[ccp4bb] Lysozyme/Xenon derivatives Phenix.
Hi all, I have a strange result using Phenix's AutoSol to look for xenon sites in lysozyme. For a few months now I have been trying to produce xenon derivatives of lysozyme using pressures in the range of 50-350psi and time ranges between 5-60min trying to find the sweet spot so that this technique can be applied to other proteins. For each dataset, I AutoSol using phenix and have it look for 2, 3, and 4 xenon sites. I haven't had much success though and typical values when asked to look for 2 sites have been Rwork/Rfree = 0.5585/0.5892, CC=0.14, Bayes-CC=6.5 and with xenon sites: (a, b, c, alpha, beta, gamma, space group) CRYST1 78.870 78.870 36.940 90.00 90.00 90.00 P 41 21 2 (columns after numbering are a, b, c, occ, B factor) HETATM1 XE XE Z 1 48.601 67.475 1.088 0.06 6.58 XE HETATM2 XE XE Z 2 54.986 76.636 2.746 0.11 7.49 XE which, to me, says that there is no binding (or at least nothing obvious). I finally got a good result though with 350psi and 5 minutes pressurization. After AutoSol, FOM=0.436, Bayes-CC=33.57, Model CC=0.83, Rwork/Rfree=0.2843/0.3023, 117/125 residues build and placed. And even though I told it to only look for 2 sites, Phenix went ahead and found 22 sites: (a, b, c, alpha, beta, gamma, space group) CRYST1 79.130 79.130 36.920 90.00 90.00 90.00 P 43 21 2 (colums after numbering are a, b, c, occ, B factor) HETATM1 XE XE Z 1 -36.117 -56.764 -1.825 0.18 9.43 XE HETATM2 XE XE Z 2 -54.805 -54.805 0.000 0.14 6.44 XE HETATM3 XE XE Z 3-8.337 -31.695 -16.766 0.08 8.26 XE HETATM4 XE XE Z 4-5.702 -64.959 -35.134 0.05 5.21 XE HETATM5 XE XE Z 5 -9.535 -22.397 -31.839 0.06 14.33 XE HETATM6 XE XE Z 6-7.319 -66.343 -35.123 0.05 6.02 XE HETATM7 XE XE Z 7-8.580 -21.153 -30.274 0.05 7.43 XE HETATM8 XE XE Z 8 -1.494 -29.583 -2.339 0.06 7.36 XE HETATM9 XE XE Z 9-0.518 -16.483 -16.776 0.05 6.56 XE HETATM 10 XE XE Z 10 -0.464 -29.847 -4.386 0.06 6.04 XE HETATM 11 XE XE Z 11 -1.222 -16.234 -18.952 0.04 6.51 XE HETATM 12 XE XE Z 12 -6.001 -20.380 -4.532 0.05 7.22 XE HETATM 13 XE XE Z 13 -5.169 -27.522 -10.128 0.05 3.54 XE HETATM 14 XE XE Z 14 -11.931 -67.039 -4.699 0.05 7.96 XE HETATM 15 XE XE Z 15 -6.538 -10.408 -3.761 0.04 10.22 XE HETATM 16 XE XE Z 16 -0.458 -23.897 -31.221 0.05 6.85 XE HETATM 17 XE XE Z 17 -3.308 -65.181 -8.754 0.03 8.08 XE HETATM 18 XE XE Z 18 -11.080 -29.129 -2.144 0.04 5.61 XE HETATM 19 XE XE Z 19 -5.720 -73.029 -2.035 0.04 11.24 XE HETATM 20 XE XE Z 20 10.236 60.097 33.822 0.03 5.38 XE HETATM 21 XE XE Z 21 5.980 68.581 3.532 0.03 7.49 XE HETATM 22 XE XE Z 22 11.107 13.277 27.334 0.03 7.10 XE I'm pretty sure there aren't 22 sites for xenon to bind in lysozyme but it looks to me like the top 2 (maybe top 3) are actual sites which is great. The strange part is that when I use the same data and have Phenix look for 3 or 4 sites it then only finds 3 or 4 very low occupancy sites and the CC's/R's are again all very poor. Has anyone had this problem or does anyone know what's going on? This is the first success that I'm having with xenon derivatization and it seems to me that if Phenix can find 22 sites when asked to find 2 it should have no problem when asked to find 3 or 4. Thanks in advance, ~Brennan~
Re: [ccp4bb] Lysozyme/Xenon derivatives Phenix.
