Re: [ccp4bb] Saving distances drawn in Coot 0.6.1
On 04/09/11 19:58, Romain Talon wrote: I would like to know if there is any way to save dotted lines distances drawn in Coot in order to recover the information then opening a new session, even if the program was closed before ? If you mean the dotted lines from environment distances, then those should be saved in the state file (and recovered after restarting). If that doesn't happen then that's a bug (you might like to try with the latest stable release, it works for me). If you mean the dotted lines from Calculate Distances, then that is not saved in the state file. (It can be, of course, with a bit of effort.. should it be?) Paul.
[ccp4bb] How to help crystal grow bigger
Dear All: Recently I am working on a protein which can already grow nice pyramid-like crystals after the condition was optimized, while the crystals are too small to be picked up. The crystals grew quite fast and densely, so I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put the plate under 16 degree to slow down the evaporation, while the crystals were still the same. I also tried macro or micro seeding with or without the paraffin oil. Macroseeding would give a larger crystal (not very nice) with many small crystals in the drop even I washed the seeds carefully. For microseeding, the same small crystals grew. I don't have many experiences in crystallography, so I have no idea how to make it grow bigger... Any suggestion is most welcome. Thanks. SnowDeer
Re: [ccp4bb] twinning in hexagonal system
Dear John,
In a hexagonal crystal class the possible point groups are
P3 (only a 3-fold axis)
P321(3-fold plus 2-fold relating hkl and kh-l)
P312 (3-fold and 2-fold relating (hkl and -k,-h,-l)
P6 ( 6 fold axis only)
P622 (6-fold plus 2-fold relating hkl and k,h,-l)
pointless will tell you which of these symmetry operators is well
observed, and suggest a pooint group.
truncate gives graphs of indicators of possible twinning.
The safest indicators are:
the moments of E - if the moment of E*4 is 2.0 twinning is likely.
However if you have non-crystallographic translation this can be misleading.
The L test done with the safe well-measured data
The cumulative intensity test with the safe well-measured data.
If these are satisfactory in your assignment of point group to P622 then
that is probably the correct one.
If they are not then you need to choose which symmetry element to drop.
ctruncate will suggest which is the most likely twin operator, and the
point group must be changed to remove that sym op (or sym ops)
The H test is not very reliable. it is only useful when you have the
point group right..It looks at the agreement between symmetry pairs so
obviously if you really have point group P6 but you assign the point
group as P3, it will tell you that the symmetry pair h,k,l and -h,-k,l
agree very well and give an H score of ~ 0.5.
On 08/30/2011 08:45 PM, Garib N Murshudov wrote:
Hello
Space groups with point groups 622 and 432 merohedral twinning is not possible
(they are the highest groups possible for proteins).
If you could merge in 622 it means that Rmerge was very small. It is very
likely that point group is either 622 or 6 with very strong rotational symmetry
that is perpendicular to 6 fold axis. In these cases H test (sfcheck based on
this) and N(z) test (truncate is based on this) will overestimate (H) or
underestimate (N(z)) if twin is present. At the moment better test for these
cases seems to be L-test (that is in ctruncate I think).
Solving structure using molecular replacement in the presence of twinning does
not seem to be a problem (unless data are very noisy and/or model is too far).
Pointless gives very good idea about true space group.
I would solve in P6_{x} space groups and then run zanuda (from ysbl server) to
correct space group.
I hope it helps
regards
Garib
On 30 Aug 2011, at 20:31, john peter wrote:
Hello All,
This is regarding twinning in a data set.
I collected a native data set to resolution, 1.8 A. I used XDS suite
to process and scale the data set. It scaled well in P622 and I found
systematic absence (l=6n present).
Hence thought the space group may be P6122/P6522. SFCHECK did not
show any twinning and also it did not detect pseudo-translation. Twin
test in http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin
Detector: Padilla-Yeates Algorithm ) showed perfect twinning.
Scaled in P61/65 and SFCHECK reported twinning fraction 0.272 no
pseudo-translation.
http://nihserver.mbi.ucla.edu/pystats/ ( Merohedral Twin Detector:
Padilla-Yeates Algorithm ) showed perfect twinning.
Another server from ucla http://nihserver.mbi.ucla.edu/Twinning/
showed partial twinning with twin fraction 0.23 as follows.
2 along a, b, a*, b*
No. Twin Law Related Reflections = 18033 (pairs)
No. Twin Law Pairs Considered = 9016
H = 0.266149
H2 = 0.095303
Twin Fraction = 0.233249 +/- 0.000602
(SHELXL Commands: TWIN 1 0 0 -1 -1 0 0 0 -1 and BASF 0.233249)
In P61/65, I got the following matthews-coeffs
mol/asym Matthews Coeff %solvent P(1.73) P(tot)
_
19.7587.39 .00 .00
24.8874.79 .00 .01
33.2562.18 .06 .14
4 2.4449.57 .57 .63
5 1.9536.97 .36 .21
6 1.6324.36 .00 .00
7 1.3911.75 .00 .00
_
May I ask what could be the real twin fraction and what is the
likelihood of solving the structure by molecular replacement by models
with 25 % sequence identity and 30 % sequence similarity.
