Re: [ccp4bb] Same protein, different molecule numbers per ASU
They must represent a different packing - the volume of Shape 1 is 10% > than 2*Vol-Shape2 But it isnt uncommon to get different forms - check your symmetry contacts (PISA will do that) and see how the two forms pack. eleanor On Mon, 11 Jul 2011 18:09:52 +0800, ferrol shariff wrote: > Hi Alenxander, > Thanks for the reply. Here's the details on the unit cell parameters > > [image: image.png] > > [image: image.png] > > [image: image.png] > > Thank you. :) > > Regards, > > Fairolniza > On Mon, Jul 11, 2011 at 2:47 PM, Alexandre OURJOUMTSEV > wrote: > >> Dear Ferrol, >> >> ** ** >> >> Could you let us know the unit cell parameters for both these crystals ? >> (did I miss them somewhere in your previous mails?). I wonder if in fact >> this is not the same packing with minor variation. >> >> Sacha >> >> ** ** >> >> *De :* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *De la part >> de*ferrol shariff >> *Envoyé :* dimanche 10 juillet 2011 10:10 >> *À :* CCP4BB@JISCMAIL.AC.UK >> *Objet :* [ccp4bb] Same protein, different molecule numbers per ASU >> >> ** ** >> >> Hello and good day to everyone! :) >> >> ** ** >> >> I have some general questions on crystallography work. I hope you don't >> mind giving me some ideas. >> >> I have solved my lipase protein both ground-grown crystals and >> space-grown >> crystals with good resolutions (1.4A and 2.2A). They are the same protein >> from the same source, same purification methods, and produced crystals >> from >> the same crystallization conditions (except the gravity part). >> >> From the data, it shows that both of them belong to the same space group >> P212121. But they have different number of molecule per asymmetric unit. >> Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the >> moment i have problem explaining this issue. Is it normal to have such >> results? Same protein with different number of molecule/ASU? >> >> I've been trying to get some references on this matter but so far i don't >> really get anything that can directly explain it. Furthermore, do i need >> to >> relate this with the gravity effect? >> >> I hope you don't mind sharing some experiences on crystallography >> especially regarding this matter. >> >> Thank you very much >> >> -- >> FAIROLNIZA >> >> >>
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Fairolniza, my experience with multiple crystal forms growing from the same drop (I had 4 that I was able to identify, there could have been more) was that there were similarities in the way protein molecules packed into "fibers" that were subsequently arranged into distinct crystal forms. You may want to consider looking at the protein-protein interfaces in your two crystal forms using PISA server and see if there are similarities. IMHO, any theory explaining why microgravity would favor one of the two forms would be pure speculation (with additional caveat that there is no way you can make two experiments completely identical in all other respects anyway), but what's wrong with that? :) Cheers, Ed. -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Hi Alenxander, Thanks for the reply. Here's the details on the unit cell parameters [image: image.png] [image: image.png] [image: image.png] Thank you. :) Regards, Fairolniza On Mon, Jul 11, 2011 at 2:47 PM, Alexandre OURJOUMTSEV wrote: > Dear Ferrol, > > ** ** > > Could you let us know the unit cell parameters for both these crystals ? > (did I miss them somewhere in your previous mails?). I wonder if in fact > this is not the same packing with minor variation. > > Sacha > > ** ** > > *De :* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *De la part > de*ferrol shariff > *Envoyé :* dimanche 10 juillet 2011 10:10 > *À :* CCP4BB@JISCMAIL.AC.UK > *Objet :* [ccp4bb] Same protein, different molecule numbers per ASU > > ** ** > > Hello and good day to everyone! :) > > ** ** > > I have some general questions on crystallography work. I hope you don't > mind giving me some ideas. > > I have solved my lipase protein both ground-grown crystals and space-grown > crystals with good resolutions (1.4A and 2.2A). They are the same protein > from the same source, same purification methods, and produced crystals from > the same crystallization conditions (except the gravity part). > > From the data, it shows that both of them belong to the same space group > P212121. But they have different number of molecule per asymmetric unit. > Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the > moment i have problem explaining this issue. Is it normal to have such > results? Same protein with different number of molecule/ASU? > > I've been trying to get some references on this matter but so far i don't > really get anything that can directly explain it. Furthermore, do i need to > relate this with the gravity effect? > > I hope you don't mind sharing some experiences on crystallography > especially regarding this matter. > > Thank you very much > > -- > FAIROLNIZA > > > -- FAIROLNIZA "The advantage of the emotions is that they lead us astray, and the advantage of science is that it is not emotional" -Oscar Wilde, The Picture of Dorian Gray, 1891 <><><>
