Re: [ccp4bb] Circular Dichoism 280nm range signal
''Signals in the region from 250-270 nm are attributable to phenylalanine residues, signals from 270-290 nm are attributable to tyrosine, and those from 280-300 nm are attributable to tryptophan. Disulfide bonds give rise to broad weak signals throughout the near-UV spectrum. '' http://www.ap-lab.com/circular_dichroism.htm From: JinSoo.Bae jiroz...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 9:41 AM Subject: [ccp4bb] Circular Dichoism 280nm range signal Dear all, Sorry for the off topic. We are comparing the high order structure between Fc engineered mAbs using CD methods. There are some different (noise ?) signal in the 280nm range each antibodies. Other region is almost close match with respect of peak shape and theta value. Is there any specific reason why the wavelength 280nm area have a little different signal ? That will be really thanks if someone explain the reason or give a some references papers. Thanks in advance. Jin Soo Bae 790-784 room 204, Dept of Life Science, POSTECH, san31, Hyoja-dong, Nam-gu, Pohang, Gyungbuk, Korea tel:82-54-279-8627 fax:82-54-279-8111
[ccp4bb] Protein concentration form chromatograms
Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
Re: [ccp4bb] Protein concentration form chromatograms
Step1: visit Protparam tool and CP your sequence, scroll down until you find the extinction coefficient part. If OD280 is not close to 1 then make sure to take that into account in your chromatogram say it is 0.7 step2: look at your mAU's If your peak is 1000 mAU then you have 0.7 mg/ml in that fraction Step3: you are pretty lazy google for it Jürgen On Jan 15, 2014, at 10:09 AM, Karel Chaz karel.c...@gmail.commailto:karel.c...@gmail.com wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Protein concentration form chromatograms
Hi Karel - To add to what Jurgen said, a few points on the measurement of the protein peak from the chromatogram. - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) - If your peak is a strong one, watch out for non-linearity in the absorbance measurement - my Akta UV monitor doesn't give a reliable reading once the A280 goes above 1.8. This will cause you to underestimate your total protein amount. - You also need to apply a correction factor since your UV cell path length isn't 1 cm. You could look up what the path length is in the manual, but the easiest way to do this is to compare UV readings for a set of fractions from your FPLC monitor and a standard spectrophotometer. Figure out the ratio of the two (it'll be a simple whole number, probably 2 or 5, or maybe 1 if your UV monitor does the correction automatically), then put it on a post-it next to the UV monitor so you won't have to do this again. Now multiply your chromatogram's integrated peak area by this factor to give you the standard (1 cm path length) A280 measurement. Hope that helps, Matt On 1/15/14 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Protein concentration form chromatograms
On 1/15/14, 9:34 AM, Matthew Franklin wrote: - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) GE's UNICORN for AKTAs (and probably the new Biorad FPLC software) integrates and tells you the area under whatever peak. Baselines and peak boundaries are user adjustable.