Thank you so much for reading this mail during your busy hours and
all suggestions, comments would be gratefully welcome appreciated.
thank you ccp4 mailing list.
John
Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Temperature Factor statistics
On 08/31/2011 01:32 AM, Yuri Pompeu wrote: Quick newbie question, After i get my output file from baverage containing the average b-factor and rms by residues, How can I calculate and display the average (and or mean) B-factors? Is there a way of calculating it by protein, ligands and solvent separately? thank you If these are in different chains - Yes. Look at the plots.. Eleanor
Re: [ccp4bb] Trying to digest PISA results
Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] question about sfall output
Hmm - Firstly: your final FC PHIC calculation are just wrong. I cant tell why without more details - is the SG set correctly, because those phases cannot reflect the SG symmetry.. Can you send the log file? For the scaling against Fobs Qs. It looks to me as though you are using values of Fobs already scaled to match the model by some program.. The scaling algorithms in different programs are subtly different - there is no clear correct answer in to how to handle a dolvent continuum for instance - so I suspect the differences reflect that. In sfall (and I think most programs) the model B factors are changed to give best agreement with the Wilson scale for the observed data, and a scale factor is applied to the Fobs to bring it to match the fcalc. So I guess sfall has changed the overall B for the model slightly changing the higher resolution Fcs a bit.. Eleanor On 09/03/2011 08:07 PM, Pawel wrote: Dear all, First of all just hello to everyone since I am new to this forum. Not being a crystallographer, bear with me if I say something crazy. I was recently tinkering with Sfall and noticed that the Fcalc output by Sfall have different values depending on if one feeds Sfall with an input mtz file containing Fobs (mode sfcalc xyzin vs. mode sfcalc xyzin hklin) and also depending on if one tells Sfall to scale the Fobs or not (noscale on or off). This puzzles me as I would have thought that since Fcalc are calculated from the atomic coordinates, they should be affected by neither the Fobs fed to Sfall nor the scaling of the Fobs. As an example, Sfall calculations on PDB:1AHO. In each case the Fcalc column is the penultimate one. Here the difference in Fcalc between scaling or not scaling the Fobs is not so pronounced, but it still puzzles me that there would be any change at all. Also, in this case when the Sfall is run without hklin, there is a substantial change in PHIC. 1. HKLIN was provided and scaling turned on. LIST OF REFLECTIONS === H K L FP SIGFP FC PHIC 0 1 2 162.32 3.86 238.41 270.00 0 1 4 97.72 2.34 190.32 90.00 0 1 5 230.11 12.78 256.90 270.00 0 1 6 205.83 7.00 322.93 90.00 0 2 2 53.28 2.14 198.16 0.00 0 2 4 137.66 5.46 2.63 0.00 0 2 5 249.32 5.84 293.85 0.00 0 2 6 203.96 5.37 303.16 180.00 2. HKLIN was provided but scaling was turned off. LIST OF REFLECTIONS === H K L FP SIGFP FC PHIC 0 1 2 158.53 3.77 238.28 270.00 0 1 4 95.44 2.29 189.92 90.00 0 1 5 224.74 12.48 256.07 270.00 0 1 6 201.02 6.84 321.43 90.00 0 2 2 52.04 2.09 198.01 0.00 0 2 4 134.45 5.33 2.63 0.00 0 2 5 243.50 5.70 292.83 0.00 0 2 6 199.20 5.24 301.69 180.00 3. HKLIN with Fobs was not provided (both FC and PHIC are quite different) LIST OF REFLECTIONS === H K L FC PHIC 0 1 2 260.16 -108.18 0 1 4 122.43 -7.47 0 1 5 38.13 81.93 0 1 6 209.85 125.96 0 2 2 386.47 -78.65 0 2 4 145.47 -82.02 0 2 5 107.70 -15.80 0 2 6 101.94 138.75 If anyone could explain, I'd be very grateful. Thanks! Pawel Janowski
Re: [ccp4bb] Trying to digest PISA results
720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
[ccp4bb] MOLREP TFcntrst
Dear all, Does anyone know the definition of the TFcntrst value in MOLREPv10.2.35 and the difference of TFcntrst to Cntrst? an example from a MOLREP log-file: Time_elapsed: 0h 0m 18s Remained: 0h 0m 40s Sol_ RF TF Tf/sig TFcntrst PFind PFPFmin wRfac Scor Scor_max Cntrst Sol___1__1 25.38 2.848 1.00 1.00 1.00 0.725 0.143 0.143 0.00 Sol___2_12 30.78 2.136 1.00 1.00 1.00 0.736 0.120 0.143 0.00 Sol___3_11 24.33 2.538 1.00 1.00 1.00 0.733 0.124 0.143 0.00 Sol___4__1 29.08 8.246 1.00 1.00 1.00 0.665 0.266 0.266 0.00 Sol___5__1 34.66 7.999 1.00 1.00 1.00 0.666 0.265 0.266 0.00 Sol___6_14 27.33 2.200 1.00 1.00 1.00 0.724 0.135 0.266 0.00 ... If anyone could help me, I'd be very grateful. Thanks! Sven -- Empfehlen Sie GMX DSL Ihren Freunden und Bekannten und wir belohnen Sie mit bis zu 50,- Euro! https://freundschaftswerbung.gmx.de
Re: [ccp4bb] Trying to digest PISA results
I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] How to help crystal grow bigger
