Re: [ccp4bb] Solving the structure
On 06/24/2011 08:50 AM, mullapudi edukondalu wrote: Dear Members, I have my first data set on one of my protein crystals, that diffract to 2.7 A, and the space group is I222. According to Mathews coefficient, there should be 4 molecules in the asymmetric unit. But, when I run molecular replacement programme it found only 3, and the crystal packing is not that good. I have 100% similar model. And Ref mac gave me an Rfree of 46%. Could any one of you please suggest me how to go further with this problem? Looking forward to hear from you. Best Regards Edukondalu Mullapudi I guess you tried I 212121 to as a possible spacegroup? Eleanor
Re: [ccp4bb] MR question
Well - it isnt surprising that all your geometry is good at the start. You have fitted a refined structure against a a different crystal form, so the first geometry report relates to your starting model which will not be the true model which fits your new data. Refinement has to push that model about to get to the truth, and inevitably that will degrade the geometry. That is good - not bad.. My usual approach is to let the refinement rip as long as the Rfactors are falling, then use the bad geometry indicators to pinpoint where things need to be changed. You will find many such regions where side chains have moved, and even loops - often very easy to correct in coot. If you cant see what to do set the occupancies of the changed reegions to 0.00, do a few more cycles of refinement and see if the difference maps indicate where that part of the structure should go.. Eleanor On 06/22/2011 07:25 AM, Careina Edgooms wrote: Dear ccp4 members I have something that surprises me with molecular replacement. I have obtained a solution for a single point mutation using Phaser, the solution seems ok. I do one round of refinement with refmac and I check the structure using molprobity before I even start really to refine it. The structure looks very good. MolProbity gives all green lights. Then I start to fix the structure, I add waters, I check fit to density, rotamers, geometry etc, I do some more refinements. I check with MolProbity and it looks a lot worse... many clashes, many bad ramachandran and rotamers, many red lights. I do not understand. How can each successive round of refinement make the structure worse and worst? Is there a fundamental problem, perhaps, like undiagnosed NCS or incorrect space group or incorrect MR solution? Could they be giving these strange result do you think? Thanks Careina
Re: [ccp4bb] Kd's in Crystals
Jacob, In case if the hint that I sent yesterday was not clear, below is the solution for the equation Kd=[P][L]/[PL] in terms of ligand occupancy: O=[ PL]/[Po]= 1/(Kd/L+1) You see, it does not depend on [Po] Alex On Jun 26, 2011, at 10:05 AM, aaleshin wrote: The concentration of a protein in a crystal [Po] and the volume of a crystal V are needed only to calculate the total amount of a ligand [Lo] required for soaking. [Lo] [Po]*V The occupancy of the active sites in a crystal will depend only on the ligand concentration in solution and Kd. It does not depend on protein concentration in the crystal. Indeed: Kd=[P][L]/[PL] Assuming total concentration of the protein = Po, Kd= 1mM and S= 1 mM, the active site occupancy will be: 1= P/Po-P; P/Po=1/2 So the concentration of the ligand in solution should be Kd to get the full occupancy. Alex
Re: [ccp4bb] Kd's in Crystals
Yes, I think you are right--the somewhat counterintuitive case I was thinking of was, for example, when: Kd = 20nM [L] = 20uM [Po in crystal] = 20mM In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the occupancy should be ~100%, and [PL] at equilibrium should be about 20mM, so in the crystal, the total [L] should be ~20mM. This explains, among other things, why bromophenol blue makes crystals bluer than the surrounding solution--the Kd is probably significantly lower than the BB concentration in the drop. Thanks for your clarifications! Jacob The question would remain, then, whether there is any utility in titrating ligands into crystals, and monitoring occupancies as a readout for binding. Although crystallization conditions are horribly non-physiological, perhaps there would be utility in the case where there are multiple known binding sites of various affinities, and other methods would have trouble resolving the binding events. One could start with: 1. totally saturated conditions, set occ=1 for all sites, refine B's, then 2. fix B's at this value, and refine the occ's in a subsequent series of dilutions. All of this is not totally theoretical--I am considering a set of experiments along these lines, where there really are multiple sites of varying affinity. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Way off topic.. less than 96 well culture plates in black?