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Dear Ferrol, Could you let us know the unit cell parameters for both these crystals ? (did I miss them somewhere in your previous mails?). I wonder if in fact this is not the same packing with minor variation. Sacha De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de ferrol shariff Envoyé : dimanche 10 juillet 2011 10:10 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Same protein, different molecule numbers per ASU Hello and good day to everyone! :) I have some general questions on crystallography work. I hope you don't mind giving me some ideas. I have solved my lipase protein both ground-grown crystals and space-grown crystals with good resolutions (1.4A and 2.2A). They are the same protein from the same source, same purification methods, and produced crystals from the same crystallization conditions (except the gravity part). >From the data, it shows that both of them belong to the same space group >P212121. But they have different number of molecule per asymmetric unit. >Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the moment >i have problem explaining this issue. Is it normal to have such results? Same >protein with different number of molecule/ASU? I've been trying to get some references on this matter but so far i don't really get anything that can directly explain it. Furthermore, do i need to relate this with the gravity effect? I hope you don't mind sharing some experiences on crystallography especially regarding this matter. Thank you very much -- FAIROLNIZA
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Thank you for the clarification. So you have two independent crystal forms from the 'same' condition and one of them does not grow in space. Sounds like there is some basis to the theory that lack of gravity caused the first form to disappear. Unfortunately it can also be the subtle changes in apparatus, differences for example how the temperature *varied* during incubation (yes, averaging on 20 but with what fluctuation and duty cycle, etc.) or the slightly different (differently aged even) batches of solution used for crystallization. When you say that you've not found any 'major' structural changes - did you find some 'minor' ones that are truly there (i.e. not artefacts of refinement with different resolution and data quality)? Are they interesting in any way? Otherwise it sounds like you've got the 'too many variables, but something is different enough to change the outcome' scenario that is oh so typical of crystallization, with a side note that space flight was one of the variables. In this case, I would avoid the term 'explain' and use 'rationalize' or 'speculate' instead :) Artem On Sun, Jul 10, 2011 at 2:56 PM, ferrol shariff wrote: > Thank you very much everyone for the replies. > > Ok. Here are more details on the work: > > 1) Crystallization was done using counter diffusion method- in a capillary > (for both Space and Ground experiments) > > 2) Vapor diffusion method was done only for Ground experiments, and > produced both Shape 1 and Shape 2 > > 3) The ground crystals usually grow in two forms- as nice single crystal > (Shape 1) and another one as needle-like long rods (Shape 2). > > 4) In space, only Shape 1 crystal was formed > > 5) Both Ground and Space experiments- temperature was fixed at 20 deg. > Space experiment- temperature was reported to us once a week. > > 6) Shape 2 crystals (the rods) diffracted much better, with much lower > mosaicity range, produced good data processing as compared to Shape 1 (Shape > 1 Space crystal's mosaicity range= 0.5-0.96! ) > > 7) Both Shape 1 and Shape 2 have different cell dimensions, with different > volumes (Shape 1= ~ 2X of Shape 2) > > 8) Both structures from Shape 1 and Shape 2- solved by molecular > replacement methods- no major structural changes between them > > Thank you. > > Regards, > > Fairolniza > > > p/s: And thank you, Dr. Snell for the paper. I already have the paper and > it has been one of my favorite references :) > > > > > On Sun, Jul 10, 2011 at 10:58 PM, Artem Evdokimov < > artem.evdoki...