dear SnowDeer, The first step to see if bigger crystals can be grown is to use bigger protein drops and vary the precipitant/protein ratio at the beginning of the vapour diffusion experiment. You can work out for yourself why this should give you all the informations that you need in subsequent experiments. Enrico. On Mon, 05 Sep 2011 10:06:22 +0200, SnowDeer huanxu...@gmail.com wrote: Dear All: Recently I am working on a protein which can already grow nice pyramid-like crystals after the condition was optimized, while the crystals are too small to be picked up. The crystals grew quite fast and densely, so I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put the plate under 16 degree to slow down the evaporation, while the crystals were still the same. I also tried macro or micro seeding with or without the paraffin oil. Macroseeding would give a larger crystal (not very nice) with many small crystals in the drop even I washed the seeds carefully. For microseeding, the same small crystals grew. I don't have many experiences in crystallography, so I have no idea how to make it grow bigger... Any suggestion is most welcome. Thanks. SnowDeer -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] Workshop on Dynamic X-ray Scattering in Structural Biology 2011
Second Announcement – Registration is now open at http://cars9.uchicago.edu/biocars/pages/DXSworkshop11/index.shtml Workshop on Dynamic X-ray Scattering in Structural Biology 2011 November 2 - 4, 2011 Hosted by BioCARS, The University of Chicago Held at Advanced Photon Source, Argonne National Laboratory Organized by Zhong Ren,r...@uchicago.edu, 630-252-0498 Administrative contact: Katie Tietz,ti...@cars.uchicago.edu, 630-252-0450 This workshop will provide hands-on training in designing and conducting time-resolved experiments principally in macromolecular crystallography, but also in fiber diffraction and solution scattering from biological samples. BioCARS and BioCAT users will learn about the exciting capabilities in dynamic scattering at beamlines 14-ID and 18-ID of the APS. The workshop will feature three Lecture Sessions. The first will focus on the experimental techniques of time-resolved crystallography using either Laue or monochromatic diffraction. The second concerns a notable challenge of dynamic crystallography lying in data analysis and structure refinement. The third will showcase recent examples of structural dynamics that go beyond classical time-resolved crystallography. Four hands-on sessions (Crystallization, Experiment, Software and Case Study) are designed to offer an overview of various experimental approaches to capturing dynamic structures. In the Crystallization Session, we will demonstrate novel crystallization techniques for room temperature diffraction, often a key requirement for dynamic crystallography. The Software Session will provide training on data analysis and structure refinement of time-dependent structural changes. In the Experiment Session, attendees are invited to bring their own crystal samples and to conduct preliminary testing of their diffraction quality, often the first step in developing time-resolved projects. After the participants have familiarized themselves with a variety of theoretical and practical aspects of time-resolved experiments, we encourage attendees to present their specific cases in structural biology and prospective projects in the Case Study Session. Through one-on-one discussions with BioCARS and BioCAT staff or in a group discussion, this session will provide an opportunity to identify promising experimental approaches, potential difficulties and feasible solutions to the participants’ specific problems. Please note that due to time limitation, hands-on sessions will be able to accommodate up to 25 participants for the BioCARS experiment and software sessions and up to 10 participants for the BioCAT experiment session. Lecture sessions are open to all registered participants. Please send questions and comments to Zhong Ren (r...@uchicago.edu) or Katie Tietz (ti...@cars.uchicago.edu). ~~~ Vukica Srajer Ph.D. University of Chicago/CARS 9700 South Cass Ave, Bldg 434B Argonne, IL 60439 tel: (630) 252-0455 fax: (630) 252-0443
Re: [ccp4bb] Protein preps become a jelly
Hi Aidong, I've seen this in protein samples that have been snap frozen then thawed on ice. Thawing at room temperature (or in the hand) stopped this state forming. Could temperature be the issue? Maybe try 10C or leave the samples on the bench for a while and see if the state is reversible? David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of aidong Sent: 30 August 2011 16:31 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein preps become a jelly Dear Buddies, Sorry for bothering you with an off-ccp4 question. We recently are experiencing a very strange phenomena. A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C. All of them have been purified by gel filtration. Some of these proteins behave like this from very first preps but some of them had been very kind to us previously. We have googled extensively in CCP4BB and www but it appears this only happens to us. It would be highly appreciated that you could exchange their experiences or provide your suggestions. Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University Xiamen, Fujian 361005 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/
Re: [ccp4bb] Aging PEGs