When you crystallized your lysozyme, did you have any other anomalous scattering atoms in the conditions? I am thinking of chloride ions in particular, but there could be many others. How did you distinguish between sites of xenon and sites of other kinds of atoms in your analysis? Did you do a control experiment without xenon? Maybe with air? Maybe with and without pressurization? Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Brennan Bonnet [brennan.bon...@lightsource.ca] Sent: Thursday, February 02, 2012 10:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lysozyme/Xenon derivatives Phenix. Hi all, I have a strange result using Phenix's AutoSol to look for xenon sites in lysozyme.
Re: [ccp4bb] Lysozyme/Xenon derivatives Phenix.
Dear Brennan, Since you accidentally sent your request to the CCP4bb rather than the Phenix email list, I suggest that you try SHELXC/D/E, e.g. using Thomas Schneider's HKL2MAP, to see if it gets the same answers. These programs have solved lysozyme hundreds of times. HKL2MAP will also automatically correct the space group for you! Best wishes, George On 02.02.2012 17:43, Brennan Bonnet wrote: Hi all, I have a strange result using Phenix's AutoSol to look for xenon sites in lysozyme. For a few months now I have been trying to produce xenon derivatives of lysozyme using pressures in the range of 50-350psi and time ranges between 5-60min trying to find the sweet spot so that this technique can be applied to other proteins. For each dataset, I AutoSol using phenix and have it look for 2, 3, and 4 xenon sites. I haven't had much success though and typical values when asked to look for 2 sites have been Rwork/Rfree = 0.5585/0.5892, CC=0.14, Bayes-CC=6.5 and with xenon sites: (a, b, c, alpha, beta, gamma, space group) CRYST1 78.870 78.870 36.940 90.00 90.00 90.00 P 41 21 2 (columns after numbering are a, b, c, occ, B factor) HETATM1 XE XE Z 1 48.601 67.475 1.088 0.06 6.58 XE HETATM2 XE XE Z 2 54.986 76.636 2.746 0.11 7.49 XE which, to me, says that there is no binding (or at least nothing obvious). I finally got a good result though with 350psi and 5 minutes pressurization. After AutoSol, FOM=0.436, Bayes-CC=33.57, Model CC=0.83, Rwork/Rfree=0.2843/0.3023, 117/125 residues build and placed. And even though I told it to only look for 2 sites, Phenix went ahead and found 22 sites: (a, b, c, alpha, beta, gamma, space group) CRYST1 79.130 79.130 36.920 90.00 90.00 90.00 P 43 21 2 (colums after numbering are a, b, c, occ, B factor) HETATM1 XE XE Z 1 -36.117 -56.764 -1.825 0.18 9.43 XE HETATM2 XE XE Z 2 -54.805 -54.805 0.000 0.14 6.44 XE HETATM3 XE XE Z 3-8.337 -31.695 -16.766 0.08 8.26 XE HETATM4 XE XE Z 4-5.702 -64.959 -35.134 0.05 5.21 XE HETATM5 XE XE Z 5 -9.535 -22.397 -31.839 0.06 14.33 XE HETATM6 XE XE Z 6-7.319 -66.343 -35.123 0.05 6.02 XE HETATM7 XE XE Z 7-8.580 -21.153 -30.274 0.05 7.43 XE HETATM8 XE XE Z 8 -1.494 -29.583 -2.339 0.06 7.36 XE HETATM9 XE XE Z 9-0.518 -16.483 -16.776 0.05 6.56 XE HETATM 10 XE XE Z 10 -0.464 -29.847 -4.386 0.06 6.04 XE HETATM 11 XE XE Z 11 -1.222 -16.234 -18.952 0.04 6.51 XE HETATM 12 XE XE Z 12 -6.001 -20.380 -4.532 0.05 7.22 XE HETATM 13 XE XE Z 13 -5.169 -27.522 -10.128 0.05 3.54 XE HETATM 14 XE XE Z 14 -11.931 -67.039 -4.699 0.05 7.96 XE HETATM 15 XE XE Z 15 -6.538 -10.408 -3.761 0.04 10.22 XE HETATM 16 XE XE Z 16 -0.458 -23.897 -31.221 0.05 6.85 XE HETATM 17 XE XE Z 17 -3.308 -65.181 -8.754 0.03 8.08 XE HETATM 18 XE XE Z 18 -11.080 -29.129 -2.144 0.04 5.61 XE HETATM 19 XE XE Z 19 -5.720 -73.029 -2.035 0.04 11.24 XE HETATM 20 XE XE Z 20 10.236 60.097 33.822 0.03 5.38 XE HETATM 21 XE XE Z 21 5.980 68.581 3.532 0.03 7.49 XE HETATM 22 XE XE Z 22 11.107 13.277 27.334 0.03 7.10 XE I'm pretty sure there aren't 22 sites for xenon to bind in lysozyme but it looks to me like the top 2 (maybe top 3) are actual sites which is great. The strange part is that when I use the same data and have Phenix look for 3 or 4 sites it then only finds 3 or 4 very low occupancy sites and the CC's/R's are again all very poor. Has anyone had this problem or does anyone know what's going on? This is the first success that I'm having with xenon derivatization and it seems to me that if Phenix can find 22 sites when asked to find 2 it should have no problem when asked to find 3 or 4. Thanks in advance, ~Brennan~
[ccp4bb] Off-topic: DPI and SAXS
Hi all. Does anyone have experience in using dual polarization interferometry (DPI) to study conformation change of protein when binding ligand? How do this compare to SAXS? I exclude NMR because the protein is large 90 kDa and no crystal structure is available. Thank you. Theresa
