Re: [ccp4bb] monomeric coiled coil
Hi, 'The Role of Diffusion in Enzyme Kinetics' http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1367934/pdf/biophysj00672-0040.pdf Mark ~~~~~ Dr Mark Agacan, Scientific Officer for the Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH Tel: +44 1382 386095Fax: +44 1382 345764Mobile: 07525 451 117 ~ Please consider the environment. Do you really need to print this email? >>> Anastassis Perrakis 7/5/2010 13:08 >>> Hi Jacob - Sorry if I caused any misunderstanding, but I am not saying that kD is not mattering in cells. Of course it should matter. I am making the distinction that the concentration dependent component of it, k(on), is of less importance, since in the viscous environment of a cell, the chance of two molecules to find each other should be limited by diffusion rates before concentration becomes the limiting factor for their chance to meet and form a complex. The concentration independent component of kD, k(off), the half time that two molecules need to break a complex once formed, I think is what is most relevant in the cell. Although I came across this argument a few times, and I find it convincing, I am not aware of a paper that proves the first derivation, that indeed diffusion rates will be the limiting factor. Unless someone posts the reference, I just got myself an evening project to find it ... A. On Jul 5, 2010, at 0:18, Jacob Keller wrote: 4. The physiological concentration is a bit misleading. First, its clear now that cells have microenvironments, and 'physiological' concentrations are hard to define. Also, in a cell, I think (and I think others tend to agree) that kD plays little role at the end. kD is a combination of k(on) - which is concentration dependent but in a cell very likely diffusion limited - and of k(off) which I think is what matters most in the cell. Can you provide some references about kD not mattering in cells? I had thought it a basic tenet that kD was a major indication of physiological relevence. After all, one could presumably determine a kD for any two proteins, so the order of magnitude of the kD would seem to matter. I hear that your argument is more subtle in considering the elements of kD, but isn't kD usually a rough indicator of k(off), and of course more readily-accessible experimentally? I am intrigued by the idea... Jacob Keller P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 The University of Dundee is a registered Scottish charity, No: SC015096
[ccp4bb] X-Stream 2000 problem - SUMMARY
I would like to thank everyone who replied with information regarding the X-Stream icing problem, and a summary is given below. The problem is now almost cured, and I found the most important factors to be: - the accurate positioning of the cryostream nozzle so that the crystal is the first thing the cold stream hits, rather than the pin - moisture / humidity - turbulence around the crystal - air-conditioning system blowing cold air close to the cryostream Thanks again Mark From: Mark Agacan Monday - January 12, 2009 4:31 PM To: Mark Agacan Subject: X-Stream summary Apologies for this slightly off topic question: I am having a great deal of trouble with my X-Stream 2000 cryostream system and I wondered if other users have similar problems. I've replaced almost all components (new GAST compressors, helium recharges, filters, etc., etc.) in the last couple of months but there is almost always icing of any cryo within 10 - 20 minutes of mounting a loop, and it is adversely affecting data collections. It appears like there is too much moisture in the cold or wam streams but the tubes have been fully dried out as per Rigaku advice. This X-Stream is attached to a generator with inverted phi axis and and i'm wondering if this could be the source of the problem, as the X-Stream for another generator in the same laboratory with normal phi axis does not ice up. Can some sort of turbulence around the loop caused by backdraft from the cryo hitting the inverted phi axis / camera mount cause excess humidity and lead to icing on the pin, loop and crystal? Has anyone else got this problem? Any suggestions would be very gratefully appreciated. Best Wishes, Mark ** Hi Mark We had a LOT of pain with icing, and it really comes down to one thing: water in the gN2. And don't expect to measure some other way whether you have it, because your X-stream (or Cobra) is the most sensitive water meter there is. In our case, the symptom was the X-stream (and later Cobra) blocking up after between several days and several hours. And we solved it by ditching the gN2 generators we were using, and organising boil-off gN2 (much purer). The secondary effect of sample