (say 100%), 3.6A data actually have
 higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For
 refinement, better data/parameter should give more accurate structure, ie.
 3.6A data is better. But higher resolution should give a better resolved
 electron density map. So which crystal form really give a better (more
 reliable and accurate) protein structure?





-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] first use of synchrotron radiation in PX

2013-03-13 Thread Jacob Keller
Did anyone see this prescient line in the PNAS paper? Seems that the MAD
concept was suggested way back then...

JPK


While the enhancement of anomalous scattering
has not yet been examined in detail, it is in principle
possible to use data collected at three wavelengths (15) to
completely solve the phase problem. The synchrotron source
is uniquely suited for these applications.



On Wed, Mar 13, 2013 at 11:04 AM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote:

 Hi

 Not sure if this is strictly speaking the first protein *solved* on a
 synchrotron, but I think this is the first report of shooting protein
 crystals at a synchrotron in the widely available literature -

 http://www.pnas.org/content/73/1/128.full.pdf+html

 Phillips J C, Wlodawer A, Yevitz M M and Hodgson K 0 1976 Proc.
 Nat. Acad. Sci. USA 73 128-32

 Applications of synchrotron radiation to protein crystallography:
 Preliminary results


 On 13 Mar 2013, at 14:38, Alan Cheung wrote:

  Hi all - i'm sure this many will know this : when and what was the first
 protein structure solved on a synchrotron?
 
  Thanks in advance
  Alan
 
 
  --
  Alan Cheung
  Gene Center
  Ludwig-Maximilians-University
  Feodor-Lynen-Str. 25
  81377 Munich
  Germany
  Phone:  +49-89-2180-76845
  Fax:  +49-89-2180-76999
  E-mail: che...@lmb.uni-muenchen.de

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




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] validating ligand density

2013-03-12 Thread Jacob Keller

 One final quote that is not in the twilight paper summarizes it nicely:

 The scientist must be the judge of his own hypotheses, not the
 statistician.
  A.F.W. Edwards (1992) in Likelihood - An account of the statistical
 concept
 of likelihood and its application to scientific inference , p. 34.


There must be a lot of thinking behind this statement--while it seems
plausible, it seems far from proven prima facie. Also, it assumes that the
scientist is not a statistician.

Jacob








 Btw, the book is good reading.

 Best, BR

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Robbie
 Joosten
 Sent: Tuesday, March 12, 2013 10:03 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] validating ligand density

 Dear Srinivasan,

 Although the Twilight program can only look at deposited PDB entries, the
 tips about ligand validation in the paper are very useful. I suggest you
 start from there.
 You can use EDSTATS in CCP4 to get real-space validation scores. Also look
 at the difference map metrics it gives (and the maps themselves of course),
 they will tell you whether you misidentified your ligand. Occupancy
 refinement in Refmac can also help you: if the occupancy drops a lot
 something is wrong. That can be partial binding (not that much of a
 problem)
 or worse, a ligand that isn't there. By the way,  I've been playing with
 that recently and some ligands/hetero compounds in the PDB were so
 incredibly 'not there' that Refmac would crash (that bug seems to be fixed
 in the latest version).

 HTH,
 Robbie

  -Original Message-
  From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
  R.Srinivasan
  Sent: Monday, March 11, 2013 23:03
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] validating ligand density
 
  Hello all,
 
  We co-crystallized an inactive variant of our enzyme in
  the
 presence of
  substrate and have determined the structure at 1.85A.
 
  Now, we want to validate the fitting of the ligand into
  the
 electron
  density. We tried validating using the difference map (2Fo-Fc) after
 refining
  the structure without the ligand. But, it is still a bit inconclusive
  if
 the density
  fits the ligand.
 
  It would be very kind to know if there are tools for
 validating this
  electron density. We were excited about twilight but turns out it can
  only
 be
  used with deposited structure.
 
 
  We will appreciate your help and suggestions.
 
 
  Many thanks,
  Srinivasan




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] validating ligand density

2013-03-12 Thread Jacob Keller
Dear Jacob,

 You are overinterpreting, the statement is about judging, not proving a
 hypothesis. I am sure Mr. Edwards judged his statement to be ok.


I guess there is a good likelihood that you are right, but who am I to
judge?

JPK






 Herman

  --
 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
 *Jacob
 Keller
 *Sent:* Tuesday, March 12, 2013 3:44 PM

 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] validating ligand density

  One final quote that is not in the twilight paper summarizes it nicely:

 The scientist must be the judge of his own hypotheses, not the
 statistician.
  A.F.W. Edwards (1992) in Likelihood - An account of the statistical
 concept
 of likelihood and its application to scientific inference , p. 34.


 There must be a lot of thinking behind this statement--while it seems
 plausible, it seems far from proven prima facie. Also, it assumes that the
 scientist is not a statistician.

 Jacob








 Btw, the book is good reading.

 Best, BR

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Robbie
 Joosten
 Sent: Tuesday, March 12, 2013 10:03 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] validating ligand density

 Dear Srinivasan,

 Although the Twilight program can only look at deposited PDB entries, the
 tips about ligand validation in the paper are very useful. I suggest you
 start from there.
 You can use EDSTATS in CCP4 to get real-space validation scores. Also look
 at the difference map metrics it gives (and the maps themselves of
 course),
 they will tell you whether you misidentified your ligand. Occupancy
 refinement in Refmac can also help you: if the occupancy drops a lot
 something is wrong. That can be partial binding (not that much of a
 problem)
 or worse, a ligand that isn't there. By the way,  I've been playing with
 that recently and some ligands/hetero compounds in the PDB were so
 incredibly 'not there' that Refmac would crash (that bug seems to be fixed
 in the latest version).

 HTH,
 Robbie

  -Original Message-
  From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
  R.Srinivasan
  Sent: Monday, March 11, 2013 23:03
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] validating ligand density
 
  Hello all,
 
  We co-crystallized an inactive variant of our enzyme in
  the
 presence of
  substrate and have determined the structure at 1.85A.
 
  Now, we want to validate the fitting of the ligand into
  the
 electron
  density. We tried validating using the difference map (2Fo-Fc) after
 refining
  the structure without the ligand. But, it is still a bit inconclusive
  if
 the density
  fits the ligand.
 
  It would be very kind to know if there are tools for
 validating this
  electron density. We were excited about twilight but turns out it can
  only
 be
  used with deposited structure.
 
 
  We will appreciate your help and suggestions.
 
 
  Many thanks,
  Srinivasan




 --
 ***

 Jacob Pearson Keller, PhD

 Looger Lab/HHMI Janelia Farms Research Campus

 19700 Helix Dr, Ashburn, VA 20147

 email: kell...@janelia.hhmi.org

 ***




-- 
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.org

***


Re: [ccp4bb] Way off topic: Donate V. Cholera Genomic DNA?

2013-03-06 Thread Jacob Keller
Dear All, thanks very much for the various suggestions--I finally made the
connection. A good point was also made that gene synthesis is becoming ever
cheaper (~$0.28/bp) so maybe PCRing from genomic DNA is not always
advisable. I'll keep this in mind for the future, although for this one I
had my reasons for going for the gDNA.

Thanks again,

Jacob

On Tue, Mar 5, 2013 at 1:07 PM, Jacob Keller j-kell...@fsm.northwestern.edu
 wrote:

 Dear List,

 please pardon the major off-topicity, but I need a trace amount of V
 cholera genomic DNA to PCR from, and it just kills me to spend ~$300 to
 order it for this one and only use. Is anyone in the DC/Baltimore area
 willing and able to donate this? I travel on the capitol beltway daily, so
 anything nearby would work, esp. NIH.

 Jacob

 --
 ***
 Jacob Pearson Keller, PhD
 Postdoctoral Associate
 HHMI Janelia Farms Research Campus
 email: k j-kell...@northwestern.eduell...@janelia.hhmi.org
 ***




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: k j-kell...@northwestern.eduell...@janelia.hhmi.org
***


[ccp4bb] Way off topic: Donate V. Cholera Genomic DNA?

2013-03-05 Thread Jacob Keller
Dear List,

please pardon the major off-topicity, but I need a trace amount of V
cholera genomic DNA to PCR from, and it just kills me to spend ~$300 to
order it for this one and only use. Is anyone in the DC/Baltimore area
willing and able to donate this? I travel on the capitol beltway daily, so
anything nearby would work, esp. NIH.

Jacob

-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: k j-kell...@northwestern.eduell...@janelia.hhmi.org
***


Re: [ccp4bb] How to compare B-factors between structures?

2013-03-04 Thread Jacob Keller
You only entertain addition+subtraction--why not use
multiplication/division to normalize the b-factors?

JPK

On Mon, Mar 4, 2013 at 2:04 PM, James Holton jmhol...@lbl.gov wrote:

 Formally, the best way to compare B factors in two structures with
 different average B is to add a constant to all the B factors in the low-B
 structure until the average B factor is the same in both structures.  Then
 you can compare apples to apples as it were.  The extra B being added
 is equivalent to blurring the more well-ordered map to make it match the
 less-ordered one. Subtracting a B factor from the less-ordered structure is
 sharpening, and the reason why you shouldn't do that here is because
 you'd be assuming that a sharpened map has just as much structural
 information as the better diffracting crystal, and that's obviously no true
 (not as many spots).   In reality, your comparison will always be limited
 by the worst-resolution data you have.

 Another reason to add rather than subtract a B factor is because B factors
 are not really linear with anything sensible.  Yes, B=50 is more
 disordered than B=25, but is it twice as disordered? That depends on
 what you mean by disorder, but no matter how you look at it, the answer
 is generally no.

 One way to define the degree of disorder is the volume swept out by the
 atom's nucleus as it vibrates (or otherwise varies from cell to cell).
  This is NOT proportional to the B-factor, but rather the 3/2 power of the
 B factor.   Yes, 3/2 power.  The value of B, is proportional to the
 SQUARE of the width of the probability distribution of the nucleus, so to
 get the volume of space swept out by it you have to take the square root to
 get something proportional the the width and then you take the 3rd power to
 get something proportional to the volume.

 An then, of course, if you want to talk about the electron cloud (which is
 what x-rays see) and not the nuclear position (which you can only see if
 you are a neutron person), then you have to add a B factor of about 8 to
 every atom to account for the intrinsic width of the electron cloud.
  Formally, the B factor is convoluted with the intrinsic atomic form
 factor, but a native B factor of 8 is pretty close for most atoms.

 For those of you who are interested in something more exact than
 proportional the equation for the nuclear probability distribution
 generated by a given B factor is:
 kernel_B(r) = (4*pi/B)^1.5*exp(-4*pi^2/B*r^**2)
 where r is the distance from the average position (aka the x-y-z
 coordinates in the PDB file).  Note that the width of this distribution of
 atomic positions is not really an error bar, it is a range.  There's a
 difference between an atom actually being located in a variety of places vs
 not knowing the centroid of all these locations.  Remember, you're
 averaging over trillions of unit cells.  If you collect a different dataset
 from a similar crystal and re-refine the structure the final x-y-z
 coordinate assigned to the atom will not change all that much.

   The full-width at half-maximum (FWHM) of this kernel_B distribution is:
  fwhm = 0.1325*sqrt(B)
 and the probability of finding the nucleus within this radius is actually
 only about 29%.  The radius that contains the nucleus half the time is
 about 1.3 times wider, or:
 r_half = 0.1731*sqrt(B)

 That is, for B=25, the atomic nucleus is within 0.87 A of its average
 position 50% of the time (a volume of 2.7 A^3).  Whereas for B=50, it is
 within 1.22 A 50% of the time (7.7 A^3).  Note that although B=50 is twice
 as big as B=25, the half-occupancy radius 0.87 A is not half as big as 1.22
 A, nor are the volumes 2.7 and 7.7 A^3 related by a factor of two.

 Why is this important for comparing two structures?   Since the B factor
 is non-linear with disorder, it is important to have a common reference
 point when comparing them.  If the low-B structure has two atoms with B=10
 and B=15 with average overall B=12, that might seem to be significant
 (almost a factor of two in the half-occupancy volume) but if the other
 structure has an average B factor of 80, then suddenly 78 vs 83 doesn't
 seem all that different (only a 10% change).  Basically, a difference that
 would be significant in a high-resolution structure is washed out by
 the overall crystallographic B factor of the low-resolution structure in
 this case.

 Whether or not a 10% difference is significant depends on how accurate
 you think your B factors are.  If you kick your coordinates (aka using
 noise in PDBSET) and re-refine, how much do the final B factors change?

 -James Holton
 MAD Scientist


 On 2/25/2013 12:08 PM, Yarrow Madrona wrote:

 Hello,

 Does anyone know a good method to compare B-factors between structures? I
 would like to compare mutants to a wild-type structure.

 For example, structure2 has a higher B-factor for residue X but how can I
 show that this is significant if the average B-factor is also higher?
 Thank you for your help.





-- 

Re: [ccp4bb] disulfide engineering

2013-02-28 Thread Jacob Keller
Along these lines, what reagents do people use to promote disuflide bonds,
i.e., the anti-DTT?

JPK

On Thu, Feb 28, 2013 at 2:06 AM, David Briggs drdavidcbri...@gmail.comwrote:

 You might want to try Disulfide by design

 http://cptweb.cpt.wayne.edu/DbD2/

 Cheers

 Dave
 On Feb 28, 2013 6:55 AM, Careina Edgooms careinaedgo...@yahoo.com
 wrote:

 Dear CCP4 members

 I wish to engineer a disulfide bond at the dimer interface of a protein I
 am working with. Does anyone know of any available software to assist with
 this?

 Best
 Careina




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] How to compare B-factors between structures?

2013-02-25 Thread Jacob Keller
Why not normalize each to its average b-factor? And...maybe they are not
significantly different in the first place?

