Re: [ccp4bb] Redundancy vs no of frames
If you spin the crystal through a full 360 degrees then any given relp
(unique reciprocal lattice point) will pass through the Ewald sphere no
more than twice. This is because the Ewald sphere is a surface and the
relp is taking a circular path. It is either inside the Ewald sphere
or outside, and transitions once in-to-out and once out-to-in in a
closed loop. Some relps are always outside the Ewald sphere and their
circular paths are too small to ever enter into it. These tend to be
close to the rotation axis, and the reason why it is formally impossible
to get 100% completeness when your space group is P1 and you only have
one rotation axis. You can keep spinning, of course, and then your
redundancy is 2x the number of spins, but since you are using the same
pixels over and over again, it isn't really multiplicity, to use the
flippant definition from my previous post.
Now, of course, multiplicity/redundancy generally includes symmetry
mates, and even in P1 you have Friedel symmetry. The circle traced out
by the Friedel mate (-h,-k,-l) is a mirror image of the (h,k,l) circle
in the plane normal to the rotation axis. That is, -h,-k,-l is always on
the opposite side of the origin from h,k,l and also at the same
d-spacing, so its circle is the same radius and the same distance from
the origin as the h,k,l circle, just on the opposite side of the beam.
This circle also crosses the Ewald sphere twice, and always on different
pixels than h,k,l.
If your space group is not P1, then your multiplicity per revolution
will therefore be 4*n, where n is the number of real-space symmetry
operations. That is, the number of x,y,z-ish lines you see under the
space group in ${CLIB}/symop.lib is the n that you want.
If you don't do a full 360-degree rotation, then the relationship to
multiplicity gets noisier, you may see some spots more than once long
before you have even one of another. But, if you want a rule of thumb,
your coverage of reciprocal space is roughly 4*n*revolutions, where
revolutions can be a fraction.
As for the right definition of redundancy vs multiplicity, people seem
quite adamant to stick with whatever term is used in the log file of
their favorite processing program denzo uses redundancy, but
scala/aimless use multiplicity. Funny how terms get defined this
way. Perhaps the best way to change terminology for good is to write a
really amazing computer program that everyone will have to use and make
it print out the term you like?
-James Holton
MAD Scientist
On 1/17/2015 1:05 PM, rohit kumar wrote:
Dear all,
Can anyone tell me how to calculate number of frames from redundancy
or vica versa
Thank you
[ccp4bb] phosphoprotein crystallization
I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj
Re: [ccp4bb] phosphoprotein crystallization
Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] coot 0.8.1 freezes my graphics
On 18/01/15 18:29, Kenneth A. Satyshur wrote: Coot is our best program there ever was for fitting electron density. It is very simple to use and easy to teach. But sometimes improvements are made that seem to just slow things down. Having used 0.7.1 for a long time, i noticed that rotation and scaling of density is very quick, but translations are much slower (cntrl left mouse). Now in 0.8.1, translations have become impossibly slow. They freeze the whole graphics system for as long as 10 seconds. It is recalculating a map, and this can be seen using top to display the cpu usage. Let me encourage you to read section 9.12 of the manual. Here's the link: http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/docs/coot.html#Slow-Computer-Configuration Also, we are now given a spherical density, which is annoying, You are not the first disapprove of the change - others have been somewhat less forthright, however. really, since cell edges are not spherical and its hard to examine 3D space in a bubble. But there must be a way of speeding up the recalculation of the map during translation. I have a dual quad core with 15 idle cores while coot recalculates its new map in one core. Maybe multi core is the way to go. I hope this can be fixed. I believe that there have been advances in rendering 3D scalar data (such as medical imaging) using the GPU. It would be nice if an implementation of such were ported to Coot https://github.com/smistad/GPU-Marching-Cubes Otherwise I will stick with the 0.7 version that makes a cuboid map. I really enjoy the rapid examination of density that coot provides especially in 3D. I'll consider adding a display-density-as-the-old-box configuration flag. thanks to the Coot team. You're welcome. Paul.