Hi all Sorry for the off topic post, but given the breadth of experience on this bb, I would like to ask. I need to measure fluorescence from bacterial cell cultures in a 12/24/48 cell culture plate (the number of wells is unimportant). As this is a fluorescence measurement, I require them to be black on the walls. I'm looking for a supplier / catalog number of such plates. To save bb spam, I'll take replies privately. Thanks! F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Kd's in Crystals
Hi, We had a paper where we looked at Kd of arginine in the arginine repressor-DNA complex (p. 248-249). JMB,2010, *399*, pp.240-254. Maia Jacob Keller wrote: Yes, I think you are right--the somewhat counterintuitive case I was thinking of was, for example, when: Kd = 20nM [L] = 20uM [Po in crystal] = 20mM In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the occupancy should be ~100%, and [PL] at equilibrium should be about 20mM, so in the crystal, the total [L] should be ~20mM. This explains, among other things, why bromophenol blue makes crystals bluer than the surrounding solution--the Kd is probably significantly lower than the BB concentration in the drop. Thanks for your clarifications! Jacob The question would remain, then, whether there is any utility in titrating ligands into crystals, and monitoring occupancies as a readout for binding. Although crystallization conditions are horribly non-physiological, perhaps there would be utility in the case where there are multiple known binding sites of various affinities, and other methods would have trouble resolving the binding events. One could start with: 1. totally saturated conditions, set occ=1 for all sites, refine B's, then 2. fix B's at this value, and refine the occ's in a subsequent series of dilutions. All of this is not totally theoretical--I am considering a set of experiments along these lines, where there really are multiple sites of varying affinity. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Kd's in Crystals
Jacob, In the formula: Kd=[P][L]/[PL] [P] and [L] are concentrations of UNBOUND protein and ligand, and [PL] is that in the complex. Since the occupancy of the ligand in the crystal is [ PL]/[Po]= 1/(Kd/L+1), varying [L] around Kd like from 0.1Kd to 10Kd will make the titration of occupancy. You can calculate from the provided formula which [L] will give 0.25, 0.5 and 0.75 occupancies. Forget that the protein is crystallized. We assume that its behavior has not changed due to it. In reality, ligand affinity of conformationally flexible proteins can change by many orders of magnitude in both directions. This is why soaking does not work sometimes and you have to do co-crystallization. If you decide to titrate a crystal with a ligand, you should collect data and refine the ligandless and fully-ocupied crystals first, then use the superimposition of their structures for refinement of all other cases. Take care of waters that substitute for the partially bound ligand, they should have occupancies =1-Occ_of_ligand. Good luck. Alex On Jun 27, 2011, at 10:04 AM, Jacob Keller wrote: Yes, I think you are right--the somewhat counterintuitive case I was thinking of was, for example, when: Kd = 20nM [L] = 20uM [Po in crystal] = 20mM In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the occupancy should be ~100%, and [PL] at equilibrium should be about 20mM, so in the crystal, the total [L] should be ~20mM. This explains, among other things, why bromophenol blue makes crystals bluer than the surrounding solution--the Kd is probably significantly lower than the BB concentration in the drop. Thanks for your clarifications! Jacob The question would remain, then, whether there is any utility in titrating ligands into crystals, and monitoring occupancies as a readout for binding. Although crystallization conditions are horribly non-physiological, perhaps there would be utility in the case where there are multiple known binding sites of various affinities, and other methods would have trouble resolving the binding events. One could start with: 1. totally saturated conditions, set occ=1 for all sites, refine B's, then 2. fix B's at this value, and refine the occ's in a subsequent series of dilutions. All of this is not totally theoretical--I am considering a set of experiments along these lines, where there really are multiple sites of varying affinity. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Kd's in Crystals
Hi, You may want to have a look at the two papers below. Experimental determination of van der waals energies in a biological system. Wear MA, Kan D, Rabu A, Walkinshaw MD. Angew Chem Int Ed Engl. 2007;46(34):6453-6. The First Direct Determination of a Ligand Binding Constant in Protein Crystals. Wu Sy S, Dornan J, Kontopidis G, Taylor P, Walkinshaw MD. Angew Chem Int Ed Engl. 2001 Feb 2;40(3):582-586. Regards Iain Quoting Jacob Keller j-kell...@fsm.northwestern.edu on Mon, 27 Jun 2011 12:04:35 -0500: Yes, I think you are right--the somewhat counterintuitive case I was thinking of was, for example, when: Kd = 20nM [L] = 20uM [Po in crystal] = 20mM In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the occupancy should be ~100%, and [PL] at equilibrium should be about 20mM, so in the crystal, the total [L] should be ~20mM. This explains, among other things, why bromophenol blue makes crystals bluer than the surrounding solution--the Kd is probably significantly lower than the BB concentration in the drop. Thanks for your clarifications! Jacob The question would remain, then, whether there is any utility in titrating ligands into crystals, and monitoring occupancies as a readout for binding. Although crystallization conditions are horribly non-physiological, perhaps there would be utility in the case where there are multiple known binding sites of various affinities, and other methods would have trouble resolving the binding events. One could start with: 1. totally saturated conditions, set occ=1 for all sites, refine B's, then 2. fix B's at this value, and refine the occ's in a subsequent series of dilutions. All of this is not totally theoretical--I am considering a set of experiments along these lines, where there really are multiple sites of varying affinity. