[ccp4bb] Nonlinearities and Thresholds
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 + To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] am I doing this right?
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?"
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?"
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?"
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 + To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"
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?"
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 + To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Contagious, Self-Distributing "Vaccines?"
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
>
--
+
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?"
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 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Contagious, Self-Distributing "Vaccines?"
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 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] 3D Printer Model of the Coronavirus
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 > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > -- + 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 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] MAX IV Life Science Director position
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
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
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
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
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
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
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
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
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
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
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
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?
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?
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?
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
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
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
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?
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?
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?
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
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?
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
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
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
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
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
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 -- *** Jacob Pearson Keller, PhD Postdoctoral Associate HHMI Janelia Farms Research Campus email: j-kell...@northwestern.edu ***
[ccp4bb] Crystallography Near Absolute Zero?
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
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
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
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
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
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
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
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
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
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
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
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?
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
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
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
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
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
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
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??
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??
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
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
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)
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
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
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
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
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
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 *** -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] estimate of effective concentration
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
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
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?
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?
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?
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?
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?
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?
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?
...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?
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?
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
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
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
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
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
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
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?
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. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Propane still?
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
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
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
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
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
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 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Powder Rings in Single Crystals
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
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
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- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPqpVlUxlJ7aRr7hoRAhZqAJ9M49u+cYsX+AhzRlYKOCTX8YCsbQCgzFBi ZV+cZCyQgGLusrnufngyZ5A= =cuQy -END PGP SIGNATURE- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Powder Rings in Single Crystals
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
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
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
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 ***
