Re: [ccp4bb] Cryo-protectant
Hallo, there is quite a large number of cryo-protectants other than MPD or glycerol. If I remember correctly, the Methods of Enzymology, (176) list some of the, and you might check the recipes of the Hampton cryo screen. As suggested by Li, you can try oils. You can use small sugars (fructose, glucose...), PEG400, or 1,6-butanediol (maybe this was 2,5-butanediol) and mixtures of the above, e.g. 15%PEG400 + 5% glycerol etc. It also matters how you freeze the crystals - whether you freeze them in the nitrogen stream at the X-ray machine or dip them into a container with liquid nitrogen (I prefer the latter) Also, you can - dip them directly into the cryo solution - move them step by step to higher concentrations and hereby observe the crystals' health under the microscope - transport the crystals with a pipette rather than using the cryo-loop. this techniques reduces their exposure to air and exsiccation. etc. pp With an inhouse machine you can also try a measurement at room temperature in a capillary. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 24 Apr 2009, Liew Chong Wai wrote: Hi all, Good day I used MPD as a cryoprotectant (20%, 30%) for my crystal. However, there is no diffraction signal at all. Without the MPD cryo, i still manage to get 5angstrom, but it has very strong ice-ring signal. I used glycerol (15%, 20%, 25% and 30%) before, but it cracked the crystal. Please advice. Thanks Liew
Re: [ccp4bb] Twinning or not?
-Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Eleanor Dodson Sent: 23 April 2009 15:59 To: Kumar Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Twinning or not? Look at the moment plots after scalepack2mtz; if these are normal it seems very unlikely you have twinning.. Eleanor I know this subject has been beaten to death in previous BB discussions, but is it always as clear-cut as this? If for example you had a NCS 2-fold co-incident with the twinning axis wouldn't that bias the moment stats? The derivation of the moment stats in the twinned case assumes that the pairs of twin-related intensities (assuming we're talking about the hemihedral case) are statistically independent, but that assumption is clearly invalidated if there's NCS parallel to the twinning axis, and in that situation the moment stats would tend towards the untwinned case (depending of course on the exactness of the NCS and the resolution). It's of no consolation to someone to tell them that their situation is very unlikely if it actually happens to them! It seems reasonable to say that if the moment stats conform to the twinned case, then twinning is almost certainly present (barring data processing blunders); however if the moment stats conform to the untwinned case then you can't say for sure that it's not twinned, there's still a chance (maybe small) that it's twinned even if the data has been correctly processed. I've read on several occasions that twinning and NCS are quite likely to occur together, but I wonder if anyone has done a proper analysis and in particular looked at cases where the twinning NCS axes coincide to see the effect on the moment stats? Cheers -- Ian Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Cryo-protectant
I usually try the following, in order: 1. Transfer crystals to mother liquor + 30% glycerol or ethylene glycol (sometimes lower depending on crystallization solution). This did not work for you. 2. Transfer crystals to mother liquor + 30% glucose (or try sequential soaks in M.L. + 15% and then 30% glucose. Just a few sec/min is usually enough). Glucose or other sugars often work. when glycerol or EG fails. 3. Try the "no-fail" in situ cryo method, which is a gradual buildup of cryoprotectant. See http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo . This is very gentle, and often works when #1 and #2 does not, but in our hands nearly always increases mosaicity. (But mosaic is better than no diffraction.) 4. Try dragging crystals though paratone-N to remove surface water from the crystal. This actually nearly always works for us, but is more fussy than #1 or #2, and it is easier to damage crystals during manipulation because of the viscosity of the oil. I normally plunge protected crystals into LN2 after mounting. Ice rings are a good indication of poor cryoprotection, but lack of diffraction could just be your crystals, too. For our latest dataset, we just sorted through 38 crystals until we found a good one. The key, as it turned out, is that all of our beautiful large crystals were apparently difficult to visualize disordered stacks of plates (we didn't notice this until some fractured during cryo-soaks) whereas some of the small crystals were actually single crystals. We selected a decently diffracting small one and took lng frames to get a good data set. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Liew Chong Wai wrote: Hi all, Good day I usedMPD asa cryoprotectant (20%, 30%) for my crystal. However, there is no diffraction signal at all. Without the MPD cryo, i still manage to get 5angstrom,butit hasvery strongice-ring signal. I used glycerol (15%, 20%, 25% and 30%) before, but it cracked the crystal. Please advice. Thanks Liew
Re: [ccp4bb] Cryo-protectant
What is the crystallization condition? On Fri, 2009-04-24 at 11:46 +0800, Liew Chong Wai wrote: Hi all, Good day I used MPD as a cryoprotectant (20%, 30%) for my crystal. However, there is no diffraction signal at all. Without the MPD cryo, i still manage to get 5angstrom, but it has very strong ice-ring signal. I used glycerol (15%, 20%, 25% and 30%) before, but it cracked the crystal. Please advice. Thanks Liew -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Cryo-protectant