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** Dr. Iain McNae School of Biological Sciences Institute of Structural and Molecular Biology The University of Edinburgh Kings Building?s Mayfield Road Edinburgh EH9 3JR Fax 0131 650 7055 Telephone 0131 650 7052 Mobile 07969 304 852 -- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
[ccp4bb] Post-doctoral position available
A post-doctoral position is available in the group of Dr. Petri Kursula, to study structure-function relationships in proteins specifically expressed in the myelin sheath. The targets of the project are membrane-associated myelin proteins, which have functions e.g. in the interactions between the myelin sheath and the axon, and in the compaction of the multilayered myelin membrane. We are specifically interested in the structures of these proteins and their complexes, their interactions with macromolecular and small-molecule ligands, as well as membranes. The project will involve e.g. large-scale recombinant expression of myelin proteins or domains thereof, their biophysical characterization, X-ray crystallography and other structural biology methods, as well as more specific methods, such as oriented CD spectroscopy and other membrane interaction assays. The ideal candidate will have: - PhD in biochemistry/structural biology or a closely related field - a good publication record - significant hands-on experience in recombinant protein expression and large-scale purification - a genuine interest in structural biology - fluency in English - experience in supervising junior colleagues Previous work with extracellular domains or peripheral membrane proteins will be considered an asset, as will experience in versatile structural biology and biophysical methods. The position is initially available for 1 year, with possibilities for extension for up to 3 years. The selected candidate will be affiliated with the Department of Biochemistry, University of Oulu, Finland, but a large part of the work is expected to be carried out at the Centre for Structural Systems Biology (CSSB-HZI), on-site the DESY synchrotron campus, Hamburg. More information about our group can be found at www.biochem.oulu.fi/kursula. To apply, please send your cv, including list of publications, plus the names and email addresses of 2-3 referees by email to petri.kurs...@oulu.fi. The application deadline is July 31st, 2011. --- Petri Kursula, PhD Group Leader and Docent of Neurobiochemistry (University of Oulu, Finland) Visiting Scientist (CSSB-HZI, DESY, Hamburg, Germany) www.biochem.oulu.fi/kursula www.desy.de/~petri petri.kurs...@oulu.fi petri.kurs...@desy.de ---
Re: [ccp4bb] Y-Chi2 running out of chart
Jim, that's the first thing I tried - triclinic gave similar Y-Chi2 profile. Shiva, not sure what you mean by collecting the same crystal again (thaw and remount?). But I collected 180 degrees, Chi2 seems to correlate with rotation angle. Cheers, On Fri, Jun 24, 2011 at 1:39 PM, Jim Pflugrath jim.pflugr...@rigaku.comwrote: ** Instead of an imperfect crystal, this can also occur if one chooses the wrong Bravais lattice type (or spacegroup) to integrate. For example, if you choose tetragonal when it is really orthorhombic with a ~ b, or if you choose orthorhombic and beta is 90.2, then you can see that trying to force that unit cell will lead to higher residuals during positional refinement.If one reciprocal lattice is oriented mostly along Y, then I think what Bing observes can happen in such cases. This is easily tested by integrating in triclinic. -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Shiva Bhowmik *Sent:* Friday, June 24, 2011 11:42 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Y-Chi2 running out of chart Hi Bie, Curious to know what are the cell parameters obtained after scaling? You mention observing perfect Chi2 statistics with lysozyme crystals. But are you observing the same Chi2 statisctis with the crystal that yielded unusual Y-Chi2 if you collect another dataset. If there is a consistency of observing the unusual Y-Chi2 with that crystal again then it is likey that the crystal maybe highly imperfect. If not, then there could have been a one time un-nailed problem occurred during that collection. Cheers, Shiva On Thu, Jun 23, 2011 at 8:59 AM, bie gao gao...