@gmail.com> wrote: > >> I would not rule out pure chance. Crystals of the same protein can and >> often do grow in two (or more!) different forms, from 'the same conditions' >> and often in the very same drop. In this case it's the same space group, but >> presumably different cell dimensions? Until a significant number of crystals >> from each condition (lab vs space) are analyzed, there's no reason not to >> believe that it was just luck :) >> >> Arten >> >> On Sun, Jul 10, 2011 at 9:53 AM, Jim Pflugrath >> wrote: >> >>> ** >>> OK, same space group, but you didn't indicate what the unit cells were. >>> They are different, right? >>> >>> -- >>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of >>> *ferrol shariff >>> >>> *Sent:* Sunday, July 10, 2011 3:10 AM >>> *To:* CCP4BB@JISCMAIL.AC.UK >>> *Subject:* [ccp4bb] Same protein, different molecule numbers per ASU >>> >>> Hello and good day to everyone! :) >>> >>> I have some general questions on crystallography work. I hope you don't >>> mind giving me some ideas. >>> >>> I have solved my lipase protein both ground-grown crystals and >>> space-grown crystals with good resolutions (1.4A and 2.2A). They are the >>> same protein from the same source, same purification methods, and produced >>> crystals from the same crystallization conditions (except the gravity >>> part). >>> >>> From the data, it shows that both of them belong to the same space group >>> P212121. But they have different number of molecule per asymmetric unit. >>> Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the >>> moment i have problem explaining this issue. Is it normal to have such >>> results? Same protein with different number of molecule/ASU? >>> >>> I've been trying to get some references on this matter but so far i don't >>> really get any
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Thank you very much everyone for the replies. Ok. Here are more details on the work: 1) Crystallization was done using counter diffusion method- in a capillary (for both Space and Ground experiments) 2) Vapor diffusion method was done only for Ground experiments, and produced both Shape 1 and Shape 2 3) The ground crystals usually grow in two forms- as nice single crystal (Shape 1) and another one as needle-like long rods (Shape 2). 4) In space, only Shape 1 crystal was formed 5) Both Ground and Space experiments- temperature was fixed at 20 deg. Space experiment- temperature was reported to us once a week. 6) Shape 2 crystals (the rods) diffracted much better, with much lower mosaicity range, produced good data processing as compared to Shape 1 (Shape 1 Space crystal's mosaicity range= 0.5-0.96! ) 7) Both Shape 1 and Shape 2 have different cell dimensions, with different volumes (Shape 1= ~ 2X of Shape 2) 8) Both structures from Shape 1 and Shape 2- solved by molecular replacement methods- no major structural changes between them Thank you. Regards, Fairolniza p/s: And thank you, Dr. Snell for the paper. I already have the paper and it has been one of my favorite references :) On Sun, Jul 10, 2011 at 10:58 PM, Artem Evdokimov wrote: > I would not rule out pure chance. Crystals of the same protein can and > often do grow in two (or more!) different forms, from 'the same conditions' > and often in the very same drop. In this case it's the same space group, but > presumably different cell dimensions? Until a significant number of crystals > from each condition (lab vs space) are analyzed, there's no reason not to > believe that it was just luck :) > > Arten > > On Sun, Jul 10, 2011 at 9:53 AM, Jim Pflugrath > wrote: > >> ** >> OK, same space group, but you didn't indicate what the unit cells were. >> They are different, right? >> >> -- >> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of >> *ferrol shariff >> >> *Sent:* Sunday, July 10, 2011 3:10 AM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* [ccp4bb] Same protein, different molecule numbers per ASU >> >> Hello and good day to everyone! :) >> >> I have some general questions on crystallography work. I hope you don't >> mind giving me some ideas. >> >> I have solved my lipase protein both ground-grown crystals and space-grown >> crystals with good resolutions (1.4A and 2.2A). They are the same protein >> from the same source, same purification methods, and produced crystals from >> the same crystallization conditions (except the gravity part). >> >> From the data, it shows that both of them belong to the same space group >> P212121. But they have different number of molecule per asymmetric unit. >> Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the >> moment i have problem explaining this