JPK

On Mon, Feb 25, 2013 at 3:08 PM, Yarrow Madrona amadr...@uci.edu wrote:

 Hello,

 Does anyone know a good method to compare B-factors between structures? I
 would like to compare mutants to a wild-type structure.

 For example, structure2 has a higher B-factor for residue X but how can I
 show that this is significant if the average B-factor is also higher?
 Thank you for your help.


 --
 Yarrow Madrona

 Graduate Student
 Molecular Biology and Biochemistry Dept.
 University of California, Irvine
 Natural Sciences I, Rm 2403
 Irvine, CA 92697




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


[ccp4bb] Improving Homology Models

2013-02-20 Thread Jacob Keller
Dear Crystallographers,

it has been my experience that homology modelling programs get folds pretty
well, but sometimes the details are pretty obviously bad, like too-close
contacts. One might think that the modelling software would put in a sort
of polishing step, but they don't seem to. Is there any way to trick the
CCP4 or other software to fix these things, such as by simulated annealing
or otherwise, I guess without any weight on the [non-existent] structure
factors?

Thanks,

Jacob

-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] protein crystals or salt crystals

2013-02-08 Thread Jacob Keller
I'd have to disagree on that.  Protein crystals are fragile but not

 soft.  If your crystals are like gelatine it's unusual.  It has been
 demonstrated that elastic properties of protein crystals are similar to
 organic solids


Interesting--do you have a reference quickly on hand for those
measurements? I have always been somewhat curious about that

JPK



 Cheers,

 Ed.

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




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Off Topic- Cystine Detection

2013-02-07 Thread Jacob Keller
Even so, it should run somewhat differently, and probably visibly on
SDS-PAGE. Even a single phosphorylation changes mobility, and I think I
remember having seen intramolecular disulfides change mobility (anyone else
have experience on this?)

JPK

On Wed, Feb 6, 2013 at 10:33 PM, Yuri yuri.pom...@ufl.edu wrote:

 **

 The disulfide bond is intramolecular. I do not have reasons to believe it
 is cross linking my protein





 On Wed, 6 Feb 2013 22:16:36 -0500, Jacob Keller wrote:

 Couldn't you just run reducing/non-reducing SDS-PAGE lanes and see the
 difference?
 JPK

 On Wed, Feb 6, 2013 at 11:10 AM, Yuri Pompeu yuri.pom...@ufl.edu wrote:

 Dear All,
 I am trying to probe the existence of a disulfide bond on the surface of
 my protein.
 I have attempted Ellman´s and my results were not as clear as I would
 have hoped for.
 I am not a sulfur/cysteine chemist and would appreciate the advice on
 what experiments to try!
 Thanks a bunch
 YAP



 --
 ***
 Jacob Pearson Keller, PhD
 Postdoctoral Associate
 HHMI Janelia Farms Research Campus
 email: j-kell...@northwestern.edu
 ***



 --
 Yuri Pompeu




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Off Topic- Cystine Detection

2013-02-06 Thread Jacob Keller
Couldn't you just run reducing/non-reducing SDS-PAGE lanes and see the
difference?

JPK

On Wed, Feb 6, 2013 at 11:10 AM, Yuri Pompeu yuri.pom...@ufl.edu wrote:

 Dear All,
 I am trying to probe the existence of a disulfide bond on the surface of
 my protein.
 I have attempted Ellman´s and my results were not as clear as I would have
 hoped for.
 I am not a sulfur/cysteine chemist and would appreciate the advice on what
 experiments to try!
 Thanks a bunch
 YAP




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] need some suggestions for crystallization

2013-02-06 Thread Jacob Keller
Does that CA have a metal center (I think they all do)? If so, doesn't the
citrate compete for the metal?

JPK

On Mon, Feb 4, 2013 at 2:25 PM, Roger Rowlett rrowl...@colgate.edu wrote:

 Human carbonic anhdyrase II can be easily crystallized from 1.3 M sodium
 citrate/0.1 M TrisCl pH 8.5 at 10 mg/mL protein concentration. Crystals are
 P21 and easily diffract to beyond 2.0 A on a home source. We cryopreserve
 in ML + 30% glucose. Sulfonamide ligands are easy to soak into the crystals
 in a few minutes during cryopreservation, and provide teaching
 opportunities for protein-ligand model building and refinement.

 Cheers,

 __**_
 Roger 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 02/04/2013 12:31 PM, Jim Pflugrath wrote:

 A number of protein crystal recipes are available on the Rigaku web site:
 http://www.rigaku.com/**products/protein/recipeshttp://www.rigaku.com/products/protein/recipes

 Lysozyme is nice because it is so cheap, grows quickly, cryoprotectants
 in a straightforward way, and the unit cells are not large nor are the
 crystals fragile.  One can get triclinic, monoclinic, orthorhombic, and
 tetragonal crystals relatively quickly.

 With hen egg white lysozyme if you are not up for sulfur-SAD phasing,
 then co-crystallizing or quick-soaking in iodide (say 100 mM) gives a very
 nice anomalous signal at just about any wavelength.  That is, no need to
 worry about going to a so-called edge.

 While there are many other easy-to-go crystals, I have found that none of
 them combine all the properties of hen egg white lysozyme has a good
 teaching tool.

 Jim

 ==
 Can you all suggest some protein crystallization conditions (along with
 cryo conditions) for some commercially available proteins?  I'm looking
 to get 6-8 different ones (and we'll just take them and see how it
 goes).  I wouldn't mind knowing unit cell parameters as well (just a
 citation works, I can have them figure it out).  I have about 7 weeks to
 get everything grown and frozen and ready to go.

 Any help would be greatly appreciated.  It always amazes me how helpful
 this group is.  Thank you very much.

 Dave




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***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


[ccp4bb] Crystallography Near Absolute Zero?

2013-01-23 Thread Jacob Keller
Is anyone aware of any datasets taken at near absolute zero? I was
wondering what would happen...

Jacob

-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Golden Jubilee of Ramachandran Plot

2013-01-19 Thread Jacob Keller
Were there really no computers in 1963?

JPK

On Fri, Jan 18, 2013 at 9:51 AM, David Schuller dj...@cornell.edu wrote:


 http://eventheodd.blogspot.in/2013/01/golden-jubilee-of-ramachandran-plot.html

 Golden Jubilee of Ramachandran 
 Plothttp://eventheodd.blogspot.in/2013/01/golden-jubilee-of-ramachandran-plot.html

 Exactly fifty years from now i.e. in the year 1963, G. N. Ramachandran
 et. al published breakthrough original research in Journal of Molecular
 Biology. Ramachandran plot still remains a touchstone for protein form and
 structure (example, in validating a homology models). This plot is
 remarkable because it came ahead of time (it was proposed in 1963 when
 there were no computers, mechanical calculators were the cutting edge of
 technology)
 ...

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




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition

2013-01-05 Thread Jacob Keller
I have always wondered what the contribution is from the pH-ing
counter-ions, because the buffers are always, e.g., Tris-Cl or
Na-HEPES. I have often thought it might be more ideal to screen with
TRIS-HEPES as the protein buffer, but probably over the years these
counter-ions have fortuitously aided crystallization, so maybe it's
not so bad. Also, proteins often have salt in the protein stock as
well. Bottom line is that I wonder whether in Cale's case it might
have been the Na or the Cl that was the key, and perhaps not the
hepes/tris.

Jacob Keller




-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Script for removing/gathering select rows of data

2012-11-09 Thread Jacob Keller
Dear all, thanks for your help--I made a very simple little script using
awk, and it works excellently (anyone is welcome to it, of course). Thanks
so much for taking the time.

Jacob





On Wed, Nov 7, 2012 at 9:49 PM, Jacob Keller j-kell...@fsm.northwestern.edu
 wrote:

 Dear Crystallographers,

 does anyone have a script on hand which can:

 -read in a tab-delimited dataset
 -determine whether each row in column X is above or below some input
 cutoff value Y,
 -outputs all such rows to a new file?

 if someone already has this on hand, I would appreciate it--seems like it
 should be a very generally useful script to have around, and I need such
 right now...

 Thanks very much,

 Jacob Keller




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Script for removing/gathering select rows of data

2012-11-07 Thread Jacob Keller
Dear Crystallographers,

does anyone have a script on hand which can:

-read in a tab-delimited dataset
-determine whether each row in column X is above or below some input cutoff
value Y,
-outputs all such rows to a new file?

if someone already has this on hand, I would appreciate it--seems like it
should be a very generally useful script to have around, and I need such
right now...

Thanks very much,

Jacob Keller


Re: [ccp4bb] On maps and doubts

2012-10-10 Thread Jacob Keller
btw, this thread has one of my favorite titles ever...

JPK

On Tue, Oct 9, 2012 at 4:11 AM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote:

 This is interesting.  In principle m and D should provide an optimum map,
 and at high resolution they do a reasonable job.
 The answer about occupancy is a good point.

 You don't say what resolution your data is at, but maybe it is rather low?
 I suspect that below ~ 3A the estimates of both m and D are somewhat
 unreliable - they are fitted to resolution curves and are influenced
 severely by scaling problems, all of which are more serious at low
 resolution.   What about consulting Garib! He must be near by..
 Eleanor
 On 4 Oct 2012, at 20:17, Israel Sanchez wrote:

  Hello everyone,
 
  I would like to share my experience with one dataset and request some
 advice on which is the best way to prove a conformational change seen in a
 density map.
 
  The first issue arose when we were looking for an extra ribosomal factor
 added to a crystalized ribosome. After careful data collection and
 refinement (I/sigma last shell 1.2, 3.1A and CC1/2 around 22%) the
 sigma-A-weighted maps 2mFo-DFc and Fo-Fc does not show any clear difference
 density that we could interpret as the expected factor. Interestingly, a
 computed map with coefficients 3mFo-2DFc started to show some features that
 clearly could be explained as a fragment of the factor. The density
 improved even more with a B-sharpened map. We have seen this behavior
 before and I was wondering if someone else is using this kind of maps and
 may could explain the reason behind this density improvement. Is it a crazy
 idea to go even higher like 4mFo-3DFc?
 
  The second query has to do with which is the best way to prove that a
 conformational change is present in an specific residue (in this case and
 RNA base) in your structure. To my knowledge, a classic omit map with
 simulated annealing would do the job regarding removing the model bias.
 Actually, I found an interesting alternative in PHENIX called a Kick map,
 were a series of maps computed from a ramdoinised set of models yields a
 averaged map ideally free from model bias. Does anyone has a preference for
 any of those schemes? Are there more alternative to prove a conformational
 change in a model phased with a molecular replacement solution?
 
  Thank you very much in advance.
  --
   Israel Sanchez Fernandez PhD
  Ramakrishnan-lab
  MRC Laboratory of Molecular Biology,
  Hills Road, Cambridge, CB2 0QH, UK
 
 




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Direct crystallization of Lysozyme from eggs

2012-09-25 Thread Jacob Keller
Alhtough perhaps a little more macabre--maybe you could crystallize
hemoglobin or myoglobin from blood or muscle (meat?). I recollect some
story of whale myo- or hemo-globin crystallizing on the salty decks of a
whaling ship...

Jacob



On Tue, Sep 25, 2012 at 11:59 AM, David Smith dsm...@ls-cat.org wrote:

 Dear crystallographers,

 I am working on an outreach program through the APS to get motivated high
 school students some/more research experience.  As I work at a
 crystallography beamline (LS-CAT), we are trying to get a
 crystallographically centered experiment in place that will be interesting
 to a high school student.  Among other ideas, I had mentioned to the
 teacher the possibility of crystallizing lysozyme directly from egg
 whites.  The teacher picked up on this idea as the one he and his students
 would like to pursue.  I have done a bit of reseach and I have found the
 Alderton and Fevold paper from '46 about direct crystallization from egg
 whites.  However I am unable to find any papers that refine or expand upon
 this experiment.  While there appears to be some literature about lysozyme
 and crystallizing the same, these papers do not use lysozyme directly from
 hen egg whites.  Do any of you know of any more recent papers or procedures
 that would be relevant or easily adapted to an high school environment?

 Our goal is to get diffraction quality crystals, expose the crystals at
 the beamline, and present a poster of the experience.

 Cheers and thanks,

 David Smith

 --
 David W Smith
 Research Scientist
 LS-CAT
 APS, Argonne IL
 W:(630)343-9811
 F:(630)252-4664




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Off-topic: Pore-forming Substances

2012-09-24 Thread Jacob Keller
Dear Crystallographers:

#off topic

I am trying to calibrate some microscopic imaging intracellular pH
measurements, but the usual nigericin (small pore-forming K+/H+ exchanger)
technique does not seem to be working well. Maybe it actually never works
well? Anyway, I was think of trying to use something which forms larger
pores to allow all things to equilibrate quickly, but not allow the bigger
pH-sensitive dye (SNARF) to escape? I don't care, by the way, if it kills
the cells, as the calibration is done it the end, provided that the cells
don't float away off the coverslip. Does anyone know of a candidate for
such?

JPK

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] off topic: protein peptide binding

2012-09-14 Thread Jacob Keller
For the gel, it might be really hard to see the peptide as small things
tend to blur terribly--better to look for shifts in the protein band. If I
were you, I would do serial dilutions of the peptide at some constant, very
visible protein concentration. Also you have to be sure that the
protein/peptide charges work--and you can run basic (positive) proteins on
native gel by simply switching the electrodes. Pay attention to what you
put in your loading buffer as well, as this may alter binding.

JPK

On Fri, Sep 14, 2012 at 8:46 AM, anita p crystals...@gmail.com wrote:

 Hi All,
 I wanted some advice regarding mapping out Protein-peptide interaction.
 The peptide is a 12 mer and the protein is 15kDa.
 Invivo studies suggest that the peptide is binds the protein and helps in
 transport.
 Hence I feel it would perhaps transient binding.
 I know that I should do ITC or BIAcore to show binding, but before going
 to those techniques, I feel, running a native gel would perhaps help. So
 the native gel can have lane1: protein, lane 2: peptide, lane 3:
 protein+peptide.