Re: [ccp4bb] Protein concentration form chromatograms
I am not sure, but probably path length not 1 cm in a detector cuvette . You must refer to your HPLS system manual for this value 15.01.2014 19:09, Karel Chaz пишет: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration form chromatograms
Thanks for all the replies. And I would not call this laziness, rather crowdsourcing I should have added a few more details; it is easy to export from say Unicorn to excel, a list of pairs of values, Abs280 vs elution times, with which one can recreate the chromatogram. I wanted to use these for my calculations. K On Wed, Jan 15, 2014 at 10:49 AM, Evgeny Osipov e.m.osi...@gmail.comwrote: I am not sure, but probably path length not 1 cm in a detector cuvette . You must refer to your HPLS system manual for this value 15.01.2014 19:09, Karel Chaz пишет: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration form chromatograms
I don't think it possible for protein purification purpose. There would be no linear correlation between A280 and the protein concentration. For pure protein analysis, as for minor amount protein used, it is possible. Acoot From: Karel Chaz karel.c...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 11:09 PM Subject: [ccp4bb] Protein concentration form chromatograms Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
Re: [ccp4bb] Protein concentration form chromatograms
If using Unicorn, 1)Open your chromatogram in Evaluation window. 2)Go to Operations- Pool 3)Choose which baseline estimation suits you, define the pools (numbered rulers that appear under the chromatogram) 4)type in the path length (2 or 10 mm for UV-900 and 2 mm for UPC-900) 5)type in the mass extinction coefficient (Molar extinction coefficient/Mw in daltons) 6) get your answer from the table at the bottom of the screen The reading will make sense for pure protein. Also note that for the most accurate result you need to know the exact path length which varies a little bit from cell to cell (or so they say). Regards, Dmitry On 2014-01-15, at 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K smime.p7s Description: S/MIME cryptographic signature
[ccp4bb] Best compounds for heavy atom soaks
Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
free cysteines? - pCMB phosphate-binding? - tungstate Best, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 1/15/2014 5:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
You don't mention the condition, which is important (esp pH). Best pH values are 6-8. From personal experience: try K2PtCl4 and OsCl3. Try ~5 mM for 30-60' (quick soak). The advantage of osmium salts is that they give a nice color to your crystals so you know something is binding. HTH and GL, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Wednesday, January 15, 2014 5:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Best compounds for heavy atom soaks Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
There is quite a bit of literature on this, but my favorite paper is this: http://www.ncbi.nlm.nih.gov/pubmed/18391402 Towards a rational approach for heavy-atom derivative screening in protein crystallography. Spoiler: The overall winner is ethyl mercury phosphate (Figure 7). Of course, chemistry will dictate your success (as every article and book chapter on this topic stresses). Check out quick soak literature, but not halides like sodium bromide (less likely to work at your resolution). Engin On 1/15/14, 11:18 AM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
More references to consider… You asked about soaking times - here are two articles advocating quick soaking at relatively high heavy atom concentration, which has worked well for us. We've had good luck with thimerosal. Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1099-103. Epub 2002 Jun 20. Generating isomorphous heavy-atom derivatives by a quick-soak method. Part II: phasing of new structures. Sun PD, Radaev S. Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1092-8. Epub 2002 Jun 20. Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases. Sun PD, Radaev S, Kattah M. Petsko has a good discussion about the chemistry of heavy atom derivatization. Methods Enzymol. 1985;114:147-56. Preparation of isomorphous heavy-atom derivatives. Petsko GA. PMID: 4079763 Beck et al. would suggest you consider the triiodo magic triangle. Acta Cryst. (2008). D64, 1179-1182[ doi:10.1107/S0907444908030266 ] A magic triangle for experimental phasing of macromolecules T. Beck, A. Krasauskas, T. Gruene and G. M. Sheldrick John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: tanne...@missouri.edumailto:tanne...@missouri.edu http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html On Jan 15, 2014, at 11:32 AM, Engin Özkan eoz...@stanford.edumailto:eoz...@stanford.edu wrote: There is quite a bit of literature on this, but my favorite paper is this: http://www.ncbi.nlm.nih.gov/pubmed/18391402 Towards a rational approach for heavy-atom derivative screening in protein crystallography. Spoiler: The overall winner is ethyl mercury phosphate (Figure 7). Of course, chemistry will dictate your success (as every article and book chapter on this topic stresses). Check out quick soak literature, but not halides like sodium bromide (less likely to work at your resolution). Engin On 1/15/14, 11:18 AM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
