Re: [ccp4bb] X-ray photon correlation length
Dear James, what an interesting discussion! Am 30.01.2009 um 19:42 schrieb James Holton: ... I think the coherence length is related to how TWO different photons can interfere with each other, and this is a rare event indeed. It has nothing to do with x-ray diffraction as we know it. No matter how low your flux is, even one photon per second, you will eventually build up the same diffraction pattern you get at 10^13 photons/s. Colin is right that photons should be considered as waves and on the length scale of unit cells, it is a very good approximation to consider the electromagnetic wave front coming from the x-ray source to be a flat plane, as Bragg did in his famous construction. This is also my current understanding, since no matter what the longitudinal coherence (spectral purity) or transversal coherence (size of the source and detector distance) of the X-ray beam is, there is no time coherence in the beam, neither for rotating anode generators, nor for undulator beamlines (see Lengeler, Naturwissenschaften, Vol. 88, p 249-260; it is in English). Apparently, even a single photon sees the whole crystal as a wave and deposits its energy as a particle with a probability according to Bragg's law. ... Now, if a perfect crystal is really really small (much smaller than the interaction length of scattering), then there is no opportunity for the re-scattering and extinction and all that weird stuff to happen. In this limiting case, the scattered intensity is simply proportional to the number of unit cells in the beam and also to |F| ^2. This is the basic intensity formula that Ewald showed how to integrate over all the depleting beams and re-scattering stuff to explain a large perfect crystal. ... I'm not sure where this rumor got started that the intensity reflected from a mosaic block or otherwise perfect lattice is proportional to the square of the number of unit cells. This is never the case. The reason is explained in Chapter 6 of M. M. Woolfson's excellent textbook, but the long and short of it is: yes the instantaneous intensity (photons/steradian/s) at the near- infinitesimal moment when a mosaic domain diffracts is proportional to the number of unit cells squared, but this is not useful because x-ray beams are never perfectly monochromatic nor perfectly parallel. Hmm, I don't have Woolfson's book at hand, so I can't read this chapter. My current understanding is: if N unit cells scatter in phase, the scattered total amplitude is N times the scattered amplitude of the unit cell in that direction. Since the recorded intensity is proportional to the square of the amplitude, the scattered total intensity is proportional to N^2 (for simplicity, I assume perfect sources, crystals and detectors, so I don't discuss spot shapes). Now, if the coherence lengths is limited by the size of the mosaic blocks, each block scatters like a tiny crystal independent of the other mosaic blocks. Thus, if we have m N mosaic blocks (of equal size), each block results in a scattered intensity ~(N/m)^2, and the m blocks add their intensities, yielding a total intensity ~N^2/m. Dependent of the perfection of the crystal, the total scattering intensity for the two extremes between m=N (which wouldn't make any sense) and m=1 is proportional to between N and N^2, respectively. Please, correct me, if I'm wrong. Best regards, Dirk. *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@lmb.uni-muenchen.de ***
[ccp4bb] correlation length - reloaded
Thanks Colin, Yours is one of several lessons I have received on coherence and optics in the past few days. I do appreciate all effort and the input. However, as I mentioned before I am a biologist and I don't so much care about the fundamental nature of the universe as I do about how it relates to helping people solve protein structures. So, I think I would like to perhaps re-phrase Bernhard's question, but primarily ask a question of my own: __ Is there a way to use a fancy x-ray beam to overcome lattice pathologies ... such as, shall we say: turn a nightmare like a lattice translocation defect into a simple merohedral twin? __ That is, I think this whole discussion started with those streaky spots Margriet Ovaere posted last week. We have all seen streaky spots and wondered what they mean, but far more important than that we wanted to get rid of them. So, let me pose the following 4 situations: 1) The easiest kind of merohedral twin to think about is when you have two good crystals stuck together. They are both in the beam and oriented so that their lattices line up and there is no practical detector distance that will resolve the spots produced from crystal A vs crystal B. This is not a problem so long as the overlapping