[ccp4bb] protein complex crystallisation
Dear CCP4 Community, My apologies for the off topic question to the bb. I am trying to crystallise a small protein-protein complex. I purify as a complex protein after expression in BL21DE3 cells through NI-NTA affinity chromatography. pI of one of the protein is around 10.6 where as another component have 4.0. I am in the screening stage of the crystallisation. During crystallistion process it precipitate very quickly as i add the protein into the crytallisation solution drop. This happens in almost all the condition of the hampton INDEX and crystal screens. I purify this complex in Tris-Cl buffer at pH=8.0. it is happily soluble and donot prcipitate at this pH in the same buffer. I can concetrate this protein upto ~10-15mg/ml. could any one suggest the solution, that will be most appreciatable. Thanks in advance Ron
Re: [ccp4bb] protein complex crystallisation
What protein concentration are you using in your screen? It sounds like you need to try lower protein concentration, or a screen with lower precipitant concentration, or both. Hampton makes a Crystal Screen Lite with the precipitant concentrations half of what they are in the normal screen (or you can dilute the regular screen). Don't forget that protein concentration is one of the important variables to optimize. For some complexes, I have had best results with protein concentration as low as 2.5-3 mg/mL. Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ron hudson Sent: Saturday, September 20, 2008 11:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein complex crystallisation Dear CCP4 Community, My apologies for the off topic question to the bb. I am trying to crystallise a small protein-protein complex. I purify as a complex protein after expression in BL21DE3 cells through NI-NTA affinity chromatography. pI of one of the protein is around 10.6 where as another component have 4.0. I am in the screening stage of the crystallisation. During crystallistion process it precipitate very quickly as i add the protein into the crytallisation solution drop. This happens in almost all the condition of the hampton INDEX and crystal screens. I purify this complex in Tris-Cl buffer at pH=8.0. it is happily soluble and donot prcipitate at this pH in the same buffer. I can concetrate this protein upto ~10-15mg/ml. could any one suggest the solution, that will be most appreciatable. Thanks in advance Ron
Re: [ccp4bb] protein complex crystallisation
Dear Ron -- On 20 Sep 2008, at 15:59, Ron hudson wrote: it is happily soluble and donot prcipitate at this pH in the same buffer. Just a quick question. How does your complex look like under DLS? You might still have soluble aggregates, which will precipitate... you can screen for soluble non-aggregates by varying the pH, and check all of this under DLS. HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
[ccp4bb] Postdoctoral position available at University of Wisconsin at Madison
A postdoctoral position is immediately available in the structural biology lab of McArdle Cancer Institute of University of Wisconsin at Madison to study structures of signaling complexes related to cancer (http://mcardle.oncology.wisc.edu/faculty/bio/xing.html). Strong background in crystallography is required and background in protein biochemistry is a plus. Please send your application to [EMAIL PROTECTED]
[ccp4bb] Crystallogrphy today
Dear friends and crystallographers, During One of my lab meeting , I told twinning in crystals are ok, because ccp4's recent releases just need the keyword TWIN to solve them, As a new generation research student, I am now confused, is that I need to learn and understand all programs(so many...but research does not mean relaying on them) to solve my crystallographic problems(is that all) if you see all the queries in ccp4BB is just about undocumented or misunderstood program oriented questions. is that all i have to learn in crystallography in future. Still upto what limitations we are now in crystallography. this is my very naive and prime question. 1.Phase problem 2.twin problem 3.solving intrinsically disordered proteins 4.hetro multimeric proteins 5.high order oligomers 6.cryo crystallography 7.automation in high through put crystallography 8.radiation damage 9.kinetic crystallography 10. crystal growth research (antigravity, pressure ) 11.stereo graphics if i am right all the above has been studied (what we are not clear still about them), I need an answer to motivate me in doing my research in Crystallography. S.Jayashankar (A confused new generation research student) Research Student Institute for Biophysical Chemistry Hannover Medical School Germany.