 If the protein binds to the peptide then I should not see a band
 corresponding to the peptide in lane 3.

 But before I start this experiment, I wonder if any body has run 12 mer
 peptide on native gel, How long should I run...  How much quantity of
 peptide I should for the gel.

 I wont be able to do western or pull-down, Equipment for native gel is
 available to me.
 kindly advice,
 regards
 Anita




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Off-topic: Best Scripting Language

2012-09-13 Thread Jacob Keller

 It turns out that the syntax and semantics of all reasonable programming
 languages are very similar, or fall into only a few classes (e.g. C-like,
 S-expressions, etc.), so once you are fluent in one from a class, it's
 easy to pick up the others. This can't be said of natural languages, which
 are full of idioms and grammatical exceptions, even in closely related
 dialects.


This is more opinions than I can shake a stick at! Don't we all have other
fish to fry (or for the French, other cats to whip? Other national
equivalents?) Anyway, I was nervous as a long-tailed cat in a room full of
rocking chairs to ask this question, and look at the Pandora's box that
this has opened!

Java anyone?! I've actually noticed a bit of a dearth of Java in the
crystallography world, with some exceptions...

Thanks everybody for your suggestions--I will mull them over, since you all
make such good arguments (not meant in the programming sense, but probably
that's true too!),

Jacob





 James


 [1]
 http://www.codinghorror.com/blog/2006/08/computer-languages-arent-human-languages.html
 [2] http://daringfireball.net/2005/09/englishlikeness_monster




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Off-topic: Best Scripting Language

2012-09-12 Thread Jacob Keller
Dear List,

since this probably comes up a lot in manipulation of pdb/reflection files
and so on, I was curious what people thought would be the best language for
the following: I have some huge (100s MB) tables of tab-delimited data on
which I would like to do some math (averaging, sigmas, simple arithmetic,
etc) as well as some sorting and rejecting. It can be done in Excel, but
this is exceedingly slow even in 64-bit, so I am looking to do it through
some scripting. Just as an example, a sort which takes 10 min in Excel
takes ~10 sec max with the unix command sort (seems crazy, no?). Any
suggestions?

Thanks, and sorry for being off-topic,

Jacob

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Off-topic: Best Scripting Language

2012-09-12 Thread Jacob Keller

 For the specific purpose you list -
 input from tab-delimited data
 output to simple statisitical summaries and (I assume) plots
 - it sounds like gnuplot could do the job nicely.


I wasn't aware that gnuplot can do calculations--can it? I was probably
going to use it somewhere as a plotting option.


 Otherwise I'd recommend perl, and dis-recommend python.


Why are you dis-ing python? Seems everybody loves it...

JPK





 Ethan


 --
 Ethan A Merritt
 Biomolecular Structure Center,  K-428 Health Sciences Bldg
 University of Washington, Seattle 98195-7742




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] the lysozyme of membrane proteins?

2012-09-11 Thread Jacob Keller
Wouldn't bacteriorhodopsin be a good choice, especially since it's colored
and easy to express? Seems pretty expensive for a class, though, with all
the detergents involved...

JPK

On Tue, Sep 11, 2012 at 4:18 PM, Ho Leung Ng h...@hawaii.edu wrote:

 Hello,

   I am developing an undergraduate biochemistry lab class and
 would like to incorporate experiments with membrane proteins. Does
 anyone have suggestions on membrane proteins that are relatively easy
 to express, purify, and assay? Bonus points for crystallizable! At the
 moment, my leading candidate is aquaporin AqpZ from E. coli. I am
 planning to express the membrane protein as a GFP fusion so students
 can easily follow it through the course of the labs.


 Thank you,
 Ho

 Ho Leung Ng
 University of Hawaii at Manoa
 Assistant Professor, Department of Chemistry
 h...@hawaii.edu




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] protein interactions

2012-09-06 Thread Jacob Keller
I know this isn't exactly your question, but it doesn't really take that
long to clone, express, and purify things nowadays--a few days, even? Also,
won't you be doing this anyway? So why not cut out the middle-man? Or,
better still, in your cloning downtime, do the software stuff.

JPK



On Thu, Sep 6, 2012 at 7:20 AM, moham...@strubi.ox.ac.uk wrote:

 Hi Careina,

 In answer to your first question you could also try the iPATCH server:

 http://portal.stats.ox.ac.uk/userdata/proteins/i-Patch/home.pl

 This takes two reference structures for proteins that interact, and
 combined with multiple sequence alignments of their homologs attempts to
 predict the surface contact residues between them.

 As far as your second question is concerned, a quick google search using
 the term protein interaction prediction from sequence gave some useful
 links, one of which is Struct2Net:

 http://groups.csail.mit.edu/cb/struct2net/webserver/

 This tool attempts to predict protein-protein interactions purely from
 sequence data. However, it does use a structure-based threading approach,
 so your sequences will be run against the pdb. If they are unique to
 anything in the structural databases, it may not be useful.


 Hope this helps,

 Mohammad


 
 Dr. Mohammad W. Bahar
 Division of Structural Biology
 Wellcome Trust Centre for Human Genetics
 University of Oxford

 Hi Careina,

 For the first question, it sounds as though IBIS would do what you want:
 http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.cgi. It will also try to
 answer your second question, although sequences are compared to known
 structures, so if your sequence is dissimilar to anything in the PDB it
 won't work. It looks as though you can only put in one query
 sequence/structure and will then have to scan the results for the
 appearance of the second.

 I hope this helps.

 -- David


 On 6 September 2012 07:43, Careina Edgooms careinaedgo...@yahoo.comwrote:

 Anybody know of any software out there that can predict potential
 interaction sites between two proteins? They have been shown to interact
 via y2h screens but I have no idea if they would interact on their own in
 vitro. Before I clone them into a vector and purify them I would like some
 sort of confirmation that the interaction could occur in the absence of
 other cellular factors.
 There are 2 interactions I am looking at. For the one, the structures of
 both proteins are known. For the other only one structure is known.
 So, is there software that uses 2 known structures to predict binding
 sites and (I know this is a long shot), but is there any software around
 that could predict an interaction based on the sequences only (or one 3D
 structure and one sequence)?

 Thanks
 Careina






-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag

2012-08-27 Thread Jacob Keller
How about just co-transfecting with two different plasmids which have
different selection markers? I've done it a lot, and it seems to work
fine...

JPK

On Fri, Aug 24, 2012 at 10:39 PM, Lye, Ming ming_...@hms.harvard.eduwrote:

 Dear CCP4bb,

 We would like to co-express proteins under Se-Met conditions for de-novo
 phasing of a complex. One of the proteins expresses much better with a
 GST-tag compared with a his-tag.

 As its binding site is at its N-terminus, we are hoping to co-express it
 as a C-terminal GST tag recombinant. So far however, we haven't found any
 commercially available duet vectors with such a tag. We would truly
 appreciate if anyone knows of the availability of such vectors, or has any
 suggestions on similar vectors.

 Thanks so much!
 Ming




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Calculating I/sig when sig = 0

2012-08-23 Thread Jacob Keller
Oh, I thought the sigmas were derived from the differences in intensities
of the multiple measurements of a given reflection--I guess both the
individual-measurement counting stats and differences in measurements must
be combined in the end. But, what does the software do if somehow a sigma=0
creeps in, or more generally, what is the best statistical approach for
this?

JPK

On Thu, Aug 23, 2012 at 12:44 PM, Jim Pflugrath jim.pflugr...@rigaku.comwrote:

  Singly-measured reflections should have a sigma from Poisson counting
 statistics, so that should not be a problem.  A problem might occur if the
 X-ray background is exactly zero and the observed (sic) intensity is
 exactly zero.


  --
 *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob
 Keller [j-kell...@fsm.northwestern.edu]
 *Sent:* Thursday, August 23, 2012 12:36 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] Calculating I/sig when sig = 0

  Dear Crystallographers,

  what approach is taken to calculate I/sig when sig = 0? (This could
 happen for singly-measured reflections or perhaps some other scenario, such
 as rejection of other measurements leaving only one measurement.) I could
 imagine alternatives, but what is actually done?

  JPK


  --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Various OSes and Crystallography

2012-08-09 Thread Jacob Keller
Dear List,

I guess this is somewhat of a perennial issue, but I am faced with choosing
an OS for a new computer, and am curious about benefits and drawbacks with
regard to crystallography. So far, I have been using windows, and have
found no limitations whatsoever, but then again, maybe I don't know what I
am missing. But, since so many folks out there use Macs, I am open to using
one. Are there any really reasonable arguments for preferring Mac over
windows (or linux) with regard to crystallography? What can Mac/Linux do
that windows cannot (especially considering that there is Cygwin)? What
wonderful features am I missing?

Jacob

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] dumb software question

2012-08-07 Thread Jacob Keller
You mean cell in terms of a living cell, not a unit cell, right?

JPK

On Tue, Aug 7, 2012 at 10:24 AM, Paul Kraft haresea...@yahoo.com wrote:


 Hi guys,
 is there a program similar to ccp4/coot that allows one to visuallize an
 ideal cell (either prokaryote or eukaryote) in 3D with semi accurate
 distances. One that is windows based (as much as I detest) that would be
 good for teaching biochem 380 students emphasising distance, diffusion, etc
 measurements with links to the pdb? Thanks in advance.
 Paul

 Dr. Paul Kraft
 Structural Biologist
 cell 586-596-2770
 email: haresea...@yahoo.com




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] ADP binds to the protein in solution but not show up in the structure

2012-07-27 Thread Jacob Keller
Or similarly, the conditions in the crystallization drop (pH, ions, etc.)
may kick out the ligand.

JPK

On Fri, Jul 27, 2012 at 3:34 AM, herman.schreu...@sanofi.com wrote:

 **
 Dear Ni Shi,
 Did you test a crystal, or uncrystallized protein? It may be that due to
 crystal packing effects, only protein without ADP crystallizes.

 Best regards,
 Herman

  --
 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Ni
 shi
 *Sent:* Friday, July 27, 2012 5:17 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] ADP binds to the protein in solution but not show up
 in the structure

   Dear CCP4ers,

 We have crystallized a kinase without adding any nuceotide, and  the
 structure does not show any eletron density at the ATP binding site. Yet
 most of the  bacterial-purified proteins of this family and also  the
 structures contains ADP . So we boiled the bacterial-purified protein, and
 tested the ADP concentration with NADH-coupled ATPase Assay,  Surprisingly,
 the result shows that there is one molecule of ADP per  protein. So why
 would that happen? any suggestions are appreciated

 Best wishes

 Ni shi
  --
 Ni shi
 State Key Laboratory of Plant Phy
 siology and biochemistry
 College of Biological Sciences
 China Agricultural University
 No.2, Yuan Ming Yuan West Road
 Haidian District, Beijing, China 100193
 Tel: +86-10-62732672
 E-mail: xinghua...@126.com xhqin1...@gmail.com
  xhqin1...@gmail.com





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] How to identify unknow heavy atom??

2012-07-24 Thread Jacob Keller
Perhaps also exafs should be mentioned--I believe the various ion species,
redox states, and even binding geometry can be determined.

JPK

On Tue, Jul 24, 2012 at 12:37 PM, Roberts, Sue A - (suer) 
s...@email.arizona.edu wrote:

 Hello

 Actually, if the home source uses a copper tube, neither copper nor zinc
 have much of an anomalous signal at that wavelength (the energy is below
 the absorption edge for both).
 The best way is to check the location of the absorption edge at the
 synchrotron.  Cu+ and Cu++ can be distinguished this way, but make sure the
 absorption scan is done before you collect data since copper(II) can be
 photoreduced to copper(I) in the synchrotron x-ray beam.  Whether or not
 you can get a clue from geometry depends upon the resolution of the
 structure.

 Sue

 On Jul 24, 2012, at 10:22 AM, Nat Echols wrote:

  On Tue, Jul 24, 2012 at 10:14 AM, Haytham Wahba haytham_wa...@yahoo.com
 wrote:
  1- if i have anomalous peak of unknown heavy atom, How can i identify
 this
  heavy atom in general. (different methods)
 
  2- in my case, i see anomalous peak in heavy atom binding site (without
 any
  soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know
  which one give the anomalous peak in my protein.
 
  3- there is way to know if i have Cu+ or Cu++.
 
  You may be able to identify the element based on the coordination
  geometry - I'm assuming (perhaps incorrectly) that it is actually
  different for Cu and Zn.  Marjorie Harding has written extensively on
  the geometry of ion binding:
 
  http://tanna.bch.ed.ac.uk/
 
  The only way to be certain crystallographically, if you have easy
  access to a synchrotron, is to collect data above and below the K edge
  of any candidate element, and compare the difference maps.  (For
  monovalent ions it is more complicated, since they don't have
  accessible K edges.)  On a home source, Cu should have a larger
  anomalous map peak, but I'm not sure if this will be enough to
  identify it conclusively.
 
  -Nat

 Dr. Sue A. Roberts
 Dept. of Chemistry and Biochemistry
 University of Arizona
 1041 E. Lowell St.,  Tucson, AZ 85721
 Phone: 520 621 8171 or 520 621 4168
 s...@email.arizona.edu
 http://www.cbc.arizona.edu/xray or
 http://www.cbc.arizona.edu/facilities/x-ray_diffraction




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] How to identify unknow heavy atom??

2012-07-24 Thread Jacob Keller
I think it's done on a crystal itself, but others who know better than I
can comment.

JPK

On Tue, Jul 24, 2012 at 1:02 PM, Theresa Hsu theresah...@live.com wrote:

 Does EXAFS requires same amount of samples as ICP-MS/ICP-AES?