And quick iodide soaks may be useful in the 200 mM range. See the sped-up video: http://www.youtube.com/watch?v=45Qc3jOPaKY Quiz time: What wavelength would give iodide a similar signal to that of selenium? Can one get a better signal than selenium by choosing a different wavelength for data collection? and of course the webinar presented by Thomas Edwards of the SSGCID: http://www.rigaku.com/node/1369 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Wednesday, January 15, 2014 11:18 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Best compounds for heavy atom soaks Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
On Jan 15, 2014, at 10:27 AM, Jim Pflugrath wrote: Quiz time: What wavelength would give iodide a similar signal to that of selenium? Can one get a better signal than selenium by choosing a different wavelength for data collection? I'll bite, At ~11,000eV Iodine has about 3.8 anomalous electrons. Going to lower energies will increase the anomalous signal. At 10,000eV Iodine has ~4.6 anomalous electrons. Scott smime.p7s Description: S/MIME cryptographic signature
Re: [ccp4bb] Best compounds for heavy atom soaks
Hi do not forget the clusters like Ta6Br12 or the lanthanides. in case your interesting protein is a membrane protein there are some choices that might work better than other we have described it here. http://www.ncbi.nlm.nih.gov/pubmed/16855303 This is not exclusive to membrane proteins at all. My personal favorite is orange platinum, with an acupunture needle stick the tip in the orange platinum powder, and and gently let the needle hoover over the drop, the static electricity will pull some orange platinum crystals into your drop without disturbing it. This is really nice if you have mechanically sensitive crystals. Then just seal the drop and wait for the orange color to emerge. Its almost like cooking a restaurant dish, just more expensive. cheers Preben On 1/15/14 6:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Tel: +47 2284 0794
[ccp4bb] Postdoc position at the Research Complex at Harwell University of Bath
*** Research Officer in Dynamic Structural Science at the Research Complex at Harwell and the University of Bath (fixed term for 2 years) *** https://www.bath.ac.uk/jobs/Vacancy.aspx?ref=VH2136 Applications are invited from suitably qualified candidates for an EPSRC funded post-doctoral Fellowship in Dynamic Structural Science. The successful applicant will have proven track record in either time-resolved spectroscopy or molecular/macromolecular crystallography. They will be housed in the Research Complex at Harwell, at the Rutherford Appleton Laboratory (RAL), Didcot, Oxfordshire, and will also be associated with the Department of Chemistry, at the University of Bath. The post-doctoral Fellowship is associated with an EPSRC supported project, centred at the Research Complex at Harwell, to work on Dynamic Structural Science, using synchrotron and neutron diffraction techniques and/or XAFS methodologies. The successful applicant will join a team of chemists and biologists from six UK Universities and from the Central Facilities at RAL, led by Professor Paul Raithby from the University of Bath. The main duties of the successful applicant will be to develop new methodologies for carrying out time resolved spectroscopic and crystallographic studies on a range of molecular and macromolecular species. The work will also include some synthesis, spectroscopic characterisation, computational studies and instrument development. The post is available from 1st April 2014 and will terminate on 28th February, 2016. Salary: Starting from £30,728, rising to £36,661 Closing Date: Sunday 16 February 2014 Informal enquiries may be addressed to Professor Paul Raithby (p.r.rait...@bath.ac.uk, tel. 01225 383183), however please ensure that applications are submitted through the University of Bath website. -- Dr Arwen R Pearson Astbury Centre for Structural Molecular Biology Astbury Building The University of Leeds LS2 9JT United Kingdom (e) a.r.pear...@leeds.ac.uk
Re: [ccp4bb] Best compounds for heavy atom soaks