icing: we'd see this as well if we had something (e.g. collimator) poking into the cold stream*: that causes turbulence which draws in moisture. Worst case you see ice flakes flick onto the crystal in real time; best case you get an ice ball after a few minutes to hours. (* technically, the stationary phase between the cold and warm streams.) Hope that helps. phx *** Hi, If you have ice on the crystal (loop) but no blockages of the cryostream itself (and this seems to be what you're saying), it is unlikely to be a problem with the LN2 (although I only have experience with Oxford Crystreams, but I imagine this also applies to X-streams). Our main problems are turbulence (collimator poking into the gas stream(s) or gas 'bouncing' back from something just beyond the crystal - moving things even a little may help here) and drafts due to 'powerful' air-conditioning. As you state that one of your setups is affected and not the other, it may well be the case that an air conditioning vent is blowing onto your setup, and disturbing the gas streams. We resorted to hanging plastic sheeting around two sides of our set-up and this made a vast difference. HTH, Johan *** mark we also have an inverted phi, and so probably our layout is similar to yours (we're also rigaku). we had icing problems that were cured by directing the airflow from the a/c unit directly down, rather than across the room (and towards the x-stream). turn off your a/c, and see if a fresh cryo still ices up in 20-30 mins. if so, then re-direct the airflow or build a plastic tent! rick Hi Mark, In a similar setup (X-stream 2000, inverted phi, we have had no problem with our X-stream.) Cheers, Zsolt Dr. Zsolt Bocskei sanofi-aventis Chemical and Analytical Sciences 16 rue d'Ankara 67000 Strasbourg France ** We've been able to run months with an old Xstream 2000 system, so that shouldn't be the problem. Unlike Frank, we haven't had problems with water in the nitrogen from a nitrogen generator. If Frank is correct, that it's water, then either the molecular sieves need to be replaced, or there is ice buildup and blockage in the coldhead. Usually blockage in the coldhead means that you can't get down in temp, or can't maintain it within 1 degree or less. We do bring the temp up, run it at RT over the weekend to dry things out, and then bring it down for routine cooling. If it&#x
[ccp4bb] X-Stream 2000 problem - ICING
Apologies for this slightly off topic question: I am having a great deal of trouble with my X-Stream 2000 cryostream system and I wondered if other users have similar problems. I've replaced almost all components (new GAST compressors, helium recharges, filters, etc., etc.) in the last couple of months but there is almost always icing of any cryo within 10 - 20 minutes of mounting a loop, and it is adversely affecting data collections. It appears like there is too much moisture in the cold or wam streams but the tubes have been fully dried out as per Rigaku advice. This X-Stream is attached to a generator with inverted phi axis and and i'm wondering if this could be the source of the problem, as the X-Stream for another generator in the same laboratory with normal phi axis does not ice up. Can some sort of turbulence around the loop caused by backdraft from the cryo hitting the inverted phi axis / camera mount cause excess humidity and lead to icing on the pin, loop and crystal? Has anyone else got this problem? Any suggestions would be very gratefully appreciated. Best Wishes, Mark _____ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ The University of Dundee is a registered Scottish charity, No: SC015096
Re: [ccp4bb] RAxis & fiber diffraction
Hello, You could try HELIX or FibreFix software, put crystalline DNA fibres into a capillary, no cryostream, shoot at full power for about an hour... from then it depends on the material. Regards, Mark _ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ The University of Dundee is a registered Scottish charity, No: SC015096
Re: [ccp4bb] UV light source for protein xtal detection
I put an array of UV LEDS on a ceramic block and connected to a power source, as a means of detecting very small crystals once they are in the loop. The most difficult part was supporting the LED block so that it shone onto the loop but did not obstruct the user. The so-called UV LEDS are often black light not UV. A small, real UV torch or keyring light may actually be better. Mark _ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ The University of Dundee is a registered Scottish charity, No: SC015096
Re: [ccp4bb] crystal shipping at room temperature