 Theresa

 On Tue, 24 Jul 2012 12:55:31 -0500, Jacob Keller 
 j-kell...@fsm.northwestern.edu wrote:

 Perhaps also exafs should be mentioned--I believe the various ion species,
 redox states, and even binding geometry can be determined.
 
 JPK
 
 
 
 
 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***
 





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Regarding refinement in Refmac5

2012-07-18 Thread Jacob Keller
How about trying some ARP/WARP?

JPK

On Wed, Jul 18, 2012 at 11:54 AM, Pavel Afonine pafon...@gmail.com wrote:

 What happens if you do a round of refinement in phenix.refine after you
 have done the mutations? Note: Phenix Autobuild is a tool to build your
 model, not refine it (though it does some refinement internally but may not
 be as fine tuned as your data/model may require).

 Pavel


 On Wed, Jul 18, 2012 at 9:50 AM, Deepthi deept...@gmail.com wrote:

 I tried opening the model with other spacegroups MTZ file. The map
 doesn't fit well for other spacegroups. The initial model was refined using
 Phenix Autobuild software. I tried MR with every spacegroup possible in
 primitive hexagonal. Only p3221 worked. There is no twinning in the
 crystal. I will try using other softwares for refinement but this is
 annoying. I also tried mutating the  model to poly alanines and refine but
 this made it worse. The R-free went up to 0.546.
 I initially thought it might be a space group problem but trying other
 space groups doesn't work either.

 Thank youvery much  for the help
 Deepthi


 On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic 
 frederic.velli...@ibs.fr wrote:

 Hi there,

 Not much information provided. How was the initial model refined ?
 Phenix ? It could be a problem with the Refmac refinement protocol
 (difficult to say with so little information) if you switched from Phenix
 to Refmac.

 How certain are you 1 - of the space group; 2 - that the crystal wasn't
 twinned ? You can have both and it can be annoying.

 Further, at this resolution I think you could use one of the SHELXes
 (forgot the terminology) for refinement, that could be more appropriate.

 F.V.


 Deepthi wrote:

 Hi all

 I am working with a small mutant protein which is 56 amino acids long.
 The crystal diffracted at 1.4A0 and the space group is  p3221. I did
 molecular replacement using Phenix software with all the data (1.4A0) and
 got a solution. Phenix did auto building with waters and R-free was 0.3123.

 I mutated some residues which don't align with the model protein  to
 Alanines. When i change the residues back to their respective side chains
 Refmac5 won't  refine it well. The maps looks clear( you can guess its
 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any
 changes to the Phenix generated model. I have no idea what is going on. Can
 anyone help me?

 Thank You in advance
 Deepthi





 --
 Deepthi





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] resolution limit

2012-07-18 Thread Jacob Keller
I was [too] obliquely alluding to this thread...

http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg27056.html

JPK



On Wed, Jul 18, 2012 at 12:32 PM, Edwin Pozharski epozh...@umaryland.eduwrote:

 http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00383.html



  Rsym...what's that?
 
  JPK
 
  On Wed, Jul 18, 2012 at 9:12 AM, Edwin Pozharski
  epozh...@umaryland.eduwrote:
 
  As has been shown recently (and discussed on this board), Rsym is not
  the
  best measure of data quality (if any measure at all):
 
  http://www.sciencemag.org/content/336/6084/1030.abstract
 
 
 
   narayan viswam wrote:
   Hello CCP4ers,
   In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 
   Rsym 224.3 %
   for multiplicity 7.8 and completeness 98.2 %. I solved the structure
  by
   MAD  refined it
   to Rfree 27.3 %. Ths crystal belongs to P622 space group and it is
  not
   twinned. The water
   content is 68%. I loweredthe multiplicity to 4.1 by excluding few
   images but still the
   Rsym is  200 % and I/sigmaI  2.0. My rudimentary crystallography
   knowledge makes me
   believe it's quite Ok to use data upto 2.8 A and report the
  statistics.
   Could I request
   people's views. Thanks very much.
   Narayan
  
   After refinement, what is R-free in the last shell? If it is
  significantly
   better
   than random, say around .4 or less, that could be taken as evidence
  that
   there
   is data in the last shell.
   Also check the error model- Rsym 2 sort of implies the error is
  greater
   than
   the signal, so I/sigI 2 seems surprising.
   eab
  
 
 
  --
  Edwin Pozharski, PhD
  University of Maryland, Baltimore
 
 
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***
 


 --
 Edwin Pozharski, PhD
 University of Maryland, Baltimore




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

2012-07-13 Thread Jacob Keller
You probably already know this, but nitrogen is not at all poisonous--about
78% of the air is nitrogen. I guess you were probably worried about
asphyxiation?

JPK


On Fri, Jul 13, 2012 at 4:19 PM, Radisky, Evette S., Ph.D. 
radisky.eve...@mayo.edu wrote:

 Several have mentioned harvesting in the cold room to reduce evaporation.
 I used to do this also as a postdoc, but I worried whether I risked
 nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not
 seem very well-ventilated.  I’ve also hesitated to recommend it to trainees
 in my current lab for the same reason.  Does anyone have solid information
 on this?  I would like to be convinced that such fears are unfounded …

 ** **

 Evette S. Radisky, Ph.D.
 Assistant Professor
 Mayo Clinic Cancer Center
 Griffin Cancer Research Building, Rm 310
 4500 San Pablo Road
 Jacksonville, FL 32224
 (904) 953-6372 

 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
 *Roger
 Rowlett
 *Sent:* Thursday, July 12, 2012 2:11 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] cryo for high salt crystal

 ** **

 We frequently crystallize one of our proteins and variants of it in
 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol
 or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M)
 and ammonium sulfate (5.6 M) have enormous solubilities in water, so I
 would not expect cryoprotectant concentrations of glycerol or glucose to
 cause precipitation (We can save cryoprotectant solutions of at least 2 M
 ammonium sulfate indefinitely). How are you introducing cryprotectant? We
 use one of two methods:

1. Fish the crystal out of the mother liquor and place into artificial
mother liquor with the same composition as the well solution +
cryoprotectant. For glycerol or other liquids, you have to make this from
scratch. For glucose, we just weigh out 300 mg of glucose in a
microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix
well of course before use. Gentle heating in a block or sonication will
help dissolve the glucose.
2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to
the drop the crystals are in. You can do this all at once, or in stages,
keeping the drop hydrated by placing the hanging drop back in the well
between additions.

 If your drops are drying out during crystal harvesting (very possible in
 dry conditions), you might try harvesting in the cold room, where
 evaporation is slower. We often have problems with crystal cracking and
 drop-drying in the winter months when the humidity is very low indoors. The
 cold room is usually humid enough and cold enough to slow evaporation to
 allow crystal harvesting. (I hate working in the meat locker, though.)

 Cheers,

 ___
 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 7/12/2012 12:55 PM, m zhang wrote:

 Hi Jim, 

 ** **

 25% is w/v. Thanks for the information. Will check the webinar.

 ** **

 Thanks,

 Min
 --

 From: jim.pflugr...@rigaku.com
 To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
 Subject: RE: [ccp4bb] cryo for high salt crystal
 Date: Tue, 10 Jul 2012 17:39:56 +

 Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that
 w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in
 reservoir, or 50% saturated in reservoir.  You will have to TEST these.
  See also this webinar on cryocrystallography which shows how to make these
 solutions: http://www.rigaku.com/node/1388 

 ** **

 You could also try high salt solutions with similar technique.

 ** **

 Good luck!

 ** **

 Jim

 ** **

 ** **
 --

 *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [
 mzhang...@hotmail.com]
 *Sent:* Tuesday, July 10, 2012 11:28 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] cryo for high salt crystal

 regaentDear All, 

 ** **

 I am sure this question was discussed before. But I am wondering if anyone
 got the same experience as I do. 

 I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at
 pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil,
 or ammonium sulfate itself: The problem is that all the cryo plus original
 reagents in the reservoir precipitate the salts out. And more serious
 problem is because of high salt in the condition, while I am trying to loop
 the crystal, both the drop and cryoprotectant drop form salt crystals (not
 sure it is KCl or ammonia sulfate) significantly and very quickly, that
 cause my crystal dissolved. My crystal doesn't seem to survive paraton-N
 

Re: [ccp4bb] harvesting in cold room

2012-07-13 Thread Jacob Keller
How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller dj...@cornell.edu wrote:

  On 07/13/12 17:29, Jacob Keller wrote:

 You probably already know this, but nitrogen is not at all
 poisonous--about 78% of the air is nitrogen. I guess you were probably
 worried about asphyxiation?


 We have oxygen sensors in our X-ray hutches for precisely that reason.

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




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Crystal Optimization

2012-07-10 Thread Jacob Keller
Give a list of all you have tried?

JPK

On Tue, Jul 10, 2012 at 12:22 PM, Muhammed bashir Khan 
muhammad.bashir.k...@univie.ac.at wrote:

 Dear All;

 Could somebody give a nice suggestion how the following type crystal could
 be optimized, I almost tried everything.

 Crystal Image is attached

 Crystal condition: 20% w/v PEG3350 and 200mM NaCl.

 Thanks in advance

 Bashir


 --
 Muhammad Bashir Khan
 **
 Structural Genome Consortium (SGC). University of Toronto
 Toronto, Canada




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] information received through the AFC: iycr2014

2012-07-05 Thread Jacob Keller
On Thu, Jul 5, 2012 at 1:44 PM, Eric Williams ericwilli...@pobox.comwrote:

 I'm guessing disorder is a major limiting factor on effective resolutions.
 ;)


Now *that's* good...

JPK






 On Thu, Jul 5, 2012 at 2:36 PM, Bernhard Rupp (Hofkristallrat a.D.) 
 hofkristall...@gmail.com wrote:

 **Ø  **enjoy that the U.N.'s declaration of the International Year of
 Crystallography comes in the form of a resolution.

 ** **

 ...which tells what a UN resolution is worth…

 ** **

 BR

 ** **

 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
 *Sampson, Jared
 *Sent:* Thursday, July 05, 2012 10:23 AM

 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] information received through the AFC: iycr2014***
 *

 ** **

 I particularly enjoy that the U.N.'s declaration of the International
 Year of Crystallography comes in the form of a resolution.   

 ** **

 Cheers,

 Jared

 --

 Jared Sampson

 Xiangpeng Kong Lab

 NYU Langone Medical Center

 550 First Ave MSB 398

 New York, NY 10016

 212-263-7898

 http://kong.med.nyu.edu/

 ** **

 On Jul 4, 2012, at 8:03 AM, Vellieux Frederic wrote:



 

 ** **





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Detergent and protein oligomerization

2012-06-21 Thread Jacob Keller
First of all, isn't the choice either dimer or trimer, and second, as a
protein-detergent complex (PDC), it would be very unlikely that a trimer of
99 kD would run at 100 kD, although all is fair in love, war, and membrane
proteins.

JPK

On Thu, Jun 21, 2012 at 10:50 AM, Raji Edayathumangalam
r...@brandeis.eduwrote:

 Hi Everyone,

 Sorry for the non-CCP4 post.

 I have a very basic question about detergents, critical micelle
 concentration and behavior on gel filtration.

 A 33kDa membrane protein was purified by gel filtration in a buffer
 containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC:
 0.14mM).  So the concentrations of beta-NG and LDAO in the gel-filtration
 buffer are ~2X and ~14X that of the CMCs of the respective detergents. The
 elution volume of the protein peak (plus detergent) on Superdex200
 corresponds to a molecular mass of 100kDa.

 I think that the 100kDa mass above includes contributions from both the
 protein as well as the detergent micelles. If this is correct, is it then
 accurate to try to glean the oligomerization state of the protein (and
 conclude that it is a trimer or tetramer) without taking into account
 detergent micellar mass and its influence on elution volume?

 How should one interpret the 100kDa mass estimate from the gel filtration?

 Thanks.
 Raji





 --
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Detergent and protein oligomerization

2012-06-21 Thread Jacob Keller
There is something even better than SEC-MALS--solving the structure!

JPK

On Thu, Jun 21, 2012 at 11:16 AM, isabel.de-mor...@diamond.ac.uk wrote:

 Dear Raji,

 The best way to find out is to run a SEC-MALLS  (Size Exclusion
 Chomatography - Multi-Angle Laser Light Scattering) experiment.

 Best,
 Isabel


 -
 Dr. Isabel De Moraes, MRSC

 Membrane Protein Laboratory Facility Co-ordinator
 Membrane Protein Laboratory
 Diamond Light Source Ltd,
 Chilton, Didcot, Oxfordshire,
 OX11 ODE, UK

 Tel (direct): 01235 778664

 --
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob
 Keller [j-kell...@fsm.northwestern.edu]
 Sent: 21 June 2012 17:11
 To: ccp4bb
 Subject: Re: [ccp4bb] Detergent and protein oligomerization

 First of all, isn't the choice either dimer or trimer, and second, as a
 protein-detergent complex (PDC), it would be very unlikely that a trimer of
 99 kD would run at 100 kD, although all is fair in love, war, and membrane
 proteins.

 JPK

 On Thu, Jun 21, 2012 at 10:50 AM, Raji Edayathumangalam r...@brandeis.edu
 mailto:r...@brandeis.edu wrote:
 Hi Everyone,

 Sorry for the non-CCP4 post.

 I have a very basic question about detergents, critical micelle
 concentration and behavior on gel filtration.

 A 33kDa membrane protein was purified by gel filtration in a buffer
 containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC:
 0.14mM).  So the concentrations of beta-NG and LDAO in the gel-filtration
 buffer are ~2X and ~14X that of the CMCs of the respective detergents. The
 elution volume of the protein peak (plus detergent) on Superdex200
 corresponds to a molecular mass of 100kDa.