Re: [ccp4bb] phosphoprotein crystallization
Try running a regular SDS page gel w/o boiling your sample. Some P proteins run differently on gel due to the conformational change that is induced. I agree that you probably have a mixture in which the P form might be the minor species. I'd probably try to get the IEX to work; given that the sample sticks poorly to the column one might expect the -2 introduced by the phosphate to make a big difference. Did you try loading the IEX column at low salt (maybe as low as 20 mM or so)? Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Srivastava, Dhiraj [dhiraj-srivast...@uiowa.edu] Sent: Monday, January 19, 2015 5:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] phosphoprotein crystallization Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] phosphoprotein crystallization
Hi, there: If you just want to know the percentage of phosphorylation, I highly recommend phospho-tag gel. You can buy the phospho-tag, and make gels your self. It's very effective. Shawn Toronto On 2015-01-19, at 1:01 PM, Bert Van-Den-Berg wrote: Try running a regular SDS page gel w/o boiling your sample. Some P proteins run differently on gel due to the conformational change that is induced. I agree that you probably have a mixture in which the P form might be the minor species. I'd probably try to get the IEX to work; given that the sample sticks poorly to the column one might expect the -2 introduced by the phosphate to make a big difference. Did you try loading the IEX column at low salt (maybe as low as 20 mM or so)? Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Srivastava, Dhiraj [dhiraj-srivast...@uiowa.edu] Sent: Monday, January 19, 2015 5:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] phosphoprotein crystallization Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] phosphoprotein crystallization
an effective modification to your protein preparations would be to add phosphatase inhibitor tablets during all stages of purification. Pierce sell a combination protease and PI tablet that is effective and economical to be added during protein preps. Also you invitro buffers should have inhibitors http://www.piercenet.com/product/pierce-protease-phosphatase-inhibitor-tablets P On Mon, Jan 19, 2015 at 8:54 AM, Dhiraj Srivastava dhiraj-srivast...@uiowa.edu wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- P
Re: [ccp4bb] ligand bonds (AlF3) breaking up after refinement in refmac
Dear All, I checked the Mg2+ B factor, and it was around 22. So, were the values for F atoms. For AlF3, it was more than 50 for Al and more than 40 for F atoms. I will take care about the occupancy. I hope that will fix the small negative density. When I inserted MgF in COOT using the code MGF (get monomer), it did appear but looked fragmented. It remained fragmented after refinement as well (as shown in the previously attached fig). But, there was no warning about bond breaking in the Refmac log file. Even the density of MgF3- looked better (2Fo-Fc). If I see the structure in Chimera or CCP4MG, it does not look fragmented. So, is it a problem of display in COOT? Is the exploded MgF3 form, not actually a broken form, since the refinement log file does not show any such indication? regards,Ansuman On Monday, 19 January 2015 2:15 PM, Mark van Raaij mjvanra...@cnb.csic.es wrote: It could be a mixture of Mg2+ and Al3+Mark J van Raaij CNB-CSIC www.cnb.csic.es/~mjvanraaijOn 16 Jan 2015 20:07, ansuman biswas bubai_...@yahoo.co.in wrote: Dear users, I have a data at 2.2 A resolution. I am able to model AlF3 into the electron density (fig attached). However after one cycle of refinement the AlF3 molecule is exploding and the atoms move apart (fig2). AlF3 is already present in refmac library. First, I used that. But it broke up after refinement. Then, I extracted AlF3 coordinate from already published PDB and prepared the cif file. But, it also failed. I modified the cif file by changing the bond lengths according to the broken AlF3 structure but it was of no help.Kindly suggest how to carry out the refinement. regards,Ansuman
Re: [ccp4bb] Redundancy vs no of frames