@gmail.com wrote: Dear colleagues, thank you all for your help! I really appreciate it. I did perform a lysozyme test after the repair. I collected ~50 degree (99%). Everything seems to be fine. Maybe I should have done it for an entire round. As for the current collection, as I suspended, it did go out of the chart but then came down to normal. Overall Rmerge is 7.9% (4% square). As Zbyszek and others mention, it is probably due to imperfect crystal and also uneven cooling. If our field engineer discovers anything else, I'll post it here. Thanks again for your help. On Wed, Jun 22, 2011 at 9:00 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: As a follow up to the excellent suggestions made by others I would suggest that a close examination of x-file headers may reveal abnormalities in e.g. crystal orientation -- suspecting an unlocked or drifting goniostat. It may also indicate a precession around the phi, which should also manifest itself in a systematic deviation of average intensities (i.e. scale factors) in a similar pattern (assuming uneven illumination of the crystal). Sometimes the precession is caused by a bubble or a tiny chunk of ice trapped under the pin, it can melt unevenly and re-align the pin a few minutes into the run (something similar used to happen a lot at one or two beam lines and it drove me nuts until I figured out the need to re-align the crystal after the initial screening). Artem On Wed, Jun 22, 2011 at 11:22 AM, bie gao gao...@gmail.com wrote: Dear Colleagues, I'm collecting a dataset on our recently repaired Rigaku home source. Crystal diffracts to 2.2A. Indexing seems to be all fine. However, during integration, I realize Y-Chi2 is increasing constantly (from 2 to 4.5, almost linear) within 60 degree collection, whereas X-Chi2 stays the same. An image is attached. There are still another 60 degree to go. Although the prediction fits the images well so far, I'm afraid the Y-Chi2 will eventually run out of the chart. My question is could it be related to any hardware malfunctioning, i.e., goniometer, image plates, etc, which may be a side effect of the recent major repair? Or what else it can be? Thanks, Bing
Re: [ccp4bb] Y-Chi2 running out of chart
BTW, I've collected a better dataset with reasonable Chi2. Seems like the first crystal is imperfect. Thank everyone for your help! On Mon, Jun 27, 2011 at 7:59 PM, bie gao gao...@gmail.com wrote: Jim, that's the first thing I tried - triclinic gave similar Y-Chi2 profile. Shiva, not sure what you mean by collecting the same crystal again (thaw and remount?). But I collected 180 degrees, Chi2 seems to correlate with rotation angle. Cheers, On Fri, Jun 24, 2011 at 1:39 PM, Jim Pflugrath jim.pflugr...@rigaku.comwrote: ** Instead of an imperfect crystal, this can also occur if one chooses the wrong Bravais lattice type (or spacegroup) to integrate. For example, if you choose tetragonal when it is really orthorhombic with a ~ b, or if you choose orthorhombic and beta is 90.2, then you can see that trying to force that unit cell will lead to higher residuals during positional refinement.If one reciprocal lattice is oriented mostly along Y, then I think what Bing observes can happen in such cases. This is easily tested by integrating in triclinic. -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Shiva Bhowmik *Sent:* Friday, June 24, 2011 11:42 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Y-Chi2 running out of chart Hi Bie, Curious to know what are the cell parameters obtained after scaling? You mention observing perfect Chi2 statistics with lysozyme crystals. But are you observing the same Chi2 statisctis with the crystal that yielded unusual Y-Chi2 if you collect another dataset. If there is a consistency of observing the unusual Y-Chi2 with that crystal again then it is likey that the crystal maybe highly imperfect. If not, then there could have been a one time un-nailed problem occurred during that collection. Cheers, Shiva On Thu, Jun 23, 2011 at 8:59 AM, bie gao gao...@gmail.com wrote: Dear colleagues, thank you all for your help! I really appreciate it. I did perform a lysozyme test after the repair. I collected ~50 degree (99%). Everything seems to be fine. Maybe I should have done it for an entire round. As for the current collection, as I suspended, it did go out of the chart but then came down to normal. Overall Rmerge is 7.9% (4% square). As Zbyszek and others mention, it is probably due to imperfect crystal and also uneven cooling. If our field engineer discovers anything else, I'll post it here. Thanks again for your help. On Wed, Jun 22, 2011 at 9:00 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: As a follow up to the excellent suggestions made by others I would suggest that a close examination of x-file headers may reveal abnormalities in e.g. crystal orientation -- suspecting an unlocked or drifting goniostat. It may also indicate a precession around the phi, which should also manifest itself in a systematic deviation of average intensities (i.e. scale factors) in a similar pattern (assuming uneven illumination of the crystal). Sometimes the precession is caused by a bubble or a tiny chunk of ice trapped under the pin, it can melt unevenly and re-align the pin a few minutes into the run (something similar used to happen a lot at one or two beam lines and it drove me nuts until I figured out the need to re-align the crystal after the initial screening). Artem On Wed, Jun 22, 2011 at 11:22 AM, bie gao gao...@gmail.com wrote: Dear Colleagues, I'm collecting a dataset on our recently repaired Rigaku home source. Crystal diffracts to 2.2A. Indexing seems to be all fine. However, during integration, I realize Y-Chi2 is increasing constantly (from 2 to 4.5, almost linear) within 60 degree collection, whereas X-Chi2 stays the same. An image is attached. There are still another 60 degree to go. Although the prediction fits the images well so far, I'm afraid the Y-Chi2 will eventually run out of the chart. My question is could it be related to any hardware malfunctioning, i.e., goniometer, image plates, etc, which may be a side effect of the recent major repair? Or what else it can be? Thanks, Bing