[ccp4bb] Abstract (to be considered for oral presentation) deadline 6th Feb.2012 for 7th Int. Workshop on X-ray Radiation Damage to Biological Crystalline Samples, Diamond Light Source, 14-16th March
Dear Colleague This is a reminder that for the Seventh International Workshop on X-ray Radiation Damage to Biological Crystalline Samples, Diamond Light Source 14-16th March 2012 the closing date for submission of abstracts to be considered for Oral presentations will be Monday 6th February 2012. For all other Abstracts the closing date will be Monday 20th February 2012. Registration, and further information and Abstract submission are open at: http://www.diamond.ac.uk/Home/Events/RD7---2012.html The cost of registration is £100 (£120 after February 20th 2012). This series of workshops was originally concerned with the effects of radiation damage during investigation of protein structures by X-ray crystallography. Other techniques of structural biology are now being included to ensure greater information exchange. The workshop will therefore be of interest to all those using ionising radiation to examine biological structures at the molecular level. It will consist of around 30 talks of 20 minutes each grouped under the topics below and ample time will be left for discussion. Confirmed speakers are indicated under each one. 1. Basic understanding of radiation damage mechanisms. Ian Carmichael, Kristina Djinovic Carugo, James Holton, Alke Meents 2. Temperature-dependent (including RT) radiation damage. Sasha Popov, Robert Thorne, Martin Weik 3. Reducing and mitigating radiation damage Matt Warkentin, Elspeth Garman, Robin Owen, Kunio Hirata 4. Practical aspects of managing radiation damage. Tom Burnley, Zbyszek Otwinowski, Tatiana Petrova, Enrique Rudino-Pinera 5. Damage at new sources - XFEL Henry Chapman, Lukas Lomb 6. Radiation damage in complementary fields Dominique Bourgeois, Liz Duke, Richard Henderson, Raimond Ravelli, Eddie Snell It will consist of around 30 talks of 20 minutes each covering: Registration and Abstract submission are now open. Registration and further information can be found at http://www.diamond.ac.uk/Home/Events/RD7---2012.html The cost of registration is £100 (£120 after February 20th 2012). At registration, delegates are invited to upload abstracts for submitted talks and poster presentations. Please use the abstract templatehttp://www.diamond.ac.uk/Home/Events/RD7---2012/Abstracts.html available on the site ensuring you indicate whether you would prefer an oral or poster presentation. The closing date for submission of abstracts to be considered for Oral presentations will be Monday 6th February 2012. For all other Abstracts the closing date will be Monday 20th February 2012. The organisers are Robin Owen, Armin Wagner, Elspeth Garman, Martin Weik, John McGeehan, Sean McSweeney, Colin Nave, Raimond Ravelli, Gerd Rosenbaum, James Holton, and Soichi Wakatsuki. Professor Elspeth F. Garman, President, British Crystallographic Association Director of Systems Biology Programme, Doctoral Training Centre. Postal address: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Tel: (44)-1865-613297 South Parks Road, FAX: (44)-1865-613201 OXFORD, OX1 3QU, U.K. E-mail: elspeth.gar...@bioch.ox.ac.ukmailto:elspeth.gar...@bioch.ox.ac.uk http://lmb.bioch.ox.ac.uk/www/garman/gindex.html - The University of Oxford's Doctoral Training Centre has three available programmes for October 2012 entry: Life Sciences Interface Doctoral Training Centre; Systems Biology Doctoral Training Centre; and Systems Approaches to Biomedical Science Industrial Doctorate Centre. To find out more, visit: http://www.dtc.ox.ac.uk/
[ccp4bb] THANK YOU: Crack-resistant tubes for centrifugation
Hello Everyone, Many many thanks to all the folks who responded to my question with very good suggestions. Here's a very quick and dirty summary of the various tubes and rotors that people use without any issues: (1) 50ml Nalgene tubes for an SS-34 rotor (2) Shape-matched new Fiberlite rotors (3) Nalgene round-bottom centrifuge tubes (re-usable) (4) Beckman ultracentrifuge tubes (re-usable) (5) 50 ml Falcon tubes (red cap) (6) 50 ml Corning tubes in F13S-14x50cy rotor (7) Polyethylene tubes work but polycarbonate do not, for some folks It seems Fiberlite rotors were a common suggestion and a bunch of folks suggested that breakage may have AS MUCH to do with centrifuge and shape-complementarity (understandably) as much as with the centrifuge tubes. Many thanks for your time and help. Go CCP4BB! Raji -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu Date: Tue, Jan 31, 2012 at 11:59 AM Subject: Crack-resistant tubes for centrifugation To: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] THANK YOU: Crack-resistant tubes for centrifugation