issue. Is it normal to have such >> results? Same protein with different number of molecule/ASU? >> >> I've been trying to get some references on this matter but so far i don't >> really get anything that can directly explain it. Furthermore, do i need to >> relate this with the gravity effect? >> >> I hope you don't mind sharing some experiences on crystallography >> especially regarding this matter. >> >> Thank you very much >> >> -- >> FAIROLNIZA >> >> "The advantage of the emotions is that they lead us astray, and the >> advantage of science is that it is not emotional" >> -Oscar Wilde, The Picture of Dorian Gray, 1891 >> > > -- FAIROLNIZA "The advantage of the emotions is that they lead us astray, and the advantage of science is that it is not emotional" -Oscar Wilde, The Picture of Dorian Gray, 1891
Re: [ccp4bb] Same protein, different molecule numbers per ASU
I would not rule out pure chance. Crystals of the same protein can and often do grow in two (or more!) different forms, from 'the same conditions' and often in the very same drop. In this case it's the same space group, but presumably different cell dimensions? Until a significant number of crystals from each condition (lab vs space) are analyzed, there's no reason not to believe that it was just luck :) Arten On Sun, Jul 10, 2011 at 9:53 AM, Jim Pflugrath wrote: > ** > OK, same space group, but you didn't indicate what the unit cells were. > They are different, right? > > -- > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *ferrol > shariff > > *Sent:* Sunday, July 10, 2011 3:10 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Same protein, different molecule numbers per ASU > > Hello and good day to everyone! :) > > I have some general questions on crystallography work. I hope you don't > mind giving me some ideas. > > I have solved my lipase protein both ground-grown crystals and space-grown > crystals with good resolutions (1.4A and 2.2A). They are the same protein > from the same source, same purification methods, and produced crystals from > the same crystallization conditions (except the gravity part). > > From the data, it shows that both of them belong to the same space group > P212121. But they have different number of molecule per asymmetric unit. > Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the > moment i have problem explaining this issue. Is it normal to have such > results? Same protein with different number of molecule/ASU? > > I've been trying to get some references on this matter but so far i don't > really get anything that can directly explain it. Furthermore, do i need to > relate this with the gravity effect? > > I hope you don't mind sharing some experiences on crystallography > especially regarding this matter. > > Thank you very much > > -- > FAIROLNIZA > > "The advantage of the emotions is that they lead us astray, and the > advantage of science is that it is not emotional" > -Oscar Wilde, The Picture of Dorian Gray, 1891 >
Re: [ccp4bb] Same protein, different molecule numbers per ASU
OK, same space group, but you didn't indicate what the unit cells were. They are different, right? _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ferrol shariff Sent: Sunday, July 10, 2011 3:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Same protein, different molecule numbers per ASU Hello and good day to everyone! :) I have some general questions on crystallography work. I hope you don't mind giving me some ideas. I have solved my lipase protein both ground-grown crystals and space-grown crystals with good resolutions (1.4A and 2.2A). They are the same protein from the same source, same purification methods, and produced crystals from the same crystallization conditions (except the gravity part). >From the data, it shows that both of them belong to the same space group P212121. But they have different number of molecule per asymmetric unit. Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the moment i have problem explaining this issue. Is it normal to have such results? Same protein with different number of molecule/ASU? I've been trying to get some references on this matter but so far i don't really get anything that can directly explain it. Furthermore, do i need to relate this with the gravity effect? I hope you don't mind sharing some experiences on crystallography especially regarding this matter. Thank you very much -- FAIROLNIZA "The advantage of the emotions is that they lead us astray, and the advantage of science is that it is not emotional" -Oscar Wilde, The Picture of Dorian Gray, 1891
Re: [ccp4bb] Same protein, different molecule numbers per ASU