Hi all Thanks for your precious suggestions and ideas. The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350 Now, my crystal seem ok in 20% ethylene glycol, but only after a couple minutes of dehydration at room temperature. For sure, i will try other cryoprotectant that was suggested here. I just wondering why MPD kills the crystal. Many thanks LIEW
Re: [ccp4bb] Cryo-protectant
Hi Liew, There have already been some very good suggestions. I agree with Tim Gruene that a great starting point is to test the diffraction properties of your crystal at room temperature. This can serve as a baseline for comparing/evaluating cryoprotecting agents and methods. You can also test your potential cryoprotection solutions to see if they freeze clear or even take a few X-ray snapshots of them to confirm there are no ice rings. There are lots of publications that can be helpful. One very helpful reference is: Garman, E.F. and Doublie, S. Cryocooling of Macromolecular Crystals: Optimisation Methods. Methods in Enzymology (2003) 368, 188-216. Be aware that, as mentioned previously, the method for freezing can make a difference (e.g. freezing in cold-stream vs. plunging in nitrogen). An obvious difference is different cooling rates (you can find references for this) or less obvious reasons, for example differences in dehydration that occur during longer/shorter transfer through air from drop to cold-source for either method. Finally, you can check out the database for cryoprotecting solutions: http://idb.exst.jaxa.jp/db_data/protein/search-e.php Good luck, -Andy -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University = On 4/24/2009 10:44 AM, Liew Chong Wai wrote: Hi all Thanks for your precious suggestions and ideas. The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350 Now, my crystal seem ok in 20% ethylene glycol, but only after a couple minutes of dehydration at room temperature. For sure, i will try other cryoprotectant that was suggested here. I just wondering why MPD kills the crystal. Many thanks *LIEW* -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University =
Re: [ccp4bb] microbatch vs hanging drop
Rui Microbatch - which I take to mean crystallization under oil with no reservoir - has many advantages, but with some robots it's a little slower to set up (20 mins on ours). So most people I know start off with vapor diffusion and only move to microbatch if they have problems with VD. It seems to find as many hits as vapor diffusion, but in different conditions - see http://www.douglas.co.uk/mbnvdall.htm http://www.douglas.co.uk/mbnvdall.htm Main advantages: 1. Gives thinner skins on drops and less protein is lost on the surface 2. The oil often protects sensitive proteins such as membrane proteins or oxygen sensitive proteins 3. You can change temperature freely (no condensation etc.) 4. Very good for use with volatile organics, see e.g. http://www.douglas.co.uk/winner1.htm http://www.douglas.co.uk/winner1.htm And you can use it with MMS microseeding just like VD - very important I believe. I guess the main disadvantage is that it can be hard to harvest crystals through the oil (although some say the oil makes it easier because you can take your time) People are using smaller and smaller reservoirs so I guess one day they'll realize that you don't need a reservoir ;-) Good luck Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of rui Sent: 22 April 2009 17:06 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R
Re: [ccp4bb] microbatch vs hanging drop
Thanks all for your kind suggestions. Thanks for Janet for pointing out that I actually mess up with some terminology. The microbatch I mentioned is not real microbatch. What I meant to say is the 3 subwell plates with 96 wells, from Greiner (CrystalQuick Cat.-No.: 609101). So what i'm trying to ask is that with this type of plates, the reservoir buffer volume is 100 uL, vs hanging drop with 600 uL, that will it be harder to get crystals? On Fri, Apr 24, 2009 at 1:54 PM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Rui Microbatch – which I take to mean crystallization under oil with no reservoir - has many advantages, but with some robots it’s a little slower to set up (20 mins on ours). So most people I know start off with vapor diffusion and only move to microbatch if they have problems with VD. It seems to find as many hits as vapor diffusion, but in different conditions – see *http://www.douglas.co.uk/mbnvdall.htm* Main advantages: 1. Gives thinner skins on drops and less protein is lost on the surface 2. The oil often protects sensitive proteins such as membrane proteins or oxygen sensitive proteins 3. You can change temperature freely (no condensation etc.) 4. Very good for use with volatile organics, see e.g. * http://www.douglas.co.uk/winner1.htm* And you can use it with “MMS” microseeding just like VD – very important I believe. I guess the main disadvantage is that it can be hard to harvest crystals through the oil (although some say the oil makes it easier because you can take your time) People are using smaller and smaller reservoirs so I guess one day they’ll realize that you don’t need a reservoir ;-) Good luck Patrick -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of * rui *Sent:* 22 April 2009 17:06 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R
Re: [ccp4bb] Cryo-protectant