Re: [ccp4bb] Crystallogrphy today
On Sep 20, 2008, at 2:18 PM, Jayashankar wrote: Dear friends and crystallographers, Are they mutually exclusive? During One of my lab meeting , I told twinning in crystals are ok, because ccp4's recent releases just need the keyword TWIN to solve them, I believe the closer you get to complete twinning, the more intractable the problem gets. I don't know if the pain scales linearly with twinning fraction. As a new generation research student, I am now confused, This is both normal and proper, but has nothing to do with generation. is that I need to learn and understand all programs(so many...but research does not mean relaying on them) to solve my crystallographic problems(is that all) if you see all the queries in ccp4BB is just about undocumented or misunderstood program oriented questions. Actually there are many lively discussions about fundamental problems. These will often arise in the context of a specific program, but you still have to understand the problem the program is designed to solve. is that all i have to learn in crystallography in future. That's up to you, but I would say no. Learn the fundamentals. Programs will come and go. Still upto what limitations we are now in crystallography. this is my very naive and prime question. 1.Phase problem This is still "the" problem. Some inroads have been made toward ab initio solutions, but the traditional heavy-atom methods, variations like MAD phasing, and molecular replacement remain in practice the standard approaches for what you usually find in the PDB. 2.twin problem see above. 3.solving intrinsically disordered proteins Crystals give a spatial average, so there is nothing magical you can do to overcome intrinsic disorder. 4.hetro multimeric proteins ribosomes are I think the current upper bound 5.high order oligomers Chromatin fibers maybe? 6.cryo crystallography This is routine. 7.automation in high through put crystallography The main problem is finding strong enough amphetamines to keep one awake while reading the papers. 8.radiation damage see cryocrystallography, and take lots of vitamin C 9.kinetic crystallography Laue? There is now a fair body of work, but development for irreversible enzyme systems is probably a worthwhile future goal. 10. crystal growth research (antigravity, pressure ) Anti-gravity? 11.stereo graphics In the land of the blind, the one-eyed Macintosh user is king (as long as the program is not X-windows-based). if i am right all the above has been studied (what we are not clear still about them), I need an answer to motivate me in doing my research in Crystallography. S.Jayashankar (A confused new generation research student) Research Student Institute for Biophysical Chemistry Hannover Medical School Germany.
Re: [ccp4bb] Crystallogrphy today
I agree with Bill. After a few minutes thinking, in between "jobs working in the yard": It depends if you need to understand "everything" (I guess that's impossible these days) --- are you comfortable with publishing and defending research results that you do not understand? I am not. In quite a few labs there are crystallographers available who understand and can make sure that they can defend the results/science. If you have such people on staff, I guess (but don't like the sound of it) that you can treat macromolecular crystallography as a "service" and you can focus on other things. But you cannot (should not) publish or defend things you do not understand. A little more involved is the answer "what if the problem is too difficult for standard approaches"? We tend to see a lot of those. Problems where you have to sit and think because all "standard" approaches do not work. Of course these problems cannot be solved without a thorough understanding of standard problems and procedures. My $0.02 (soon to be $-0.02) Mark -Original Message- From: William G. Scott <[EMAIL PROTECTED]> To: CCP4BB@JISCMAIL.AC.UK Sent: Sat, 20 Sep 2008 3:44 pm Subject: Re: [ccp4bb] Crystallogrphy today On Sep 20, 2008, at 2:18 PM, Jayashankar wrote:? ? > Dear friends and crystallographers,? ? Are they mutually exclusive?? ? >? >? > During One of my lab meeting ,? >? > I told twinning in crystals are ok, because ccp4's recent releases > just > need? > the keyword TWIN to solve them,? ? I believe the closer you get to complete twinning, the more intractable the problem gets. I don't know if the pain scales linearly with twinning fraction.? ? >? >? > As a new generation research student, I am now confused,? ? This is both normal and proper, but has nothing to do with generation.? ? > is that I need to? > learn and understand