Hi Fairolniza, There is little detail to go on here - I'd double check everything including the processing. All things being equal, even with reduced acceleration, you should have the same crystal form. That said, if there is a difference, I suspect it has little to do with any reduced acceleration directly on the growth process - Brownian motion probably dominates at this scale. Not having many details to go on and assuming the growth geometry used on the ground was similar to that on the flight I'd look to temperature as a possible culprit. Temperature control in a reduced acceleration environment is difficult (greatly reduced convection) and requires power (limited on a space vehicle). If I remember correctly Lipase has a pH dependent structural change between an open and closed state. I'd check your buffer dependance on temperature, the conditions recorded on the ground and in the flight, and check if the growth has occurred at the same pH in both. If not match the ground crystal experiment to that of the on orbit one (probably negatively biasing the ground). I assume you'd tried molecular replacement and know if there are any major structural changes between the two samples? Another possibility, although I'm not sure why it should cause a difference in the number of molecules per asymmetric unit, might be the presence of Marangoni convection. In vapor diffusion this is masked on the ground (unless you have volatile organic components) but present with reduced acceleration. For a shameless plug check out Macromolecular Crystallization in Microgravity, Snell and Helliwell, Reports in Progress in Physics, 68, 799-853 (2005) (http://iopscience.iop.org/0034-4885/68/4/R02). This has extensive details of the work in this field and some of the points above. Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Senior Scientist, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! __ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of ferrol shariff [ferrol2...@gmail.com] Sent: Sunday, July 10, 2011 4:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Same protein, different molecule numbers per ASU Hello and good day to everyone! :) I have some general questions on crystallography work. I hope you don't mind giving me some ideas. I have solved my lipase protein both ground-grown crystals and space-grown crystals with good resolutions (1.4A and 2.2A). They are the same protein from the same source, same purification methods, and produced crystals from the same crystallization conditions (except the gravity part). >From the data, it shows that both of them belong to the same space group >P212121. But they have different number of molecule per asymmetric unit. >Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the moment >i have problem explaining this issue. Is it normal to have such results? Same >protein with different number of molecule/ASU? I've been trying to get some references on this matter but so far i don't really get anything that can directly explain it. Furthermore, do i need to relate this with the gravity effect? I hope you don't mind sharing some experiences on crystallography especially regarding this matter. Thank you very much -- FAIROLNIZA "The advantage of the emotions is that they lead us astray, and the advantage of science is that it is not emotional" -Oscar Wilde, The Picture of Dorian Gray, 1891
[ccp4bb] Same protein, different molecule numbers per ASU
Hello and good day to everyone! :) I have some general questions on crystallography work. I hope you don't mind giving me some ideas. I have solved my lipase protein both ground-grown crystals and space-grown crystals with good resolutions (1.4A and 2.2A). They are the same protein from the same source, same purification methods, and produced crystals from the same crystallization conditions (except the gravity part). >From the data, it shows that both of them belong to the same space group P212121. But they have different number of molecule per asymmetric unit. Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the moment i have problem explaining this issue. Is it normal to have such results? Same protein with different number of molecule/ASU? I've been trying to get some references on this matter but so far i don't really get anything that can directly explain it. Furthermore, do i need to relate this with the gravity effect? I hope you don't mind sharing some experiences on crystallography especially regarding this matter. Thank you very much -- FAIROLNIZA "The advantage of the emotions is that they lead us astray, and the advantage of science is that it is not emotional" -Oscar Wilde, The Picture of Dorian Gray, 1891