Dear Rhys, an important addendum to the magic triangle is the fact that it gives you direct evidence whether or not the substructure search has succeeded (by locating the triangle in the substructure solution) so that you can carry on with phasing via density modification. At 3.5A resolution this is an utterly time- and effort saving piece of information you do not want to miss. Best, Tim On 01/15/2014 07:21 PM, Tanner, John J. wrote: More references to consider… You asked about soaking times - here are two articles advocating quick soaking at relatively high heavy atom concentration, which has worked well for us. We've had good luck with thimerosal. Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1099-103. Epub 2002 Jun 20. Generating isomorphous heavy-atom derivatives by a quick-soak method. Part II: phasing of new structures. Sun PD, Radaev S. Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1092-8. Epub 2002 Jun 20. Generating isomorphous heavy-atom derivatives by a quick-soak method. Part I: test cases. Sun PD, Radaev S, Kattah M. Petsko has a good discussion about the chemistry of heavy atom derivatization. Methods Enzymol. 1985;114:147-56. Preparation of isomorphous heavy-atom derivatives. Petsko GA. PMID: 4079763 Beck et al. would suggest you consider the triiodo magic triangle. Acta Cryst. (2008). D64, 1179-1182[ doi:10.1107/S0907444908030266 ] A magic triangle for experimental phasing of macromolecules T. Beck, A. Krasauskas, T. Gruene and G. M. Sheldrick John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: tanne...@missouri.edumailto:tanne...@missouri.edu http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html On Jan 15, 2014, at 11:32 AM, Engin Özkan eoz...@stanford.edumailto:eoz...@stanford.edu wrote: There is quite a bit of literature on this, but my favorite paper is this: http://www.ncbi.nlm.nih.gov/pubmed/18391402 Towards a rational approach for heavy-atom derivative screening in protein crystallography. Spoiler: The overall winner is ethyl mercury phosphate (Figure 7). Of course, chemistry will dictate your success (as every article and book chapter on this topic stresses). Check out quick soak literature, but not halides like sodium bromide (less likely to work at your resolution). Engin On 1/15/14, 11:18 AM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] Best compounds for heavy atom soaks
Keith's favourites over many years (shared with Gideon): K2PtCl4 K2PtCN4 KAu(CN)2 Ethyl Mercury Thiosalicylate (gentle Hg reagent) Try for about 2-12 hours at 1 mM initially. If crystals crack, reduce concentration - at least they bind! If these don't work, then finding a derivative is probably going to be hard1 K On 15 Jan 2014, at 17:18, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys keith.wil...@york.ac.uk * Prof. Keith S. Wilson, Structural Biology Laboratory, University of York, YORK YO10 5DD UK tel 44-1904 328262 fax 44-1904 328266 ** Email disclaimer This email and its attachments may be confidential and are intended solely for the use of the intended recipient. If you are not the intended recipient of this email and its attachments, you must take no action based upon them, nor must you copy or show them to anyone. Please contact the sender if you believe you have received this email in error. Any views or opinions expressed are solely those of the author and do not necessarily represent those of The University of York.
Re: [ccp4bb] Best compounds for heavy atom soaks
Also, if you have a sample changer for easy screening, try 1 mM, 5 mM 20 mM of each for 1h, 5h, overnight, and screen all ~60 crystals for diffraction overnight.Send those that survive to the synchrotron. K On 15 Jan 2014, at 17:18, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys keith.wil...@york.ac.uk * Prof. Keith S. Wilson, Structural Biology Laboratory, University of York, YORK YO10 5DD UK tel 44-1904 328262 fax 44-1904 328266 ** Email disclaimer This email and its attachments may be confidential and are intended solely for the use of the intended recipient. If you are not the intended recipient of this email and its attachments, you must take no action based upon them, nor must you copy or show them to anyone. Please contact the sender if you believe you have received this email in error. Any views or opinions expressed are solely those of the author and do not necessarily represent those of The University of York.
Re: [ccp4bb] Best compounds for heavy atom soaks
Hi Rhys - Don't forget to try sulfur-SAD, especially the multi-crystal version published recently: http://journals.iucr.org/d/issues/2013/07/00/ba5189/index.html This seems well suited to your situation. - Matt On 1/15/14 12:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] R-factors from SfCHECK versus R-factors from PHENIX
Hi Ursula, you will find answers here: http://www.phenix-online.org/papers/he5476_reprint.pdf Pavel On Wed, Jan 15, 2014 at 1:38 PM, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote: I am submitting a structure to the PDB database. The SfCHECK summary report provided by the PDB validation shows an R-factor for model vs structure factors of 0.32, while the R-factor from the refinement program PHENIX is 0.21. I am not familiar with SfCHECK, but I am puzzled how these programs can calculate such different R-factors. I would be thankful for some explanation. Ursula -- Ursula Schulze-Gahmen, Ph.D. Assistant Researcher UC Berkeley, QB3 356 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 642 8766