spots correspond to symmetry-equivalent HKL indicies, but if you have one of those annoying space groups (like P4, P3, and others) where the a and b axes are the same length, but non-equivalent, then the a axis of A can be aligned with the b axis of B, and then you have a merohedral twin. You then feel like you are a bad crystallographer for wanting to go and grow new crystals. However, you can be saved from 1) if you have a fancy x-ray beam. That is, shoot just one of the two crystals that are stuck together (either with a small beam, or simply by translating crystal B out of the beam) and voila! De-twinning! I love this beamline and the beamline scientist should get a really really big raise for showing me how to do that! This leads us to: 2) The crystal you have in the beam contains a large number of very small twin domains. Half of them are oriented as A above and the other half as B. This is annoying because you really don't want to learn about twinning and the x-ray beam is not small enough to just shoot one of the A crystals at a time. So, you write a big grant to build a beamline with a smaller x-ray beam. Hooray! Problem solved by physics again. However, there must be a limit to how far you can push this strategy: 3) The twin domains are so small that you cannot go more than a few unit cells in the crystal without stumbling across an A to B boundary. With so few unit cells in each twin domain the scattering from A actually starts to scatter coherently with B inasmuch as the amplitudes and phases are adding instead of simply adding the intensities contributed by each type of twin domain together into each spot. To add to the headache, every other spot in the diffraction pattern is smeary and your beamline scientist tells you that you have something called a lattice translocation defect (then he goes off to lunch and never comes back). This is very annoying because noone seems to distribute a program for removing this problem from your data, and you wonder how much money it will take to build a beamline that will solve this problem for you. Perhaps if you could somehow reduce the coherence length you could graduate from having a lattice translocation defect into merely having a merohedral twin? On the other hand, what if you unit cell gets really big and starts to get comparable to the coherence length? Will that somehow mess up your data? What the heck is a coherence length anyway? 4) The twin domains are only one unit cell each and for every A unit cell there is a B unit cell right next to it and always in the same direction. This is actually a regular crystal (with NCS and twice the unit cell size of 1). It is deviations from the A always next to B rule that I think leads to the streakiness in Margriet's spots (the description I posted last week). That is, I think she is in the twilight zone between 3) and 4). This will perhaps have a pseudotranslation which is another scary word to hear at the beamline, but I don't think there will ever be a fancy x-ray beam that can solve this problem. So, it appears that somewhere between 2) and 3) there is a dark place in x-ray physics? Is there a fancy type of x-ray beam that will let us do some new science here? Perhaps the most relevant definition of correlation length for protein crystallography is: __ How far apart can
Re: [ccp4bb] difficult MAD dataset
Hello - Sorry if some of my suggestions are redundant since I jumped in late in this thread. A couple of additional comments after struggling with a similar albeit not identical case. With a pseudo translation vector like that the SG could be any of the 8 orthorhombic SGs; P222 P21 22 P21212 P212121 P2 21 2 P2 21 21 P 2 2 21 In addition to these, I would also examine lower symmetry space groups, namely the three P21, where each axis is considered successively monoclinic. (see below) Chi2 is unusually high at lower resolution (Chi2 is 3 from 3.5A as shown below) and there is a relatively high percentage of rejections (1.5 %). Chi^2 in Scalepack *must* be adjusted by changing the expected errors to be about 1. Until then, I would not reject reflections. Only then I would reject reflections and then readjust chi^2 to be 1 again. Having said that its quite a while since I used scalepack. Maybe in the age of automation scalepack does this now automagically in the context of HKL2000, but a Chi^2 of 3 at low resolution indicates that some reflections and very likely their sigmas do not have realistic intensities, and also that your rejections might be excessive. Moreover, given the apparent very high quality of your data (it goes far beyond 2.5 A !!!), I would had expected lower Rsym at low resolution (from a good beamline or home source). Thus I would be skeptical about the symmetry. I would process the data at P1 and then either let Pointless or X- triage suggest the Laue group for the scaling. Both give truly informative and clear statistics for the Rmerge upon the introduction of symmetry elements and for the space groups. (btw these two programs are so brilliant. It was taking me ages to try scaling in alternative space groups by hand in the past - reminder: free beer to Phil and Peter for the 2009 meetings!) ;-) Good luck - Tassos