all programs(so many...but research does not > mean? > relaying on them)? > to solve my crystallographic problems(is that all)? > if you see all the queries in ccp4BB is just about undocumented or? > misunderstood program oriented questions.? ? Actually there are many lively discussions about fundamental problems. These will often arise in the context of a specific program, but you still have to understand the problem the program is designed to solve.? >? >? > is that all i have to learn in crystallography in future.? ? That's up to you, but I would say no. Learn the fundamentals. Programs will come and go.? ? >? > Still upto what limitations we are now in crystallography.? > this is my very naive and prime question.? >? > 1.Phase problem? ? This is still "the" problem. Some inroads have been made toward ab initio solutions, but the traditional heavy-atom methods, variations like MAD phasing, and molecular replacement remain in practice the standard approaches for what you usually find in the PDB.? ? >? > 2.twin problem? ? see above.? ? >? > 3.solving intrinsically disordered proteins? ? Crystals give a spatial average, so there is nothing magical you can do to overcome intrinsic disorder.? >? > 4.hetro multimeric proteins? ? ribosomes are I think the current upper bound? ? >? > 5.high order oligomers? ? Chromatin fibers maybe?? ? >? > 6.cryo crystallography? ? This is routine.? ? >? > 7.automation in high through put crystallography? ? The main problem is finding strong enough amphetamines to keep one awake while reading the papers.? ? >? > 8.radiation damage? ? see cryocrystallography, and take lots of vitamin C? >? > 9.kinetic crystallography? ? Laue? There is now a fair body of work, but development for irreversible enzyme systems is probably a worthwhile future goal.? ? >? > 10. crystal growth research (antigravity, pressure )? ? Anti-gravity?? ? >? > 11.stereo graphics? ? In the land of the blind, the one-eyed Macintosh user is king (as long as the program is not X-windows-based).? >? >? > if i am right all the above has been studied (what we are not > clear? > still about them),? >? > I need an answer to motivate me in doing my research in > Crystallography.? >? > S.Jayashankar? > (A confused new generation research student)? > Research Student? > Institute for Biophysical Chemistry? > Hannover Medical School? > Germany.?
Re: [ccp4bb] libxml2
On 06:56 Thu 18 Sep , William G. Scott wrote: > CCP4 6.0.99 builds its own libxml2. It there a way to get it to use a > later version I have already installed? The reason is that this is > messing up other programs that for some reason I can't fathom (the evil > $DYLD_LIBRARY_PATH remains unset) are finding this older version. Bill, Here's the hacks I'm using to make ccp4 act very slightly more like normal software: # Rapper bundles libxml2 and boehm-gc. Don't build, use or install those. pushd src/rapper 2>/dev/null sed -i \ -e '/^AC_CONFIG_SUBDIRS(\[gc7.0 libxml2\])/d' \ configure.ac sed -i \ -e '/^SUBDIRS/s:libxml2 gc7.0::g' \ Makefile.am sed -i \ -e '/^rapper_LDADD/s:../gc7.0/libgc.la ../libxml2/libxml2.la:-lgc -lxml2:g' \ LOOP/Makefile.am sed -i \ -e '/^INCLUDES/s:-I../gc7.0/include -I../libxml2/include:-I/usr/include/gc -I/usr/include/libxml2:g' \ LOOP/Makefile.am eautoreconf popd -- Thanks, Donnie Donnie Berkholz Developer, Gentoo Linux Blog: http://dberkholz.wordpress.com pgpd7OG9u7ExQ.pgp Description: PGP signature
Re: [ccp4bb] Crystallogrphy today
Dear Jayashankar, How refreshing it is to hear someone question how things work. Bill and Mark are beginning to show you the way. You are ready to roll up your sleeves and study the subject. This list of apparently solved problems hides a wonderland of studies and hard work (some of it based on Bill's and Mark's perspiration, and that of many others) that depend on fundamentals in physics, biology, cryogenics, computer science, and on and on. Take each one and start asking questions. If your curiosity lasts, you have a chance of making some nice advances in the field. Bob On Sat, 20 Sep 2008, Jayashankar wrote: Dear friends and crystallographers, During One of my lab meeting , I told twinning in crystals are ok, because ccp4's recent releases just need the keyword TWIN to solve them, As a new generation research student, I am now confused, is that I need to learn and understand all programs(so many...but research does not mean relaying on them) to solve my crystallographic problems(is that all) if you see all the queries in ccp4BB is just about undocumented or misunderstood program oriented questions. is that all i have to learn in crystallography in future. Still upto what limitations we are now in crystallography. this is my very naive and prime question. 