Clearly, it's better to use shape-matched rotors (I sort of assumed that you do that already!); however the BD/Falcon polyethylene tubes (conical ends) will actually change shape (flow) when placed into the round-ended rotors, if the speed is high enough -- and most of the time the tubes survive the transition w/o ill effects. Polycarbonate ones will shatter every time if their end taper does not match the rotor taper (ok, at 5000g they will probably survive). Artem On Thu, Feb 2, 2012 at 4:40 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hello Everyone, Many many thanks to all the folks who responded to my question with very good suggestions. Here's a very quick and dirty summary of the various tubes and rotors that people use without any issues: (1) 50ml Nalgene tubes for an SS-34 rotor (2) Shape-matched new Fiberlite rotors (3) Nalgene round-bottom centrifuge tubes (re-usable) (4) Beckman ultracentrifuge tubes (re-usable) (5) 50 ml Falcon tubes (red cap) (6) 50 ml Corning tubes in F13S-14x50cy rotor (7) Polyethylene tubes work but polycarbonate do not, for some folks It seems Fiberlite rotors were a common suggestion and a bunch of folks suggested that breakage may have AS MUCH to do with centrifuge and shape-complementarity (understandably) as much as with the centrifuge tubes. Many thanks for your time and help. Go CCP4BB! Raji -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu Date: Tue, Jan 31, 2012 at 11:59 AM Subject: Crack-resistant tubes for centrifugation To: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] THANK YOU: Crack-resistant tubes for centrifugation
P.P.S. it also matters a LOT how you fill the tubes. Leaving too much air gap on the top is actually very likely to cause crushing of the plastic. On Thu, Feb 2, 2012 at 6:18 PM, Artem Evdokimov artem.evdoki...@gmail.com wrote: Clearly, it's better to use shape-matched rotors (I sort of assumed that you do that already!); however the BD/Falcon polyethylene tubes (conical ends) will actually change shape (flow) when placed into the round-ended rotors, if the speed is high enough -- and most of the time the tubes survive the transition w/o ill effects. Polycarbonate ones will shatter every time if their end taper does not match the rotor taper (ok, at 5000g they will probably survive). Artem On Thu, Feb 2, 2012 at 4:40 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hello Everyone, Many many thanks to all the folks who responded to my question with very good suggestions. Here's a very quick and dirty summary of the various tubes and rotors that people use without any issues: (1) 50ml Nalgene tubes for an SS-34 rotor (2) Shape-matched new Fiberlite rotors (3) Nalgene round-bottom centrifuge tubes (re-usable) (4) Beckman ultracentrifuge tubes (re-usable) (5) 50 ml Falcon tubes (red cap) (6) 50 ml Corning tubes in F13S-14x50cy rotor (7) Polyethylene tubes work but polycarbonate do not, for some folks It seems Fiberlite rotors were a common suggestion and a bunch of folks suggested that breakage may have AS MUCH to do with centrifuge and shape-complementarity (understandably) as much as with the centrifuge tubes. Many thanks for your time and help. Go CCP4BB! Raji -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu Date: Tue, Jan 31, 2012 at 11:59 AM Subject: Crack-resistant tubes for centrifugation To: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] off-topic: special format for multiple sequence (protein) alignment
Dear members, Apologize for this off-topic question. I am looking for a protein sequence alignment tool which is capable to generate a particular output file similar to the attached format (please see the attached picture). I have been looking at some popular programs but none of them can show the conserved amino acids by colored blocks as shown in the attached file. Maybe some of you have seen some programs can do this? Thank you. King regards, Wenhe attachment: alignment.png
Re: [ccp4bb] off-topic: special format for multiple sequence (protein) alignment
On Thursday, 02 February 2012, you wrote: Dear members, Apologize for this off-topic question. I am looking for a protein sequence alignment tool which is capable to generate a particular output file similar to the attached format (please see the attached picture). I have been looking at some popular programs but none of them can show the conserved amino acids by colored blocks as shown in the attached file. Maybe some of you have seen some programs can do this? Thank you. That looks similar to the output of TeXshade, with the shading mode set to hydropathy. Ethan