Title: Re: [ccp4bb] Cryo-protectant First of all, you need to find out if crystals you grew have similar quality before you conclude poor diffraction is due to cryoprotectant solutions. As others have suggested, you can test crystals using capillary mounting method. Second, there are a lot more cryoprotectant agents out there you can try. Not sure why you sticked to MPD, which is not even present in your crystallization condition. One of protein crystallized in almost the same condition as yours. What I did is just simply increasing % PEG3350, in addition to 10%-15% increment of every other ingredients (your protein buffer + well solution). I also introduced 5% glycerol to bring down % PEG3350. You can play around % PEG3350 and % glycerol to find a fine combination that is cryo-clear and makes you crystals happy. Joe Liew Chong Wai wrote: Hi all Thanks for your precioussuggestions and ideas. The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350 Now, my crystal seem ok in 20% ethylene glycol, but only after a couple minutes of dehydration at room temperature. For sure, i will try other cryoprotectant that was suggested here. I just wondering why MPD kills the crystal. Many thanks LIEW
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? By the way, while we're on the topic of comparing uv-vis fluorescence to x-ray fluorescence, does anybody know of an example of the use of FRET in x-ray fluorescence? I cannot think, off hand, of an application for such, but theoretically it could be done easily with two types of heavy atoms, such as a Se-met and some appropriate acceptor. Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 23, 2009 8:59 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence Dirk Kostrewa wrote: yes, this is certainly true for real fluorescence effects. But the anomalous scattering can be best thought of as a resonance phenomenon without any frequency change, and as such, it has a distinct phase relationship to the elastically scattered photon and does have an effect on the intensities (which, I think, was the background of the original question?). But for the lighter atoms in biological macromolecules, where in a typical experiment the measurement frequency is far away from any resonance frequency, this effect can be neglected. This leads me to my follow-up question to the experts: why is the resonance effect anomalous scattering measured by a fluorescence scan that should have all the effects mentioned by James? Don't we get as a result a mixture of signals from resonance (i.e. anomalous) and from absorption-emission (i.e. fluorescence) effects? Fluorescent photon emission happens well after the incident photon has passed, so anomalous scattering is only indirectly related to fluorescence. The relationship is that absorption induces a phase shift in scattering (this is the anomalous scattering effect), but it also induces an electronic transition in the atom, leaving a core hole or vacant orbital near the nucleus. The filling of this core hole will generate a fluorescent photon (some fixed fraction of the time), and this allows us to equate the intensity of observed fluorescence to the number of core holes produced and therefore to the absorption cross section of the atom. In actual fact, the MAD scan we do before a MAD/SAD experiment is not a fluorescence spectrum, but rather an absorption spectrum using fluorescence as a tally. A fluorescence spectrum would have the energy of the fluorescent photon on the x-axis. (Bob Sweet has corrected me several times for getting that wrong). As for the connection between absorption and anomalous scattering, I tend to think of this in the classical picture. Scattering lags the incident beam by 90 degrees because a simple harmonic oscillator driven at frequencies much higher than resonance lags behind the force upon it. An oscillator driven at resonance will move 180 degrees out-of-phase with the driving force. You can verify this yourself by playing with a weight tied to the end of a rubber band. Another way to think about it is that absorption must create a wave that is 180 degrees out of phase with the incident beam because it reduces the intensity of the incident beam. The details of the physics are much more complicated than this, but this is how I like to remember it. So, as you approach a resonance, some of the electrons in the atom will start absorbing (resonating) and therefore move out-of-phase with the other electrons in the atom (and indeed the other electrons in the crystal). It is this out of sync behavior that reduces the effective occupancy of the atom and also creates an imaginary component to the scattering. This imaginary electron density is hard to accept if you have never taken complex algebra, but the easy way to think about it is to remember than multiplying a complex number by sqrt(-1) changes its phase by 90 degrees. So the imaginary component is really just a mathematical way to represent electrons that are out-of-sync with the majority of electrons in the crystal. Yes, the majority, because a pure selenium crystal has no anomalous scattering (since no atoms lag any other atoms). The imaginary component is what leads to the breakdown of Friedel's law
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption--fluorescence? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[ccp4bb] Watertidy troubles
I'm running watertidy to move all waters to the (symmetry-related) position closest to my protein. One protein chain (A), 144 waters (chain W). Trouble is, one round of watertidy doesn't move many of the waters (e.g., a water only 2.7 Ang. from a carbonyl oxygen of a symm-related protein was not moved). Those that were moved seem OK (checked first few). What's more, if I specify 3 rounds (default in CCP4i), then I end up with a final file that has only 21 waters. Has anyone had better luck with watertidy? Is there another program that can simply move the waters (e.g., as the PDB says they do when you deposit coords)? Thanks, Dave David Borhani, Ph.D. D. E. Shaw Research 120 West Forty-Fifth Street, 39th Floor New York, NY 10036 david.borh...@deshawresearch.com 212-478-0698 http://www.deshawresearch.com