Re: [ccp4bb] Best compounds for heavy atom soaks
Did anybody mention native gel electrophoresis to select suitable HA ions? Worked for us really nicely in a situation were speed was essential. Here's a reference: PMID 14646083 Klaus === Dr. Klaus Fütterer Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 15 Jan 2014, at 20:49, Matthew Franklin wrote: Hi Rhys - Don't forget to try sulfur-SAD, especially the multi-crystal version published recently: http://journals.iucr.org/d/issues/2013/07/00/ba5189/index.html This seems well suited to your situation. - Matt On 1/15/14 12:18 PM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Best compounds for heavy atom soaks
Hello Rhys Grinter, hello to the ccp4bb community, I don't necessarily want to advertise here one of the major research topic of Eric Girard and late Richard Kahn lab (my previous lab ;-) ) but... You could maybe try lanthanide complexes ? They are composed by a chemical ligand, which can provide a non-covalent binding to protein surfaces, and a chelated lanthanoid ion, which can ensure a really high phasing power. If they bind to your protein, you should get accurate phases, wether you collect at the LIII absorption edge of the lanthanide concerned (the best) or at higher energy (Se K edge for example...). Maybe someone have some of the lanthanide complexes currently available http://www.natx-ray.com/products/catalogue_consum_CSM002.html around you ? If you can't get them before your synchrotron run, I follow Matthew Franklin for the Sulfur-SAD method. Furthermore, I should add the use of cadmium acetate for your soaking solution. Cd2+ ion bind well to glutamate and aspartate residue, but can also be really invasive... Good luck. Best regards. Romain. Le 15/01/2014 18:18, RHYS GRINTER a écrit : Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
Re: [ccp4bb] Best compounds for heavy atom soaks
Dear Rhys, You may consider Xenon derivative, which could be prepared simply pressurizing the protein crystals in a xenon chamber. It does not require any modification of mother liquor. It just needs cryo-protectant where crystals are stable for at least one to two mins. Higher Pressure (20 to 40 bar) and 1-2 min incubation time are normally sufficient for binding of Xenon to proteins. Xenon binds to pre existing hydrophobic cavities of proteins by dispersion force. Xenon derivatives are highly isomorphous to native crystals. So SIRAS phasing could be efficient. However if you consider collecting data at longer wavelengths you could get anomalous signal from sulphur too. Weaker SAD or SIRAS phases from Xenon derivative could be used to bootstrap the Sulphur phasing. Similarly Kr pressurisation could be tried. MAD experiment can be performed at any tunable beamline but the disadvantage with this derivative is, it desorps quickly during cooling after pressurisation leaving out with lower than 50-60% occupancy. Success of the Xenon/Krypton derivatisation depends on size of the proteins and how stable your crystals are under cryo-protectant. The bigger the protein, higher the chance of Xe/Kr binding. best Santosh Santosh Panjikar, Ph.D. Scientist Australian Synchrotron 800 Blackburn Road Clayton VIC 3168 Australia Ph: +61-4-67770815 (mobile) +61-3-85404276 (office)
[ccp4bb] is there any DNA?
Dear all, We all know that for purified protein, A260/A280=0.5, and for purified DNA, A260/A280=1.8. Now my protein A260/A280=0.75, So I wonder if there is DNA mixed with protein? Is there any way I can use to test if there is DNA? Thanks~
Re: [ccp4bb] Best compounds for heavy atom soaks
You may be missing a trick by not using metals as crystallisation additives if your best diffraction is only 3.5 Angstroms. Uranium has to be my favourite heavy metal, even if I've solved more structures with Pt and Hg. It gave crystals diffracting to 1.2 A from a protein that otherwise gave no crystals, and kicked off the MAD as MIR idea. In fact the scattering from the uranium atoms was strong enough the structure could be solved by arp-warp just from the heavy atom positions (and this is a 56 kDa protein). As far as I know it's still the largest structure solved by direct methods, if you allow that definition to include actinides. The anomalous is not too shabby either. On Jan 16, 2014, at 2:18 AM, RHYS GRINTER wrote: Hello message board, My group has some crystals of an interesting protein to take to the synchrotron in a couple of weeks. We won't be able to prepare and crystallise a SelMet derivative during that time period, but we have loads of crystals sitting around. The diffraction isn't great, we see maybe 3.5 at home but might be enough to get over the line. It will be a very difficult MR target, so we were thinking of soaking so crystals with heavy atomic compounds that we have lying around. I was wondering if people had any suggestions of compounds that people have used successfully for experimental phasing and maybe concentrations to use and soaking time. Cheers, Rhys