[ccp4bb] PHENIX BSS refinement
We have seen that the recent versions of phenix (1.3) the structure refinement ends with a bulk solvent correction step, something I found quite unusual (absent in other refinement programs and in older versions of phenix, 1.24.1). We have also seen that the B factor jumps up in this last step (see bellow). Is anybody aware of this? Do you know how to get rid of this final bss step? This step is redundant, as bss is also done at the beginning of each ref cycle. Header of a pdb file after refinement with phenix version 1.3: REMARK R-factors, x-ray target values and norm of gradient of x-ray target REMARK stage r-work r-free xray_target_w xray_target_t REMARK 0 : 0.3729 0.3776 5.594260e+00 5.604698e+00 REMARK 1_bss: 0.3135 0.3236 5.460255e+00 5.490057e+00 BSS refinement step REMARK 1_xyz: 0.3096 0.3226 5.451384e+00 5.487128e+00 refinement of coordinates REMARK 1_adp: 0.2608 0.2858 5.300551e+00 5.386607e+00 refinement of ADPs REMARK 2_bss: 0.2601 0.2843 5.299065e+00 5.384776e+00 REMARK 2_xyz: 0.2545 0.2847 5.285488e+00 5.386100e+00 REMARK 2_adp: 0.2460 0.2821 5.261319e+00 5.375879e+00 REMARK 3_bss: 0.2456 0.2788 5.257944e+00 5.366116e+00 REMARK 3_xyz: 0.2457 0.2809 5.261113e+00 5.371113e+00 REMARK 3_adp: 0.2427 0.2804 5.252685e+00 5.370838e+00 REMARK 3_bss: 0.2427 0.2804 5.252685e+00 5.370838e+00 ??? Bfactors REMARK stage b_max b_min b_ave REMARK 0 : 30.00 30.00 30.00 REMARK 1_bss: 30.00 30.00 30.00 REMARK 1_xyz: 30.00 30.00 30.00 REMARK 1_adp: 118.26 1.18 35.04 REMARK 2_bss: 118.26 1.18 35.04 REMARK 2_xyz: 118.26 1.18 35.04 REMARK 2_adp: 157.72 0.00 37.99 REMARK 3_bss: 157.72 0.00 37.99 REMARK 3_xyz: 157.72 0.00 37.99 REMARK 3_adp: 166.60 0.00 38.73 REMARK 3_bss: 203.00 36.40 75.12 ?? Thanks a lot for your help. Jose M Casasnovas Centro Nacional de Biotecnología (lab. B16) CSIC, Campus UAM Darwin 3 28049 Madrid Spain Ph. 34 915854917(8) Fax. 34 915854506 email: jcasasno...@cnb.csic.es !! NEW E-MAIL
Re: [ccp4bb] difficult MAD dataset
Chi2 is unusually high at lower resolution (Chi2 is 3 from 3.5A as shown below) and there is a relatively high percentage of rejections (1.5 %). Chi^2 in Scalepack *must* be adjusted by changing the expected errors to be about 1. Until then, I would not reject reflections. Only then I would reject reflections and then readjust chi^2 to be 1 again. Having said that its quite a while since I used scalepack. Maybe in the age of automation scalepack does this now automagically in the context of HKL2000, but a Chi^2 of 3 at low resolution indicates that some reflections and very likely their sigmas do not have realistic intensities, and also that your rejections might be excessive. Just a point here. The high chi^2 values you see in the scalepack output are because of the *anomalous signal*, not because of any pathology with the data. I assume scalepack was run with the 'anomalous' card specified. In this case (I believe) the + and - reflections will be treated as equivalent for scaling and calculating statistics, but will be written to the .sca file separately. Chi^2 will be screwed up because it detects a large deviation from random in the reflections (i.e. the anomalous difference between I(+) and I(-). You might actually be rejecting the reflections with the *most* anomalous signal since they are the ones that deviate furthest from your random-noise model. If you want your chi^2 values to be meaningful you need to use the 'scale anomalous' card to separate I(+) and I(-) for scaling/statistics as well as the output .sca. The scalepack manual warns against this flag, but I've never had a problem using it (so long as you have reasonably redundant data). So, re-process with 'scale anomalous' and then run autoSHARP in all sub-spacegroups of P212121 - as Tassos will tell you this works a treat! Cheers, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
[ccp4bb] putting in methionines for SeMet crystals
Dear All, I have a 9-kDa protein that crystallizes well. Since there is no structural homologue for this molecule, I intend to make Se-Met derivative of the protein. The molecule has no Met/Cys residues in its sequence. I wanted to know where in the sequence should I mutate, so that the folding/crystallizability of the protein is not compromised. Any suggestions would be of great help. Thanks in advance, -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK
Re: [ccp4bb] putting in methionines for SeMet crystals
Hey Amit, as your protein is tiny, maybe you can calculate a molecular replacement model as described in Acta Cryst. (2009). D65, 169–175. This might take longer than engineering a few methionines, though. I'd mutate leucines. Andreas amit sharma wrote: Dear All, I have a 9-kDa protein that crystallizes well. Since there is no structural homologue for this molecule, I intend to make Se-Met derivative of the protein. The molecule has no Met/Cys residues in its sequence. I wanted to know where in the sequence should I mutate, so that the folding/crystallizability of the protein is not compromised. Any suggestions would be of great help. Thanks in advance, -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London
Re: [ccp4bb] correlation length - reloaded
Re my previous post of this title: Harry Greenblatt just pointed out that I should have said that it is the reciprocal cell edges a* and b* that are non-equivalent in P4, not the real-space cell edges a and b. Hope that didn't confuse too many people. -James Holton MAD Scientist
Re: [ccp4bb] PHENIX BSS refinement
Hi Jose, We have seen that the recent versions of phenix (1.3) the structure refinement ends with a bulk solvent correction step, something I found quite unusual (absent in other refinement programs and in older versions of phenix, 1.24.1). - the software is evolving and gets better over time -:) That is, version 1.3 is supposed to be better than 1.24.1, and the latest 1.4 should be better than 1.3. - it is good to update bulk solvent correction at the end of refinement. For example, the refinement of coordinates may invalidate the mask which in turn invalidates the solvent contribution Fbulk. We have also seen that the B factor jumps up in this last step (see bellow). Is anybody aware of this? This is because the trace of overall anisotropic scale matrix is added to atomic B-factors and subtracted from that matrix. This is exactly what CNS does (at least version 1.1). Do you know how to get rid of this final bss step? It is not a good idea, so phenix.refine does not have an option to turn it off. This step is redundant, as bss is also done at the beginning of each ref cycle. It is not redundant: see above. FYI: there is PHENIX bulletin board: http://www.phenix-online.org/mailman/listinfo/phenixbb Pavel.
[ccp4bb] Obtaining relationships between two cross rotation solutions?
I have trouble with visualizing things in three dimensions so I'm trying to figure out the relationship between two cross rotation functions (given as theta1, theta2, theta3). Is there a program or webapp that'll tell me whether two rotation solutions are related by a 180/90/60/whathaveyou axis? e.g. The space group is P4(1) The top few cross rotation solutions are: ! index, theta1, theta2, theta3, RF-function (EPSIlon= 0.25) 1 173.333 2.630 186.4810.0907 3 353.426 180.000 353.4260.0879 798.807 147.925 67.3470.0608 8 279.440 30.322 292.5870.0602 10 292.696 168.784 294.1560.0562 12 233.216 30.322 246.3640.0511 Is index 1,3 related how about 7,8?? What is the relationship between the pair 1,3 and 7,8 ? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] putting in methionines for SeMet crystals
Hi Amit, Based on the original analysis of M. Dayhoff (PAM matrix, Dayhoff substitution probability, Dayhoff, et. al 1978), introduction of Leu-Met would be the best choice for production of Se-Met derivatized protein. It would be best to consider a multiple sequence alignment of your target protein with proteins of similar sequence, ignore the ones that may have functional importance (from any literature reports) and ones that may be present in loops or may be surface exposed (easily done with some prediction algorithms, would be best to have the Se-Met in well-ordered regions) and then try multiple combinations of Leu-Met substitutions. This has been successfully exploited in several cases and a partial list follows: Gassner, N. C., Baase, W. A. Matthews, B. W. (1996). Proc. Natl Acad. Sci. USA, 93, 12155-12158 Leahy, D. J., Erickson, H. P., Aukhil, I., Joshi, P. Hendrickson, W. A. (1994). Proteins, 19, 48-54 Jones, D. T., Taylor, W. R. Thornton, J. M. (1992). Comput. Appl. Biosci. 8, 275-282. Good luck! Debanu. --- Debanu Das, JCSG Structure Determination Core, SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of amit sharma Sent: Tuesday, February 03, 2009 5:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] putting in methionines for SeMet crystals Dear All, I have a 9-kDa protein that crystallizes well. Since there is no structural homologue for this molecule, I intend to make Se-Met derivative of the protein. The molecule has no Met/Cys residues in its sequence. I wanted to know where in the sequence should I mutate, so that the folding/crystallizability of the protein is not compromised. Any suggestions would be of great help. Thanks in advance, -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK
[ccp4bb] TeachSG workshop Expertise in Macromolecular Structure Refinement in Prague April 3-4
A two-day workshop focused on both the basics and the current development in macromolecular single crystal structure refinement techniques will be held in Prague on April 3-4 2009. The workshop is organized within the TeachSG project of the EC (tightly connected with the project Spine2-Complexes) to support dissemination of knowledge in the field of structural biology. Several highly qualified speakers and tutors confirmed their contributions to the workshop (see the attached preliminary program). There will be a limited number of places so hurry up if you want to be here in April. The workshop is aimed at young researchers, students, PhD students, young post-docs (exceptions can be made if reasonably justified). We expect a wide spectrum of students from beginners in refinement to experienced users seeking proficiency in the field. To apply for participation, please, send an e-mail to myself (Jan Dohnalek, cc: dusk...@imc.cas.cz) containing a motivation letter (within the e-mail message is OK), a letter of support from your supervisor or head of group, and a short professional CV (one page). It should be clear from your documents why participation in such workshop is necessary for your career. The deadline for applications is: Tuesday 24th February 2009. The selected participants will be notified by e-mail within several days after the deadline date. The organizers provide and cover the costs of conference materials, refreshments during the course including lunches and a workshop dinner on the 3rd. Workshop participants are asked to make their own travel arrangements and accommodation bookings. We are ready to give you advice on travel if necessary. You can contact Jan Dohnalek or Jarmila Duskova (duskova at imc.cas.cz) for more details. Looking forward to see you in Prague, Jan Dohnalek IMC Prague -- == Mr. Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Laboratory of Structural Analysis of Molecules Heyrovskeho nam. 2 16206 Prague 6 Tel: +420 296809390 Fax: +420 296809410 http://protein.awardspace.com/ == TeachSG_workshop_Refinement_PragueApril.xls Description: application/msexcel
Re: [ccp4bb] Obtaining relationships between two cross rotation solutions?
I forgot to mention that I think the only fail-safe direct approach will involve converting both rotations into quaternions, subtracting (inv(A)*B) said quaternions, then converting the difference into an axis-angle pair. James On Feb 3, 2009, at 9:47 AM, Francis E Reyes wrote: I have trouble with visualizing things in three dimensions so I'm trying to figure out the relationship between two cross rotation functions (given as theta1, theta2, theta3). Is there a program or webapp that'll tell me whether two rotation solutions are related by a 180/90/60/whathaveyou axis? e.g. The space group is P4(1) The top few cross rotation solutions are: ! index, theta1, theta2, theta3, RF-function (EPSIlon= 0.25) 1 173.333 2.630 186.4810.0907 3 353.426 180.000 353.4260.0879 798.807 147.925 67.3470.0608 8 279.440 30.322 292.5870.0602 10 292.696 168.784 294.1560.0562 12 233.216 30.322 246.3640.0511 Is index 1,3 related how about 7,8?? What is the relationship between the pair 1,3 and 7,8 ? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] 'Sticky Crystals' on ccp4-wiki
Dear colleagues, I have just deposited an entry on the ccp4-wiki that summarizes input/ideas on dealing with 'sticky crystals'. Please feel free to edit it further as necessary. http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Crystals#Crystal _handling best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11680 http://www.pctools.com/en/spyware-doctor-antivirus/
[ccp4bb] problem with ccp4 6.1.0 installation
Hello, After installation I tried Graphical View of Project and I got: dotgraph_render: error: graph 1.000 5.861 0.528 node Job1 0.472 0.264 0.945 0.500 1: autoSHARP\nautoSHARP filled box black #9DD05B node Job2 1.667 0.264 0.945 0.500 2: autoSHARP\nautoSHARP filled box black #9DD05B node Job3 3.194 0.264 1.611 0.511 3: refmac5\nRestrained refinement using\nisotropic B factors filled box black #9DD05B node Job4 5.056 0.264 1.611 0.511 4: refmac5\nRestrained refinement using\nisotropic B factors filled box black #9DD05B stop Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font Error: Could not find/open font --- Anyone had simmilar problem? What and where the font should be? Thanks, Witek