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:53:27 Jacob Keller wrote: Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption--fluorescence? I am not sure about that because. Let's not confuse correlation with causality. The negative f' is adequately explained by the Kramers-Kronig equation as being a result of the resonance interaction. http://www.rp-photonics.com/kramers_kronig_relations.html The maximum resonance is at the absorption energy, which is also the maximum for the fluorescence. Both effects are because of the match between incident photon energy and the energy required to kick an electron out of its current orbital state. I am uneasy saying that one effect causes the other effect. Ethan Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
[ccp4bb] MLY
Hi everyone, We just realized in the lab that the dimethyl-lysine in the ccp4 monomer database (MLY) has planar tertiary amines, instead of trigonal pyramid with ~109º degree angles (which we have fixed for our purposes). Is there a place to report such matters to, or is this a good enough place for that? Actually the cif file has only a minimal (and inaccurate) description, and libcheck/refmac generates a planar amine with that. Are these minimal descriptions in the monomer library what was intended for users to build upon, or are they more final, authoritative files? Thanks, Engin
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:53:27 Jacob Keller wrote: Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption-- fluorescence? I am not sure about that because. Let's not confuse correlation with causality. The negative f' is adequately explained by the Kramers- Kronig equation as being a result of the resonance interaction. http://www.rp-photonics.com/kramers_kronig_relations.html The maximum resonance is at the absorption energy, which is also the maximum for the fluorescence. Both effects are because of the match between incident photon energy and the energy required to kick an electron out of its current orbital state. I am uneasy saying that one effect causes the other effect. Ethan There is a very good technical description in Jens Als-Nielsen's Elements of Modern X-ray Physics in the chapter Resonant Scattering, pg 235 ff. In fact, there is also a good description of the breakdown of Friedel's Law and the MAD experiment in that chapter. I would like to iterate Ethan's comment about resonance. The effects are not anomalous at all, we know very well what is happening: the changes in f', f and mu as a function of energy are all effects of the resonance of the photon energy with transition energy of the electron. So, we really should call it resonance scattering, not anomalous scattering. I have to admit MRD and SRD aren't as euphonic at MAD and SAD and the change will probably never happen. Joe Joseph D. Ferrara, Ph.D. Rigaku Americas Corporation Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? no. For example, with selenium only ~59% of the core holes decay by emitting a fluorescent x-ray. The rest by emitting an Auger electron. The latter seldom escape the sample. On the other hand, there are generally a lot more absorbed photons than scattered ones. For Se again at 12680 eV (just above the edge) the ratio is about 120 absorption events for every elastically scattered photon. And since 59% of the absorptions make a fluorescent x-ray, there are about 70 fluorescence events for every resonant event. Anyway, the ratio is definitely not 1:1. If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Sort of. I personally like to think of the core electrons as disappearing from the normal scattering as they start to scatter out of phase. However, ALL of the electrons scatter any given photon. Even the anomalously scattering electrons don't really disappear any more than a beat-deaf member of a marching band disappears when they get out of step with the rest of the rank and file, but they do stop contributing to the total effect. Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? No. There is no anomalous scattering from crystals with only one atom type. That is, Friedel's law holds because the phase lag from every atom is the same. Friedel's law also holds for centrosymmetric crystals, despite any anomalous effects. I suppose you might be able to see the atomic form factor change as the core electrons go out of phase as you approach the absorption edge, and well, okay, technically that is an anomalous scattering effect. But Friedel's law is not broken for elemental crystals nor for centrosymmetric crystals. By the way, while we're on the topic of comparing uv-vis fluorescence to x-ray fluorescence, does anybody know of an example of the use of FRET in x-ray fluorescence? I cannot think, off hand, of an application for such, but theoretically it could be done easily with two types of heavy atoms, such as a Se-met and some appropriate acceptor. Rick Donahue (or health physicist here at ALS) told me a story once where they found a sample of what I think was some metal carbonate that was emitting fluorescent x-rays from the metal, but it was the carbon in the carbonate that was radioactive. One could consider this an example of a transfer of excitation in the x-ray regime, but I'll have to check with Rick to be sure. -James Holton MAD Scientist
[ccp4bb] hydrogen bond between aspartate side chain and main chain carbonyl group?
Hi, In a new structure we observed a hydrogen bond between an aspartate side chain and a main chain carbonyl group. Because both of them are electronegative, I am puzzled. My questions are: does this hydrogen bond make sense at all? any precedent for such a hydrogen bond? I would appreciate your help very much! Frank