1.Phase problem 2.twin problem 3.solving intrinsically disordered proteins 4.hetro multimeric proteins 5.high order oligomers 6.cryo crystallography 7.automation in high through put crystallography 8.radiation damage 9.kinetic crystallography 10. crystal growth research (antigravity, pressure ) 11.stereo graphics if i am right all the above has been studied (what we are not clear still about them), I need an answer to motivate me in doing my research in Crystallography. S.Jayashankar (A confused new generation research student) Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. -- = Robert M. Sweet E-Dress: [EMAIL PROTECTED] Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
Re: [ccp4bb] Crystallogrphy today
Hi, In addition to the excellend comments posted already, I would like to venture an opinion regarding learning, technology, and associated matters. Do not make the problem fit the techniques you know - instead try to make sure that you know (or know where to learn) enough techniques to solve the problem at hand. This rather simple principle appears to be frequently forgotten or ignored - sometimes with humorous results. As a corollary, I would like to also mention that the relatively huge area of science that is often referred to as 'macromolecular crystallography' can be approached as a basic area of science in itself, or it may be viewed as a set of sophisticated technologies suitable for answering complex questions in structural & basic biology, drug design, protein engineering, etc. etc. Regardless of what you personally decide to do, try to view it as both basic and applied science - this approach pays off later. Artem P.S. twinned crystals still suck. _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jayashankar Sent: Saturday, September 20, 2008 5:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystallogrphy today Dear friends and crystallographers, During One of my lab meeting , I told twinning in crystals are ok, because ccp4's recent releases just need the keyword TWIN to solve them, As a new generation research student, I am now confused, is that I need to learn and understand all programs(so many...but research does not mean relaying on them) to solve my crystallographic problems(is that all) if you see all the queries in ccp4BB is just about undocumented or misunderstood program oriented questions. is that all i have to learn in crystallography in future. Still upto what limitations we are now in crystallography. this is my very naive and prime question. 1.Phase problem 2.twin problem 3.solving intrinsically disordered proteins 4.hetro multimeric proteins 5.high order oligomers 6.cryo crystallography 7.automation in high through put crystallography 8.radiation damage 9.kinetic crystallography 10. crystal growth research (antigravity, pressure ) 11.stereo graphics if i am right all the above has been studied (what we are not clear still about them), I need an answer to motivate me in doing my research in Crystallography. S.Jayashankar (A confused new generation research student) Research Student Institute for Biophysical Chemistry Hannover Medical School Germany.
Re: [ccp4bb] Crystallogrphy today
6.cryo crystallography This is routine. Well maybe there are still some surprises. Consider the case of high- pressure cooling. 8.radiation damage see cryocrystallography, and take lots of vitamin C There may be some surprises here too as one looks at smaller crystals with smaller beams. Just a few other comments. Students starting out now in this field may have the opportunity to work with the next-generation x-ray sources such as FEL's and ERL's. These sources are not just brighter than the current synchrotrons. They will provide fully-coherent, high spectral brilliance x-rays in shorter pulses than are currently available. A number of novel techniques are being proposed that will take advantage of these kinds of beams, but who knows what opportunities wait in the future for the next highly-motivated student. One place to start looking is "coherent diffraction imaging" ... I think C. Riekel et. al. just published a Phys Rev Letter on this subject. Richard Gillilan MacCHESS
Re: [ccp4bb] Crystallogrphy today
Now that it is past 12:00 in Brookhaven, I want to be the first to wish Bob Sweet a happy birthday! Bob's brilliance is matched by his helpfulness, kindness and compassion; he very much lives up to his name. So on behalf of probably hundreds of us, happy birthday. It's been awhile (too long) since I broke your beamline. Bill