Don't forget, multiplicity has its own negative connotations. http://www.imdb.com/title/tt0117108/ Shane Caldwell McGill University On Sun, Jan 18, 2015 at 12:26 PM, Edward A. Berry ber...@upstate.edu wrote: Also RAID (REDUNDANT array of inexpensive disks). To me redundancy implies robustness, overdetermination, like when I measure absorbance at 1500 wavelengths to calculate the concentration of five absorbing species with a 2-parameter baseline offset. Exactly the connotation we want for our more-than-complete datasets. eab On 01/18/2015 09:29 AM, Ian Tickle wrote: PS see http://en.wikipedia.org/wiki/Cyclic_redundancy_check . I. On 18 January 2015 at 13:54, Ian Tickle ianj...@gmail.com mailto: ianj...@gmail.com wrote: At the risk of further extending this philosophical (if not etymological) discussion: in further defence of 'redundancy' I would point out that 'no longer needed' is not the only meaning of 'redundant', though admittedly it is the one that most often grabs the headlines! The meaning of 'redundant' in the context of employment is actually a relatively recent one and somewhat changed from the original meaning. In a scientific context there's a second meaning of 'redundant', and in fact this one is much closer to the original one. In information theory the term 'redundant' applies to extra information added to a message being passed down a transmission line, in order to reduce corruption and loss of information, i.e. redundancy is absolutely needed to reduce the error rate. In a crystallographic context the purpose of redundancy, i.e. measurements over and above those strictly required to obtain a structure, is also obviously to reduce errors. 'Additional' here clearly does not necessarily imply 'not needed'. 'Redundant' comes from the Latin 're', meaning 'again', and 'unda', meaning 'wave', from which of course we get 'inundated' and 'undulator', so 'redundant' means literally 'coming in waves' or 'overflowing'. So we could say that redundancy is the process of being inundated by data from an undulator! As BR points out 'multiplicity' has long been used to indicate the number of equivalent reflexions generated by the point-group symmetry (so in PG222 h00, hk0 and hkl have respectively multiplicites of 1, 2 and 4 for non-zero hkl). I googled 'reflection multiplicity' and the top hit was http://pd.chem.ucl.ac.uk/pdnn/symm2/multj.htm . Suppose I want to express the following idea: Redundancy is likely to be correlated with multiplicity. How do I express that unambiguously if 'redundancy' is redefined as 'multiplicity'? Cheers -- Ian On 18 January 2015 at 13:12, Bernhard Rupp b...@ruppweb.org mailto: b...@ruppweb.org wrote: In defense of redundancy: While the IUCr online dictionary is notably silent about multiplicity, the term itself seems already oversubscribed and used differently in various crystallographic contexts. (i) Each general or special position in a crystal structure has a certain multiplicity, defined by symmetry. (ii) General reflection multiplicity M is usually is defined by reflection symmetry, and when reflections are affected by special operations, the resulting corresponding lower multiplicity because they map onto themselves is accounted for in the epsilon factor e. Btw a useful table of M and e is Iwasaki Ito Acta Cryst. (1977). A33, 227-229 (iii) In case of Laue patterns, overlap of higher order reflections is also called Multiplicity afaik (various Helliwell/Moffat et al papers explain this). So expanding the term multiplicity to include multiple instances of measurements of the same reflections - while admittedly avoiding the connotation of obsolescence - adds to its promiscuous meaning, where context becomes part of the definition I abstain from making any suggestions because in the past this has led to interesting but time-consuming philosophical discourse, proving in general the multiplicity of my reflections and positions redundant if not obsolete. Best, BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto: CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Sonntag, 18. Januar 2015 09:28 To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Redundancy vs no of frames Dear Rohit Kumar, I prefer the term multiplicity instead of redundancy because the latter has a connotation of not really needed any more. The relation then is multiplicity = c * number_of_frames * oscillation_range where the constant c depends mainly on the space group. HTH, Kay On Sun, 18 Jan 2015 02:35:46 +0530, rohit kumar