 I think that the 100kDa mass above includes contributions from both the
 protein as well as the detergent micelles. If this is correct, is it then
 accurate to try to glean the oligomerization state of the protein (and
 conclude that it is a trimer or tetramer) without taking into account
 detergent micellar mass and its influence on elution volume?

 How should one interpret the 100kDa mass estimate from the gel filtration?

 Thanks.
 Raji





 --
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University





 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu
 ***

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Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] estimate of effective concentration

2012-06-20 Thread Jacob Keller
It seems to me that concentration is a statistical,
macroscopically-derived concept like temperature or pressure which
gets exceedingly weird when applied to microscopic phenomena. One
weirdness is, I guess, that the somewhat arbitrary size of the box you
mentioned makes a huge difference in the number one calculates for
concentration, although it does not change the actual situation at
all. Nevertheless, I guess one has sometimes to use the concept to
apply macroscopically-derived parameters to structures, such as
binding constants. Since the application of the concept of
concentration to structures involves strange, potentially
paradoxical things, then, I was wondering what was the reason for
wanting to get into the risky business in the first place?

JPK


On Wed, Jun 20, 2012 at 6:08 PM, Filip Van Petegem
filip.vanpete...@gmail.com wrote:
 Dear crystallographers,

 I have a question concerning effective concentration. Say you have a crystal
 structure whereby two loops, each part of a different domain but within the
 same molecule happen to be juxtaposed and can form an interaction.  The
 loops have some degree of flexibility, but are ordered when interacting. The
 domains on which they are attached have a rigid configuration due to the
 remainder of the structure. The interaction is potentially very weak and
 mainly driven by the fact that the effective concentration is extremely
 high.

 The question: how can one obtain a rough estimate of the effective
 concentration of these two juxtaposed loops?   The simple straightforward
 answer would be to just divide number (1 each) by volume (some box drawn
 around the loops), and convert this to molar. That's easy. However, this is
 over-simplified and really an underestimate of 'effective' concentration,
 because these loops cannot rotate freely when attached to the domains.
  Hence, there are constraints that allow them to interact more readily
 compared to the isolated loops within the same box. So I'm looking for a
 model that also takes limited conformational freedom into account.

 If anybody has any pointers to some reference text or paper that has
 performed such an analysis, I would be very interested.

 Regards,

 Filip

 --
 Filip Van Petegem, PhD
 Associate Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Model submission

2012-06-19 Thread Jacob Keller
What extra insight does the full-length protein give, i.e., why not
just chuck it?

JPK

On Tue, Jun 19, 2012 at 10:35 AM, RHYS GRINTER
r.grinte...@research.gla.ac.uk wrote:
 Dear All,

 I'm working out the finer details on a structural paper for submission to 
 JBC. I'm having a slight problem with how to present my data. I've got a high 
 resolution (1.46 A) truncated structure of the protein with the N-terminal 
 38aa removed. I've also got data from lower resolution (2.68 A) crystals of 
 the full length protein.

 There's no significant difference between the high res and low res proteins 
 in the shared region (amino acid 38+) (r.m.s.d 0.46 A), and the while there 
 is broken density for the first 38aa from the full length data it's too poor 
 to model into.

 I want to present a figure which shows the density corresponding to the first 
 38aa and where that fits with the rest if protein molecule. What I'm unsure 
 of it whether I will be required by the journal to submit a model from the 
 lower resolution data to the PDB in order to present this figure. Bearing in 
 mind the density doesn't allow any additional residues to be modelled 
 compared to the high res. structure.

 Your opinions or advice on how best to present this data would be welcomed.

 Cheers,

 Rhys



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Tool for calculating RMSD

2012-06-19 Thread Jacob Keller
Is part of your question based there being many ways to superimpose
structures, which there are?

JPK

On Tue, Jun 19, 2012 at 5:34 PM, Petr Leiman petr.lei...@epfl.ch wrote:
 Would anyone be kind enough to explain what kind of information does the
 RMSD for two not-yet-superimposed structures transmit?

 If two structures are indeed identical but are apart spatially, then it is
 more appropriate to calculate the COM and the inertia tensor for both
 structures and report the displacement and the rotation (moleman or moleman2
 do just that, right?). Reporting RMSD in this case does not seem to make
 sense because the same RMSD describes an infinite number of spatial
 configurations of the two structures.

 If two structures are not identical, one HAS to superimpose them, i.e. to
 move all (or selected) atoms to be as close in space as possible and only
 then calculate the RMSD for the superimposed or all atoms.

 Thank you in advance,

 Petr

 
 Petr Leiman
 EPFL
 BPS-415
 CH-1015 Lausanne
 Suisse

 On Jun 19, 2012, at 5:04 PM, Claudia Millán Nebot wrote:

 Hello everyone :)

 I would like to know if it exist some tool that allows to calculate RMSD
 between 2 pdbs that are identic, but just displaced in space. It should not
 make a superposition, beause if this is the case it will just say that RMSD
 is 0 .
 I know is not such a difficult problem in terms of scripting, but i was
 wondering if there are tools already.

 Claudia Millán (cmn...@ibmb.csic.es)

 Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

 Barcelona, Spain






-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] do you think it is interesting?

2012-06-18 Thread Jacob Keller
Wow, that's very cool! Can you divulge what the function of the
protein is? One thinks of some kind of mechanical spring...

JPK

On Mon, Jun 18, 2012 at 8:49 AM, anna anna marmottalb...@gmail.com wrote:
 Hi all!
 I'd like your opinion about a structure I solved.
 Apart from protein structure itself, I think that my protein xtallized in an
 odd way!
 The biological unit is a dimer while the asymmetric unit is a tetramer (red
 cartoon in the figure) resulting from domain swapping between two dimers.
 The strange thing is that swapping connects infinite monomers and, rather
 than a xtal, my diffracting object seems a multilayer of endless linear
 polymers, a kind of papyrus with greek fret-like fibers. The figure shows
 the orientation of the polymers in each layer.
 I'd like to know if some of you have already seen a similar pattern or it is
 weird as I think!
 I'm further racking my brain to figure out a biological implication of this
 behaviour, I thought something like plaque formation but I can't find
 support in literature.

 All suggestions are welcome!!

 Cheers,
 Anna





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] do you think it is interesting?

2012-06-18 Thread Jacob Keller
I have been curious and suspicious for a long time about multimer: I
always assumed it to be a more homey substitute for oligomer, as
there seems to me to be no difference in usage, and certainly not in
the etymological sense. I have often heard it used by non-experts who
don't know exactly the meaning of the prefix oligo- but do know
multi-, so they feel more comfortable I think. But anyway, what is
wrong with calling her structures polymers? Is there a subtle
covalent insinuation to polymer?

JPK


On Mon, Jun 18, 2012 at 9:48 AM, David Schuller dj...@cornell.edu wrote:
 On 06/18/12 10:43, Tim Gruene wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 [...]

 of monomers is called a multimer, not a polymer.

 [...]
 shiver - what a terrible mixture of languages. 'multi-' has got latin
 origin, whereas both poly and mer have got greek origin, and I don't
 think one should mix these. Please!!! think of a different _GREEK_
 syllable to express what you describe as 'multimer'.

 I didn't invent the term multimer, it has been in use for some decades.
 And I am writing English, not Latin or Greek.


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



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] do you think it is interesting?

2012-06-18 Thread Jacob Keller
I love myriomer, but what's wrong with boring old polymer?

JPK

On Mon, Jun 18, 2012 at 10:27 AM, Emmanuel Saridakis
esari...@chem.demokritos.gr wrote:
 Of course, oligomer (pure Greek) usually does that kind of job, but not in
 this specific case, since oligo means few and in this case we have endless
 chains.
 I can only think of the neologism myriomer for this particular case, if
 you want to stick to Greek. Myrioi can mean 1 or countless, depending on
 where you accent the word!

 If that catches on, remember you (probably) saw it here first!

 Cheers,
 Emmanuel


 - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de
 To: CCP4BB@JISCMAIL.AC.UK
 Sent: Monday, June 18, 2012 5:43 PM
 Subject: Re: [ccp4bb] do you think it is interesting?



 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 [...]

 of monomers is called a multimer, not a polymer.

 [...]
 shiver - what a terrible mixture of languages. 'multi-' has got latin
 origin, whereas both poly and mer have got greek origin, and I don't
 think one should mix these. Please!!! think of a different _GREEK_
 syllable to express what you describe as 'multimer'.

 Cheers,
 Tim

 On 06/18/12 16:21, David Schuller wrote:

 Certainly it's interesting, but I think your description is
 inaccurate.

 Endless linear polymers - Each monomer is a polymer, but a
 collection of monomers is called a multimer, not a polymer.

 I don't suppose there are any knots? That would be really
 interesting.

 On 06/18/12 09:49, anna anna wrote:

 Hi all! I'd like your opinion about a structure I solved. Apart
 from protein structure itself, I think that my protein xtallized
 in an odd way! The biological unit is a dimer while the
 asymmetric unit is a tetramer (red cartoon in the figure)
 resulting from domain swapping between two dimers. The strange
 thing is that swapping connects infinite monomers and, rather
 than a xtal, my diffracting object seems a multilayer of endless
 linear polymers, a kind of papyrus with greek fret-like fibers.
 The figure shows the orientation of the polymers in each layer.
 I'd like to know if some of you have already seen a similar
 pattern or it is weird as I think! I'm further racking my brain
 to figure out a biological implication of this behaviour, I
 thought something like plaque formation but I can't find support
 in literature.

 All suggestions are welcome!!

 Cheers, Anna





 - -- - --
 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 Mozilla - http://enigmail.mozdev.org/

 iD8DBQFP3z51UxlJ7aRr7hoRAqviAKDJXxXkeOE3Z0M14+RT8dznQhpD3gCcDKEP
 o034eyZnadpwyQRGXI4FV9w=
 =Q5GJ
 -END PGP SIGNATURE-



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] do you think it is interesting?

2012-06-18 Thread Jacob Keller
Okay, I wiki'd it, and according to them seems you're right: it says
they are typically connected by covalent chemical bonds. So either
we revert to the etymological use of polymer, or move onward to
myriomer! (assuming the cross-bred multimer is out of the
question!)

JPK

On Mon, Jun 18, 2012 at 10:37 AM, David Schuller dj...@cornell.edu wrote:
 On 06/18/12 11:17, Jacob Keller wrote:

  But anyway, what is
 wrong with calling her structures polymers? Is there a subtle
 covalent insinuation to polymer?

 subtle? No, it's not subtle.


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



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Jacob Keller
 I suspect that there was a time when the anomalous signal in data sets was 
 fictional.
 Before the invent of flash freezing, systematic errors due to decay and the 
 need
 of scaling together many derivative data sets collected on multiple crystals 
 could render
 weak anomalous signal useless. Therefore MIR was needed. Also, current 
 hardware/software
 produces much better reduced data, so weak signals can become useful.

I think that weak beam intensities (home sources), large crystals, big
HA signals (f = ~12 @ CuKa for some Lanthanides), and high symmetries
could all make measuring anomalous signals much easier even without
cryo. And...Phil Evans did it!

JPK



***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Jacob Keller
I think some have used anomalous signals since the 1930s-40s, e.g., Bijvoet!

JPK

On Wed, Jun 6, 2012 at 10:23 AM, Ronald E Stenkamp
stenk...@u.washington.edu wrote:
 There were a number of labs using anomalous dispersion for phasing 40 years
 ago.  The theory for using it dates from the 60s.  And careful experimental
 technique allowed the structure solution of several proteins before 1980
 using what would be labeled now as SIRAS.  Ron


 On Wed, 6 Jun 2012, Dyda wrote:

 I suspect that pure MIR (without anomalous) was always a fiction. I doubt
 that anyone has ever used it. Heavy atoms always give
 an anomalous signal


 Phil


 I suspect that there was a time when the anomalous signal in data sets was
 fictional.
 Before the invent of flash freezing, systematic errors due to decay and
 the need
 of scaling together many derivative data sets collected on multiple
 crystals could render
 weak anomalous signal useless. Therefore MIR was needed. Also, current
 hardware/software
 produces much better reduced data, so weak signals can become useful.

 Fred


  [32m***
 Fred Dyda, Ph.D.                       Phone:301-402-4496
 Laboratory of Molecular Biology        Fax: 301-496-0201
 DHHS/NIH/NIDDK                         e-mail:fred.d...@nih.gov
 Bldg. 5. Room 303
 Bethesda, MD 20892-0560      URGENT message e-mail: 2022476...@mms.att.net
 Google maps coords: 39.000597, -77.102102
 http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred

 ***
 [m





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Jacob Keller
...Even with such primitive techniques, I can remember an HgI4
 derivative in which you could safely refine the anomalous occupancies
 (i.e. f values) for the iodine atoms of the beautiful planar HgI3 anion to
 5 electrons.

I am surprised--f's of I and Hg are supposed to be around 8 for CuKa
(or maybe you weren't using CuKa)?

JPK


-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Jacob Keller
No offense taken (we all have our dour moments!), but grant me a
sincere question: the f occupancy value would have been just as close
at 11 as 5 if the true value were 8, am I correct? In other words, do
you imply by saying doing well that you got as *much* as 5, or that
you got as *close* as 5? I am just trying to see whether I understand
these things correctly.

Jacob



On Wed, Jun 6, 2012 at 12:21 PM, Gerard Bricogne g...@globalphasing.com wrote:
 Dear Jacob and all,

     I realise that my last statement sounds awfully dour and dismissive, in
 a way I really didn't intend. Especially as Stefan's original posting was a
 Fun Question.

     Apologies to all for this over-the-top statement. I enjoyed a lot of
 the replies.


     With best wishes,

          Gerard.

 --
 On Wed, Jun 06, 2012 at 06:09:33PM +0100, Gerard Bricogne wrote:
 Dear Jacob,

      I thought that getting 5 for each iodine was doing pretty well, given
 the circumstances - e.g. the noisy measurements, the primitive software
 running on slow computers with tiny amounts of memory, etc. .