Dear Junhua, This does indeed sound like a description of a fiber diffraction pattern. For example, as you probably already know, A-form DNA fibres give large bands close to 3.4 Angstroms at the top and at the bottom of the images. The exact positions of the bands are specific to the type of fibre. I don't know what fibres diffract to 4.8 - 5.5 Angstroms. I have never used MiTeGen mounting tools, so I can't comment on that. M >Another question about the MiTeGen mounting tools is that I always observe a stong diffraction ring at about 5 A. Well, it is not exactly a ring; it's actually two thick arches (pretty thick, roughly from 5.5 A to 4.8 A), one at the top and the other at the bottom of the diffraction patterns (nothing on the left-hand or right-hand side). Does anybody have any idea what this might be (fiber?)? > >thanks a lot for your help! > >Junhua >--- >Junhua Pan >Department of Biochemistry & Cell Biology >327 Keck Hall, Rice University >6100 Main Street MS-140 >Houston, TX 77005 >Phone: (713)348-3346 >Email: [EMAIL PROTECTED] > > _ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _
[ccp4bb] Filament Lifetime
I think the filament from one of our x-ray generators must be a record breaker. Yesterday it clocked up its 6000th hour or 250th day on the job, not counting down / holiday time. The intensity had only dropped by around 25 % of the value it was when I put it in, eight-and-a-half months ago... Really, this filament is like the horror-movie bad guy who just won't die - it survived countless power drop-outs and surges, the rough treatment of everyone from undergraduates to postdocs, and still it kept on and on... So, I decided to give it a break, as it has already smashed the Guinness world record for filament lifetimes (I checked) - I removed and replaced this beauty today, even though it's still working. I thought about framing it and keeping it on display in the generator lab for future generations to marvel at, but I got greedy - It will be up on eBay soon. Discounts available for academic institutions, etc Have a nice weekend (it's my birthday!) Cheers Rigaku! Mark _____ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _
Re: [ccp4bb] Filament lifetime on Rigaku Micromax007
Dear Pat, I too am shocked by the extra-long lifetimes the current batch of MM filaments have. I've had filaments in both our instruments (a M007 and an M007 hf) since August and they are still going strong. Not long ago I would replace a filament before it blew if I knew there was an important data collection scheduled on the same instrument. It used to be 12 - 16 weeks on average, on this occassion it's over 30 weeks for each filament. Good to see we are getting our money's worth ;) Mark _____ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ >>> Patrick Bryant <[EMAIL PROTECTED]> 28/02/07 1:18 PM >>> Dear Colleagues, During more than three years of operation, I have recorded considerable difference in filament lifetimes on my Micromax007: roughly in the range 500-2000hrs. Some of this may be accounted for by poor manufacture and Rigaku have, in the past, noticed this problem and replaced some filaments. For the last three filaments fitted, two have lasted about 500hrs and the third about 2000hrs. These filaments were replacements. My generator running protocol has been constant throughout. Interestingly, the filaments show no visible sign of wear or damage (even the 2000hr one) and are only changed when the generator starts to shut down on OL or FC limits. I recall earlier Rigaku generators that would give 2000hrs when well maintained and they ran at 5.4KW compared to the 800W of the Micromax. I would be grateful for any comments or suggestions from other Micromax operators. Sincerely, Pat Bryant Dr Pat Bryant Senior Experimental Officer Macromolecular Crystallography Core Facility Faculty of Life Sciences Michael Smith Building The University of Manchester Oxford Road Manchester M13 9PT, UK Phone: +44-161-275-5090/5658 Fax: +44-161-275-1505 email: [EMAIL PROTECTED] Intranet:http://www.intranet.ls,manchester.ac.uk/facilities/research/xraycrystallography Internet:http://www.ls.manchester.ac.uk/research/facilities/xray
Re: [ccp4bb] Good NMR Board
Hey Justin, Try the ccpn board - the same as ccp4 but for nmr. You can subscribe to this through: http://www.ccpn.ac.uk/ Hope this helps, MARKX _ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ >>> Justin Schmitz <[EMAIL PROTECTED]> 08/02/07 10:02 AM >>> Morning every body! Does anybody know a board for NMR releated questions which has a quality like this? Thx for your answers >>> Justin Schmitz <[EMAIL PROTECTED]> 08/02/07 10:02 AM >>> Morning every body! Does anybody know a board for NMR releated questions which has a quality like this? Thx for your answers