      In any case my main point, directed at the original poster, was that
 reading the early Acta Cryst. issues (RTFL) might be an alternative and
 perhaps more enlightening way of getting a picture of the evolution of
 phasing methods than finding some clever filter settings in the RCSB ;-) .


      With best wishes,

           Gerard.

 --
 On Wed, Jun 06, 2012 at 11:08:37AM -0500, Jacob Keller wrote:
  ...Even with such primitive techniques, I can remember an HgI4
   derivative in which you could safely refine the anomalous occupancies
   (i.e. f values) for the iodine atoms of the beautiful planar HgI3 anion 
   to
   5 electrons.
 
  I am surprised--f's of I and Hg are supposed to be around 8 for CuKa
  (or maybe you weren't using CuKa)?
 
  JPK
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***

 --

     ===
     *                                                             *
     * 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 *
     *                                                             *
     ===



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-06 Thread Jacob Keller
But the edges for I and Hg are pretty far from CuKa (see attached). I
am familiar with their being extra signal (white lines) very close to
the peak, but not so far away

JPK



On Wed, Jun 6, 2012 at 2:15 PM, Bernhard Rupp (Hofkristallrat a.D.)
hofkristall...@gmail.com wrote:
 There is also a relevant point from the physics of the absorption spectra -
 the XANES white lines (near edge peaks higher than the continuum transition
 or edge step) depend on the chemical environment of the anomalous atom in
 terms of available unoccupied states (which n. b. is something entirely
 different that the local neighbor environment/geometry which can be
 backtransformed - although with quite some uncertainty - from the EXAFS
 wiggles).

 Any argument about absolute f peak values in absence of experimental
 evidence (scan) might want to consider that.

 Best, BR

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob
 Keller
 Sent: Wednesday, June 06, 2012 11:30 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an
 obsolete technique?

 No offense taken (we all have our dour moments!), but grant me a sincere
 question: the f occupancy value would have been just as close at 11 as 5 if
 the true value were 8, am I correct? In other words, do you imply by saying
 doing well that you got as *much* as 5, or that you got as *close* as 5? I
 am just trying to see whether I understand these things correctly.

 Jacob



 On Wed, Jun 6, 2012 at 12:21 PM, Gerard Bricogne g...@globalphasing.com
 wrote:
 Dear Jacob and all,

     I realise that my last statement sounds awfully dour and
 dismissive, in a way I really didn't intend. Especially as Stefan's
 original posting was a Fun Question.

     Apologies to all for this over-the-top statement. I enjoyed a lot
 of the replies.


     With best wishes,

          Gerard.

 --
 On Wed, Jun 06, 2012 at 06:09:33PM +0100, Gerard Bricogne wrote:
 Dear Jacob,

      I thought that getting 5 for each iodine was doing pretty well,
 given the circumstances - e.g. the noisy measurements, the primitive
 software running on slow computers with tiny amounts of memory, etc. .

      In any case my main point, directed at the original poster, was
 that reading the early Acta Cryst. issues (RTFL) might be an
 alternative and perhaps more enlightening way of getting a picture of
 the evolution of phasing methods than finding some clever filter settings
 in the RCSB ;-) .


      With best wishes,

           Gerard.

 --
 On Wed, Jun 06, 2012 at 11:08:37AM -0500, Jacob Keller wrote:
  ...Even with such primitive techniques, I can remember an HgI4
   derivative in which you could safely refine the anomalous
 occupancies
   (i.e. f values) for the iodine atoms of the beautiful planar
   HgI3 anion to
   5 electrons.
 
  I am surprised--f's of I and Hg are supposed to be around 8 for
  CuKa (or maybe you weren't using CuKa)?
 
  JPK
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***

 --

     ===
     *                                                             *
     * 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 *
     *                                                             *
     ===



 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***
attachment: I_Hg_edges.png

Re: [ccp4bb] Death of Rmerge

2012-06-01 Thread Jacob Keller
I don't think any data should be discarded, and I think that although
we are not there yet, refinement should work directly with the images,
iterating back and forth through all the various levels of data
processing. As I think was pointed out by Wang, even an intensity of 0
provides information placing limits on the possible true values of
that reflection. It seems that the main reason data were discarded
historically was because of the limitations of (under)grad students
going through multiple layers of films, evaluating intensities for
each spot, or other similar processing limits, most of which are not
really applicable today. A whole iterated refinement protocol now
takes, what, 15 minutes?

Jacob



On Fri, Jun 1, 2012 at 1:29 PM, Ed Pozharski epozh...@umaryland.edu wrote:
 http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html
 http://scripts.iucr.org/cgi-bin/paper?S0021889800018227

 Just collect 360 sweep instead of 180 on a non-decaying crystal and see
 Rmerge go up due to increase in multiplicity (and enough with redundancy
 term - the extra data is not really *redundant*).  Is your resolution
 worse or better?

 This has been argued over before.  Rmerge has some value in comparing
 two datasets collected in perfectly identical conditions to see which
 crystal is better and it may predict to some extent what R-values you
 might expect.  Otherwise, it's unreliable.

 Given that it's been 15 years since this was pointed out in no less than
 Nature group magazine, and we still hear that Rmerge should decide
 resolution cutoff, chances are increasingly slim that I will personally
 see the dethroning of that other major oppressor, R-value.

 On Fri, 2012-06-01 at 10:59 -0700, aaleshin wrote:
 Please excuse my ignorance, but I cannot understand why Rmerge is unreliable 
 for estimation of the resolution?
 I mean, from a theoretical point of view, 1/sigma is indeed a better 
 criterion, but it is not obvious from a practical point of view.

 1/sigma depends on a method for sigma estimation, and so same data 
 processed by different programs may have different 1/sigma. Moreover, 
 HKL2000 allows users to adjust sigmas manually. Rmerge estimates sigmas from 
 differences between measurements of same structural factor, and hence is 
 independent of our preferences.  But, it also has a very important ability 
 to validate consistency of the merged data. If my crystal changed during the 
 data collection, or something went wrong with the diffractometer, Rmerge 
 will show it immediately, but 1/sigma  will not.

 So, please explain why should we stop using Rmerge as a criterion of data 
 resolution?

 Alex
 Sanford-Burnham Medical Research Institute
 10901 North Torrey Pines Road
 La Jolla, California 92037



 On Jun 1, 2012, at 5:07 AM, Ian Tickle wrote:

  On 1 June 2012 03:22, Edward A. Berry ber...@upstate.edu wrote:
  Leo will probably answer better than I can, but I would say I/SigI counts
  only
  the present reflection, so eliminating noise by anisotropic truncation
  should
  improve it, raising the average I/SigI in the last shell.
 
  We always include unmeasured reflections with I/sigma(I) = 0 in the
  calculation of the mean I/sigma(I) (i.e. we divide the sum of
  I/sigma(I) for measureds by the predicted total no of reflections incl
  unmeasureds), since for unmeasureds I is (almost) completely unknown
  and therefore sigma(I) is effectively infinite (or at least finite but
  large since you do have some idea of what range I must fall in).  A
  shell with I/sigma(I) = 2 and 50% completeness clearly doesn't carry
  the same information content as one with the same I/sigma(I) and
  100% complete; therefore IMO it's very misleading to quote
  I/sigma(I) including only the measured reflections.  This also means
  we can use a single cut-off criterion (we use mean I/sigma(I)  1),
  and we don't need another arbitrary cut-off criterion for
  completeness.  As many others seem to be doing now, we don't use
  Rmerge, Rpim etc as criteria to estimate resolution, they're just too
  unreliable - Rmerge is indeed dead and buried!
 
  Actually a mean value of I/sigma(I) of 2 is highly statistically
  significant, i.e. very unlikely to have arisen by chance variations,
  and the significance threshold for the mean must be much closer to 1
  than to 2.  Taking an average always increases the statistical
  significance, therefore it's not valid to compare an _average_ value
  of I/sigma(I) = 2 with a _single_ value of I/sigma(I) = 3 (taking 3
  sigma as the threshold of statistical significance of an individual
  measurement): that's a case of comparing apples with pears.  In
  other words in the outer shell you would need a lot of highly
  significant individual values  3 to attain an overall average of 2
  since the majority of individual values will be  1.
 
  F/sigF is expected to be better than I/sigI because dx^2 = 2Xdx,
  dx^2/x^2 = 2dx/x, dI/I = 2* dF/F  (or approaches that in the limit . . .)

Re: [ccp4bb] Death of Rmerge

2012-06-01 Thread Jacob Keller
 Let's say you collect data (or rather indices) to 1.4 Ang but the real
 resolution is 2.8 Ang and you use all the data in refinement with no
 resolution cut-off, so there are 8 times as many data.  Then your 15
 mins becomes 2 hours - is that still acceptable?  It's unlikely that
 you'll see any difference in the results so was all that extra
 computing worth the effort?

 Now work out the total number of pixels in one of your datasets (i.e.
 no of pixels per image times no of images).  Divide that by the no of
 reflections in the a.u. and multiply by 15 mins (it's probably in the
 region of 400 days!): still acceptable?  Again it's unlikely you'll
 see any significant difference in the results (assuming you only use
 the Bragg spots), so again was it worth it?

 What matters in terms of information content is not the absolute
 intensity but the ratio intensity / (expected intensity).  As the data
 get weaker at higher d* I falls off, but so does I and the ratio I /
 I becomes progressively more unreliable at determining the
 information content.  So a zero I when the other intensities in the
 same d* shell are strong is indeed a powerful constraint (this I
 suspect is what Wang meant), however if the other intensities in the
 shell are also all zero it tells you next to nothing.

 -- Ian


I envisioned a process of iteration through the various stages of
processing, so still using integration, scaling, etc. to reduce data
before refinement, but maybe feeding back model-based information to
inform the processing of the images. Something like, Refmac says to
Mosflm: kill frames 1100-1200: they're too radiation-damaged. But I
like your idea of using all the pixels--that would be the ultimate,
wouldn't it! Actually, the best would be to have the refinement
already going when collecting data, and informing which frames to
take, and for how long! In a couple years that too will take no time
at all, but then again, we'll probably have atomic-precision real-time
in vivo microscopes by then anyway, and crystallography will have
become an (interesting!) historical curiosity...

JPK

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Death of Rmerge

2012-05-31 Thread Jacob Keller
Meant to include the following link in the previous message:

http://www.youtube.com/watch?v=9Jn8K8EA7-Q

JPK



On Thu, May 31, 2012 at 1:20 PM, Jacob Keller
j-kell...@fsm.northwestern.edu wrote:
 Dear Crystallographers,

 in case you have not heard, it would appear that the Rmerge statistic
 has died as of the publication of  PMID: 22628654. Ding Dong...?

 JPK

 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Fwd: [ccp4bb] Death of Rmerge

2012-05-31 Thread Jacob Keller
Alas, how many lines like the following from a recent Science paper
(PMID: 22605777), probably reviewer-incited, could have been avoided!

Here, we present three high-resolution crystal structures of the
Thermus thermophilus (Tth) 70S ribosome in complex withRMF, HPF, or
YfiA that were refined by using data extending to 3.0 Å (I/sI = 1),
3.1 Å (I/sI = 1), and 2.75 Å (I/sI = 1) resolution, respectively. The
resolutions at which I/sI = 2 are 3.2 Å, 3.4 Å, and 2.9 Å,
respectively.

JPK



On Thu, May 31, 2012 at 1:59 PM, Edward A. Berry ber...@upstate.edu wrote:
 Yes! I want a copy of this program RESCUT.

 REMARK 200  R SYM FOR SHELL            (I) : 1.21700
 I noticed structure 3RKO reported Rmerge in the last shell greater
 than 1, suggesting the police who were defending R-merge were fighting
 a losing battle. And this provides a lot of ammunition to those
 they are fighting.

 Jacob Keller wrote:

 Dear Crystallographers,

 in case you have not heard, it would appear that the Rmerge statistic
 has died as of the publication of  PMID: 22628654. Ding Dong...?

 JPK

 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***





--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Fwd: [ccp4bb] Death of Rmerge

2012-05-31 Thread Jacob Keller
Good idea, but how to get it to catch on without publishing in Science?

JPK

On Thu, May 31, 2012 at 4:21 PM, Dale Tronrud det...@uoxray.uoregon.edu wrote:

 On 05/31/12 12:07, Jacob Keller wrote:
 Alas, how many lines like the following from a recent Science paper
 (PMID: 22605777), probably reviewer-incited, could have been avoided!

 Here, we present three high-resolution crystal structures of the
 Thermus thermophilus (Tth) 70S ribosome in complex withRMF, HPF, or
 YfiA that were refined by using data extending to 3.0 Å (I/sI = 1),
 3.1 Å (I/sI = 1), and 2.75 Å (I/sI = 1) resolution, respectively. The
 resolutions at which I/sI = 2 are 3.2 Å, 3.4 Å, and 2.9 Å,
 respectively.


   I don't see how you can avoid something like this.  With the new,
 higher, resolution limits for data (which are good things) people will
 tend to assume that a 2.6 A resolution model will have roughly the
 same quality as a 2.6 A resolution model from five years ago when
 the old criteria were used.  KK show that the weak high resolution
 data contain useful information but certainly not as much information
 as the data with stronger intensity.

   The resolution limit of the data set has been such an important
 indicator of the quality of the resulting model (rightly or wrongly)
 that it often is included in the title of the paper itself.  Despite
 the fact that we now want to include more, weak, data than before
 we need to continue to have a quality indicator that readers can
 use to assess the models they are reading about.  While cumbersome,
 one solution is to state what the resolution limit would have been
 had the old criteria been used, as was done in the paper you quote.
 This simply gives the reader a measure they can compare to their
 previous experiences.

   Now would be a good time to break with tradition and institute
 a new measure of quality of diffraction data sets.  I believe several
 have been proposed over the years, but have simply not caught on.
 SFCHECK produces an optical resolution.  Could this be used in
 the title of papers?  I don't believe it is sensitive to the cutoff
 resolution and it produces values that are consistent with what the
 readers are used to.  With this solution people could include whatever
 noisy data they want and not be guilty of overstating the quality of
 their model.

 Dale Tronrud

 JPK



 On Thu, May 31, 2012 at 1:59 PM, Edward A. Berry ber...@upstate.edu wrote:
 Yes! I want a copy of this program RESCUT.

 REMARK 200  R SYM FOR SHELL            (I) : 1.21700
 I noticed structure 3RKO reported Rmerge in the last shell greater
 than 1, suggesting the police who were defending R-merge were fighting
 a losing battle. And this provides a lot of ammunition to those
 they are fighting.

 Jacob Keller wrote:

 Dear Crystallographers,

 in case you have not heard, it would appear that the Rmerge statistic
 has died as of the publication of  PMID: 22628654. Ding Dong...?

 JPK

 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***





 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Serine

2012-05-21 Thread Jacob Keller
I believe that's an alternative conformationtry modeling as such and
see what happens.

Jacob

On Mon, May 21, 2012 at 3:57 PM, Uma Ratu rosiso2...@gmail.com wrote:

 Dear All:

 Some of serine residues in my model have extra positive Fo-Fc density at
 the edge of side chain. Some don't have. It is not like from phosphates.

 I am wondered what is the cause for these extra density. Could these
 serines be post-translational modified?

 I have the images attached. P289ser-0512-1 does not have the extra green,
 where P140ser-0512-1 has.

 Thank you for you advice and comment

 Uma




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Propane still?

2012-05-21 Thread Jacob Keller
So much easier is to simply loop your favorite tubing a couple of times
through a small N2 dewar, then direct the output into a 15mL tube immersed
in same. Or, simply point the tubing directly into the immersed tube, and
flow slowly. In both cases, the cold N2 will condense the propane. Of
course, don't cap the 15mL tube and remove from the N2 (it will obviously
explode!).

JPK

On Mon, May 21, 2012 at 4:15 PM, Prince, D Bryan 
dbryan.pri...@astrazeneca.com wrote:

  Good afternoon fellow ccp4bb’rs, 

 ** **

 I was wondering if anyone knows if a still to condense gaseous propane to
 liquid propane using dry ice is commercially available. I want to make sure
 that it is not something I can purchase before I build one fit to purpose.
 I appreciate any advice and knowledge you can share. 

 ** **

 Regards, 

 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.






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Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Propane still?

2012-05-21 Thread Jacob Keller
Well, not to be a downer, but wikipedia has some comments on the hazards...
Environmental effects

Tetrafluoromethane is a potent greenhouse
gashttp://en.wikipedia.org/wiki/Greenhouse_gas that
contributes to the greenhouse
effecthttp://en.wikipedia.org/wiki/Greenhouse_effect.
It is very stable, has an atmospheric lifespan of 50,000 years, and a
high greenhouse
warming potentialhttp://en.wikipedia.org/wiki/Greenhouse_warming_potential of
6500 (CO2 http://en.wikipedia.org/wiki/Carbon_dioxide has a factor of 1);
however, the low amount in the atmosphere restricts the overall radiative
forcing http://en.wikipedia.org/wiki/Radiative_forcing effect.

Although structurally similar to
chlorofluorocarbonshttp://en.wikipedia.org/wiki/Chlorofluorocarbons
(CFCs),
tetrafluoromethane does not deplete the ozone
layerhttp://en.wikipedia.org/wiki/Ozone_depletion.
This is because the depletion is caused by the chlorine atoms in CFCs,
which dissociate when struck by UV radiation. Carbon-fluorine bonds are
stronger and less likely to dissociate. According to Guinness World
Recordshttp://en.wikipedia.org/wiki/Guinness_World_Records
Tetrafluoromethane
is the most persistent greenhouse gas.
[edithttp://en.wikipedia.org/w/index.php?title=Tetrafluoromethaneaction=editsection=7
]Health risks

Depending on the concentration, inhalation of tetrafluoromethane can cause
headaches, nausea, dizziness and damage to the cardiovascular
systemhttp://en.wikipedia.org/wiki/Cardiovascular_system (mainly
the heart). Long-term exposure can cause severe heart damage.

Due to its density, tetrafluoromethane can displace air, creating an
asphyxiation http://en.wikipedia.org/wiki/Asphyxiation hazard in
inadequately ventilated areas.

On Mon, May 21, 2012 at 5:08 PM, Jim Pflugrath jim.pflugr...@rigaku.comwrote:

  Here is a trick which I will attribute to Cambridge:

  Fill balloon with gas.  Put end of balloon over 15 ml Falcon tube.  Put
 Falcon tube in LN2.  No wasted gas.

  I would recommend CF4 or carbon tetrafluoride instead of propane though.
  CF4 is cheap and non-dangerous.

  Jim


  --
 *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prince,
 D Bryan [dbryan.pri...@astrazeneca.com]
 *Sent:* Monday, May 21, 2012 4:15 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] Propane still?

Good afternoon fellow ccp4bb’rs,



 I was wondering if anyone knows if a still to condense gaseous propane to
 liquid propane using dry ice is commercially available. I want to make sure
 that it is not something I can purchase before I build one fit to purpose.
 I appreciate any advice and knowledge you can share.



 Regards,

 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.






-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] completeness in scala

2012-05-17 Thread Jacob Keller
Maybe it's including covert structure factors? See recent ccp4bb post
subject...

JPK

On Tue, May 15, 2012 at 4:28 PM, case c...@biomaps.rutgers.edu wrote:

 On Tue, May 15, 2012, Toth, Eric wrote:

  In sports, maximal effort is considered to be 110%, so you're actually
  9.9% short of getting everything you could out of that crystal at its
  resolution limit.  All things considered, that's not bad.

 In (U.S.) politics, the standard continues to be the 1000% support given
 by presidential candidate George McGovern to vice-presidential candidate
 Thomas Eagleton, shortly before dropping him from the ticket.

 Sounds to me like you need to re-examine the statistics of this particular
 crystal to try to see why it is diffracting so poorly.

 ...dave case




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Strange Density

2012-05-16 Thread Jacob Keller
How about chloride? I know, there are negative electrostatics, but I think
such is seen sometimes for halides. Or, is it possible that there is a side
chain flipped?

Jacob

On Tue, May 15, 2012 at 9:51 AM, RHYS GRINTER 
r.grinte...@research.gla.ac.uk wrote:

 Dear Community,

 As I'm a relatively new to protein crystallography this might turn out to
 be an obvious question, however.

 I'm working on the structure of a enzyme requiring Ca2+ for activity and
 with calcium coordinated in the active site by Asp and 2x backbone carbonyl
 groups, in a crystal structure with Ca in the crystallisation conditions (
 http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg).
 When Ca is omitted from the crystallizing conditions and a divalent
 chelator (EGTA) is added the crystals are of significantly lower resolution
 (3.13A). Refinement of this data reveals density for a molecule coordinated
 by the Ca coordinating Asp and backbone, however this density is
 significantly further away (3.4-3.8A) too far away for water or a strongly
 coordinated divalent cation(
 http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The
 density is also much weaker than for Ca in the previous model disappearing
 at 3.5 sigma.

 The crystallisation conditions for the Ca free condition is:

 0.1M Tris/Bicine buffer [pH 8.5]
 8% PEG 8000
 30% Ethylene Glycol
 1mM EGTA

 The protein was purified by nickel affinity/SEC and dialysed into:
 20mM NaCl
 20mM Tris [pH 8.0]


 A colleague suggested that sulphate or phosphate could fit at these
 distances, but these ions have not been added at any stage of the
 crystallisation process.


 Could anyone give me some insight into what this density might represent?

 Thanks in advance,

 Rhys Grinter
 PhD Candidate
 University of Glasgow




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] zinc with HEPES

2012-05-11 Thread Jacob Keller
Try adding water first, so that you are not mixing concentrated Zn with
concentrated HEPES. Also it depends what else is in your cocktail.

JPK

On Fri, May 11, 2012 at 11:26 AM, Rajesh Kumar ccp4...@hotmail.com wrote:

  Dear All,

 This question sounds simple but I dont know the answer.
 I was preparing a 24 well crystal screen. When I try to use 10 mM  ZnSO4
 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn
 acetate the effect is same.
 I dont know why this Zn in not compatible with HEPES.
 Could you please tell me why is this?
 I appreciate your help.

 Thanks
 Rajesh




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] zinc with HEPES

2012-05-11 Thread Jacob Keller
Just to make sure I understand pH correctly: isn't it true that the [OH-]
should always be the same at a given pH (by definition)?

JPK

On Fri, May 11, 2012 at 11:48 AM, Katherine Sippel 
katherine.sip...@gmail.com wrote:

 That is probably because you pH Tris with HCl rather than HEPES with NaOH.
 The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably what
 are killing your solution.

 Cheers,

 Katherine


 On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar ccp4...@hotmail.comwrote:

  If there is no cure , then fine.
 pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6.
 Thanks to every one
 Rajesh


 --
 Date: Fri, 11 May 2012 12:35:45 -0400
 From: dj...@cornell.edu

 Subject: Re: [ccp4bb] zinc with HEPES
 To: CCP4BB@JISCMAIL.AC.UK



  There is no cure for HEPES.




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





-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] zinc with HEPES/seeding

2012-05-11 Thread Jacob Keller
mitegen loops might help, particularly micromesh...

JPK

On Fri, May 11, 2012 at 12:35 PM, Rajesh Kumar ccp4...@hotmail.com wrote:


 Dear Patrick,

 You along with others had made some suggestions last time. May be its a
 good time to update.

 With classical screening, I got a crystal like appearances/shower with
 HEPES 7.5 and LiSo4 1.5M.  Trying to vary the pH of Hepes or using Tris and
 with different conc of Lithium I could only get very very thin needles
 which shower and difficult to pick even with 0.05 loop. changing conc of
 protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs,
 glycerol, ethylene glycol, didnt reduce shower.

  I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS
 screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG)
 100nl+100nL+50nL seeds using Phoenix.

 Got several nice hits which very good size individual crystals in
 conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400.
 When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but
 no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after
 plates were set and there was no luck. Crystals started to appear from 20
 min onwards and keep growing in next couple of hours.  I thought of trying
 dehydration but they were already in dehydrating conditions them selves. I
 wanted to ask if anyone ever failed with MMS but thought not
 waste others time on this. Cross seeding to full length protein and Se met
 protein also gave beautiful crystal but again no diffraction. I have not
 checked SeMet as they were bit small.

  I am still open to ideas if you have any thing on seeding. I did try in
 microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul
 seeds, same as sitting drop which gave me very good looking crystals) but I
 didn't get any thing.  I tried streaking with reduced LiSO4 to 1.1 M but
 it didn't give me anything.

 Currently, I am making entropy mutations,  new constructs
 of different lengths to solve the above problem.

 I still want to improve this condition with needles, because I collected a
 4.5A data on one of the needle (just one). I need phases. I have sent some
 Iodide soaks to synchotron (yet to collect data) but manipulating these
 crystal drops with more than 100 tiny needles with a tough membrane on it
 has been frustrating as I end up loosing several drops  to just to fish out
 1-2 needles. I am ready to try   if there any trick left.

 Thanks for lots and lots of help.

 Regards,
 Rajesh
 --
 Date: Fri, 11 May 2012 18:09:54 +0100
 Subject: Re: [ccp4bb] zinc with HEPES
 From: patr...@douglas.co.uk
 To: ccp4...@hotmail.com

 Rajesh

 How did you do the MMS?  By hand or with a robot, and what screens did you
 use?

 and why did you change to HEPES out of interest?

 Patrick


 On 11 May 2012 18:05, Rajesh Kumar ccp4...@hotmail.com wrote:

  The rationale was to see if Zn could make differences in crystal
 morphology. This is because the protein has CxxC and CxxH similar to a zinc
 finger motif.
 All my efforts, additive screening, MMS, streaking, micro batch, hanging
 drop, changing drop ratio, drop shape, did not help me to either increase
 thickness  or change the shape of very very thin needle crystals.
 Yes, I will try very less, 50uM.
 Thanks for helping me to understand.
 Rajesh

  Date: Fri, 11 May 2012 12:53:53 -0400
  Subject: Re: [ccp4bb] zinc with HEPES
  From: liehy...@gmail.com
  To: ccp4...@hotmail.com
 
  Rajesh,
  10mM zinc seems a bit too high. I normally used it at 50uM conc.
  ray
 
  On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar ccp4...@hotmail.com
 wrote:
   Dear All,
  
   This question sounds simple but I dont know the answer.
   I was preparing a 24 well crystal screen. When I try to use 10 mM
  ZnSO4
   with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn
   acetate the effect is same.
   I dont know why this Zn in not compatible with HEPES.
   Could you please tell me why is this?
   I appreciate your help.
  
   Thanks
   Rajesh




 --
  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
  Regd. England 2177994, VAT Reg. GB 480 7371 36




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Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


[ccp4bb] Powder Rings in Single Crystals

2012-05-09 Thread Jacob Keller
Dear Crystallographers,

the saxs on crystals thread reminded me of a question I have had for a
while, and never having collected data better than ~1.6 Ang or so, cannot
answer myself from experience: I would think that there might be
powder-like diffraction rings at distances corresponding to the various
covalent bond lengths in proteins (1.2-1.5 Ang), but have never heard of
such. My thinking is that the protein itself is essentially a powder sample
within the unit cell consisting of many small, randomly-oriented molecules
(amino acids) with their covalent bonds. Do the rings in fact exist, and if
not, why not? Maybe the electron density is not as atomic, or discrete,
as the nuclei are? I wonder whether generally data collected to beyond ~1
Ang have an intensity bump at those covalent bond lengths, as I believe
is seen in nucleic acid-containing structures at the base-stacking distance
(at the right orientation)?

Jacob

-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Postdoc postion at SLS

2012-05-09 Thread Jacob Keller
I saw something online about the EIGER 16M: 201 GB of data per second! Is
that number correct?

JPK


On Wed, May 9, 2012 at 9:58 AM, Meitian Wang meitian.w...@psi.ch wrote:

 Postdoctoral Fello*Next Generation Detector for Protein Crystallography*
 Your tasks
 Built on the success of PILATUS detector technology, PSI and Dectris Ltd.
 are developing the next generation single-photon counting detector (EIGER)
 featuring smaller pixel size, higher frame rate and dynamic range. The
 proposed project is to exploit PILATUS and EIGER detectors in protein
 crystallography applications:

- Systematic data acquisition and processing optimization using
PILATUS 2M/6M detectors
- Development of fast data acquisition and processing methods with
EIGER 1M detector in protein micro-crystallography
- Commissioning of the EIGER 16M detector at a protein crystallography
beamline

 Your profile
 You hold a PhD degree in (bio-)chemistry or physics, and have several
 years of experience in protein crystallography. Working knowledge for data
 processing programs, and various phasing and refinement software is a must.
 Experience in computer programming would be a significant advantage. If you
 are a good team player with fine communication skills and sense of
 responsibility, this position will offer a great opportunity for you to
 develop your research career in an exciting and highly multidisciplinary
 environement.

 For further information please contact Dr Meitian Wang, phone +41 56 310
 41 75, or Dr. Clemens Schulze-Briese, clemens.schulzebri...@dectris.com

 Please submit your application online (including list of publications and
 addresses of referees) for the position as Postdoctoral Fellow (index no.
 6112-00).

 Paul Scherrer Institut, Human Resources, Elke Baumann, 5232 Villigen PSI,
 Switzerland


 http://www.psi.ch/pa/offenestellen/0329-2


 __
 Meitian Wang
 Swiss Light Source at Paul Scherrer Institut
 CH-5232 Villigen PSI - http://www.psi.ch/sls/
 Phone: +41 56 310 4175
 Fax:  +41 56 310 5292




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Powder Rings in Single Crystals

2012-05-09 Thread Jacob Keller
Well, what about the original DNA fiber diffraction images--no
microcrystals there, as far as I know, but one can clearly see the stacking
distances and the phosphate backbone.

JPK

On Wed, May 9, 2012 at 11:03 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 Dear Jacob,

 A protein would only scatter but not diffract, the latter - in my
 understanding - being the result of constructive interference from a
 regular array of unit cells .

 A powder pattern is the superposition of many small crystals amongst
 which you don't observe interference.

 Tim

 On 05/09/12 16:16, Jacob Keller wrote:
  Dear Crystallographers,
 
  the saxs on crystals thread reminded me of a question I have had
  for a while, and never having collected data better than ~1.6 Ang
  or so, cannot answer myself from experience: I would think that
  there might be powder-like diffraction rings at distances
  corresponding to the various covalent bond lengths in proteins
  (1.2-1.5 Ang), but have never heard of such. My thinking is that
  the protein itself is essentially a powder sample within the unit
  cell consisting of many small, randomly-oriented molecules (amino
  acids) with their covalent bonds. Do the rings in fact exist, and
  if not, why not? Maybe the electron density is not as atomic, or
  discrete, as the nuclei are? I wonder whether generally data
  collected to beyond ~1 Ang have an intensity bump at those
  covalent bond lengths, as I believe is seen in nucleic
  acid-containing structures at the base-stacking distance (at the
  right orientation)?
 
  Jacob
 

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

 GPG Key ID = A46BEE1A

 -BEGIN PGP SIGNATURE-
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-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Powder Rings in Single Crystals

2012-05-09 Thread Jacob Keller
Yes, I just looked up the paper--seems right on topic--a powder-type ring
at ~4.2 Ang, corresponding to Calpha-Calpha distances! But no 1.2-1.5 Ang
ring, from what I saw. Maybe it gets swamped out by other things. I am
thinking that the variety/distribution of bonds/distances of length 1-3 Ang
in the crystal/mother liquor combo is so high/broad that you can't see them
anymore. I wonder whether when people soak in various heavy atom clusters,
they see powder rings for the HA-HA distances in the unbound clusters?

JPK

On Wed, May 9, 2012 at 11:51 AM, Philip Kiser p...@case.edu wrote:

 Hey Jacob,

 There was a paper by Robert M. Blessing et al (Acta Cryst D 1996) that
 at least partially attributed the diffuse ring that one sees around
 3-4 A to something similar to what you are describing (scattering
 between amide oxygen and nitrogen  for example).

 Philip

 --
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Powder Rings in Single Crystals

2012-05-09 Thread Jacob Keller
It seems to me that spherical forms of Wilson plots could be used to
determine how many bonds of what nature were oriented in which direction,
and this may have been what Bricogne's micro molecular replacement
technique was capitalizing on? For example, one might be able to orient a
straight DNA molecule by finding the direction at which the ~3.2-3.5 Ang
bin signal was greatest. But I guess this is probably implicit in molecular
replacement anyway...

JPK

On Wed, May 9, 2012 at 2:08 PM, Nat Echols nathaniel.ech...@gmail.comwrote:

 On Wed, May 9, 2012 at 11:58 AM, Garib N Murshudov
 ga...@mrc-lmb.cam.ac.uk wrote:
  As far as I know there are several bumps: around 3.5-4 (there are some at
  low resolution related with molecular shapes also) - secondary
 structures,
  ~2.2 related with angles and around 1.2 related with covalent bonds. For
  DNA/RNA there is one more bump around 1.6-1.7 ( I thought that is
 because of
  Phosphor bonds). They are visible with normalised data better.

 It has been pointed out to me that my example cut off the data at too
 low a resolution to see the peak for covalent bonds.  Here is a
 different version that shows a distinctive peak around 1.15A.

 -Nat




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] saxs on xtals

2012-05-08 Thread Jacob Keller
Dear Colin,
the table you gave seems to have been from  Fe2+Fe3+2O4  or from Fe3-xTixO4.
I am curious what the nature of the Fe inside the ferritin is (I don't
think it has Ti in it, though...). Is it elemental iron?

Also, to Anna: can you send a picture of that diffraction pattern with spot
predictions superimposed? There seem to be some really bright spots which
are outliers, and maybe multiple lattices.

JPK

On Tue, May 8, 2012 at 12:21 PM, Colin Nave colin.n...@diamond.ac.ukwrote:

 Anna
 Yes, you have understood the suggestion.
 Could be the 220 and 311 reflections. See for example
 http://rruff.info/magnetite/R080025
 and

 http://rruff.info/repository/sample_child_record_powder/by_minerals/Magnetite__R080025-1__Powder__DIF_File__9448.txt

 Trying to index powder patterns from 2 rings is risky and the intensities
 don't seem to agree.  I guess you don't have higher angle data.

 Should be able to evaluate a particle size from the breadth of the rings
 though. For example a 57A crystal examined with 1A radiation would give
 broadening of about a degree.

 There do seem to be other spots - I guess these are ice rings but you
 should check. Also be nice to know if apoferritin crystallised under the
 same conditions (if it can be) shows these rings

 Regards
 Colin

 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 anna anna
 Sent: 08 May 2012 16:55
 To: ccp4bb
 Subject: Re: [ccp4bb] saxs on xtals

 Dear all,
 first of all I want to thank you for your attention and all your brilliant
 suggestions that really cleared my head!!!
 Thanks to you (or because of you!!) now I have many ideas and very much to
 do.

 Colin,
  I was just re-considering my diffraction images. Who knows if they are
 single xtals indeed!
 Let's see if I understood your point. Assuming that they are single xtals,
 if they are located at independent positions in the protein-cage it would
 be like powder diffraction, with rings at diffraction angles corresponding
 to magnetite lattice. If they are ordered they should give a diffraction
 pattern. The corresponding lattice can differ from the protein lattice, do
 you agree? If this is true, what would I see? Two superimposed diffraction
 patterns?
 Actually, I am not able to evaluate it... I attached one of the
 diffraction images. It seems to me that there are two diffused rings at
 about 2.5 and 2.9 A.

 Michael, I just read your reply. I think that the eventual periodicity of
 the partcles can't be completely independent of the protein periodicity (I
 attached a hypotethical scheme), as you suggest I will try P1.
 Once I tryed a naive version of what you suggest: I put a magnet over the
 xtallization plate. All my collegues made fun of me... :) !!

 I will check the literature that you all quoted (hard work!)

 Thank you again, new suggestions will be really appreciated.

 Cheers,
 anna

 2012/5/8 R. M. Garavito rmgarav...@gmail.commailto:rmgarav...@gmail.com
 
 Dear Anna,

 I know that you already have gotten replies from some top experts, but
 your intriguing problem brought up some issues I have run across in the
 past.

 First, from you experience with single crystal diffraction, your results
 are not that much different from those seen in virus structures where the
 nucleic acid structure is averaged out.  As the nucleic acid doesn't (and
 mostly can't) adopt the symmetry of the protein shell, the crystallization
 process alone does the averaging.   Just because that ferritin and
 magnetite have cubic symmetry elements, if they don't line up, the
 magnetite structure can be averaged out upon crystallization.  So,
 working at lower symmetry may not help, unless there is some directional
 correlation of the magnetite symmetry and position with the crystal axes.
  But try P1 and see what happens.

 A second comment is why not try neutron scattering (SANS or single crystal
 neutron diffraction), particularly as you can match out the protein with
 D2O and see just the magnetite.  While the same concerns apply for single
 crystal neutron diffraction, you see more clearly regions of higher average
 density inside the protein shell.

 And lastly, have you tried crystallizing your ferritin/nanoparticle
 complexes in the presence of a magnetic field?  It would be a neat trick,
 and people have tried such things in the past, such as for orienting
 biomolecules.  Some even used old NMR magnets.  Would be wild, if it worked.

 Good luck,

 Michael

 
 R. Michael Garavito, Ph.D.
 Professor of Biochemistry  Molecular Biology
 603 Wilson Rd., Rm. 513
 Michigan State University
 East Lansing, MI 48824-1319
 Office:  (517) 355-9724tel:%28517%29%20355-9724 Lab:  (517) 353-9125
 tel:%28517%29%20353-9125
 FAX:  (517) 353-9334tel:%28517%29%20353-9334Email:
 rmgarav...@gmail.commailto:garav...@gmail.com
 



 On May 7, 2012, at 12:30 PM, 

Re: [ccp4bb] saxs on xtals

2012-05-07 Thread Jacob Keller
It might be that the bulk solvent correction is nullifying the interior
of the ferritin structure, and there should be a way to tell the refinement
software not to treat the interior as solvent. Perhaps then you might find
your Fe? Also, I would think there should be some powder-like diffraction
rings in the background of the ferritin crystal diffraction corresponding
to the scattering from the jumbled but crystalline Fe in the inside--did
you see any extra rings in the background of your original diffraction
data? Not sure what the Fe-Fe distance is in this case in particular, but
there should be a ring (or rings) corresponding to that (those)
distance(s)

JPK

On Mon, May 7, 2012 at 11:30 AM, anna anna marmottalb...@gmail.com wrote:

 Dear all,
 I'd like some suggestions/opinions about the sense of an experiment
 proposed by a collaborator expert in saxs.
 In few words, he wants to collect SAXS data on a suspension of protein
 xtals to investigate low resolution periodicity of the xtal (more details
 below).
 The experiment requires a very huge number of xtals to obtain the circles
 typical of saxs and it is very time-consuming to me (I know nothing about
 saxs, I have only to prepare the sample). I proposed to measure a single
 rotating xtal (like in XRD) but he told they don't have a goniometer on
 saxs beamline.
 Here is my concern: does it make sense to measure many xtals together?
 Don't we lose information with respect to single xtal? And, most of all,
 what can I see by *s*axs that I can't see by* w*axs??
 Sorry for the almost off-topic question but I think that only someone who
 knows both the techniques can help me!!


 Some detail for who is intrigued by my story:
 we prepared doped magnetite nanoparticles using ferritin as bioreactor. I
 crystallized this spheres filled with metal and solved the structure at
 3.7A but I can see only the protein shell while there is no density inside,
 even if I know that the nanoparticles are there. A simple explanation is
 that the particles are free to move in the cavity(note that the diameter of
 the nanoparticle is shorter then the inner diameter of the protein shell),
 ie are disordered, and do not contribute to diffraction, in fact, to my
 knowledge, nobody have ever seen the metal core inside ferritin or dps
 proteins. However, since they are magnetic particles they must see each
 other through the protein wall, ie they can't be completely free to move in
 the cavity. Maybe, but this is just my opinion, I don't see the particle
 because the period of the particle in the xtal is different/longer than
 the period of the protein shell.
 Anyway, we are interested in the relative distance between the magnetic
 particles in the xtal to study the effects of magnetostatic interactions in
 nanoparticles 3D arrays. We are going to do this by saxs since, they told
 me, lower resolution is useful in studying this long range periodicity (the
 diameter of ferritin is about 120A) but it seems fool to me using a
 suspension of so many xtals to obtain a scattering curve while I could
 collect diffraction images from a single xtal!!! I know that saxs is used
 when you don't have xtals but if you have xtals, ie your system is ordered,
 xtallography is much more powerful!!

 Another question: how can I handle my diffraction data at 3.7A resolution
 to look for nanoparticles? Should I try a lower symmetry? Maybe the
 anomalous signal? Have you any reference for a similar case?

 Thank you very much!!

 anna








-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


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