I went through the peg solid stocks in our cupboard and noticed some of them smelled acrid. I probably won’t use them. Not sure whether they’re chemically decomposing. Maybe fossilising? Dave David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: 24 August 2011 22:21 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Aging PEGs And now, does anybody know of systematic data indicating how consistently all this matters? phx On 24/08/2011 21:45, Prince, D Bryan wrote: For those of us truly controlling types :), I used to make the PEG solutions and filter them over a Bio-Rad resin that filtered out all the junk added to stabilize the PEG solution. Then, of course I had to freeze all my PEG solutions in aliquots, or wrap them in foil and store at 4C in the dark. This would take several days, depending on the FW of the PEG. If you are really sensitive about what is in your PEG solutions, try GC-grade PEG's. The FW profile is much more restricted around the reported value (i.e. PEG 3350 molecular biology grade has a broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter peak profile.) Back before you could buy Crystal Screen I, II or HT, you had to make the stock solutions, then make the screen. But at least when you did that, you had all the stocks. Now, I just buy pre-made solutions, and keep them in a drawer with a date opened written on the bottle. Isn't progress grand? :) Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, August 24, 2011 3:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Aging PEGs A while ago I measured the pH's of old and new PEGs and found them very different, and internally attributed all old vs new PEG issues to pH. Upon reflection, this seems too simplistic. Are there other known mechanisms of crystallization capacities of PEGs of various ages? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Group Leader position in Structural Biology
The Beatson Institute for Cancer Research is committed to carry out world-class research into the biology of cancer and to help develop better treatments to improve the quality of life for cancer patients. We are core funded by Cancer Research UK and provide an outstanding environment for our scientists. We are now recruiting a Group Leader with an excellent track record and expertise in X-ray crystallography to develop and head a programme in Structural Biology. We invite applications from successful and motivated scientists who would like to develop an independent research programme in this area and join our well-established structural biology department. We offer either a tenure track and tenured position, depending on previous experience, and extremely generous support that includes core funding for staff and consumables as well as access to leading-edge facilities and research support (including state-of-the art X-ray crystallography, DNA sequencing, IT and proteomics). Further information on the Beatson's research activities, infrastructure and facilities is available on our website www.beatson.gla.ac.uk Applications, including a one to two page statement of research interests and goals, CV, list of publications and the names of three referees, should be sent to Professor Karen Vousden, Director, the Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, Scotland (email: k.vous...@beatson.gla.ac.uk). Applications should be received before 31st October 2011.
Re: [ccp4bb] Trying to digest PISA results
Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] Trying to digest PISA results
mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen jubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** .. 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://web.mac.com/bosch_lab/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] Trying to digest PISA results
NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Free flow electrophoresis would be another option, by the way anybody on the East Coast who has one of those instruments ? I'd be interested to get an email directly. Thanks, Jürgen On Sep 5, 2011, at 1:00 PM, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote: I get confused by these figures. As I understand it the interface area given in Pisa is half the loss of accessible area on forming the complex: is that right? We're working on a complex with interface area ~500A^2, where the complex is stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa estimate of DelG -2.3. Does that sound sensible? Phil On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: 720 is not an impressive size for a stable interface, but it may do depending on molecule size and exact chemistry of the interface (h-bonds, salt bridges, disulphides, charges etc etc). Everything is subject to chemical environment and concentration, as usual. For these entries, PISA gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol estimated (guessed) accuracy of PISA, this may or may not be a stable thing. And yes, it has about 70-80% chances to be simply an artefact of crystal packing, according to some sort of derivations that I did in 2nd PISA paper in J.Comp.Chem. in January last year. Having said all this, PISA is not an oracle and does not pretend to be correct in 100% of instances. Eugene. On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: Like Jan, I find it very useful to sort out the clear cut cases. Otherwise it is easy to get things wrong.. But isnt a buried surface area of 720 rather small for a stable interface? If there is other confirming evidence like 2 diff space groups then you feel more secure!! On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. 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://web.mac.com/bosch_lab/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] Trying to digest PISA results
Jacob, You may also try DARTS (drug affinity responsive target stability). In this method, small molecule (or protein) binding leads to specific protection of its target protein from protease digestion. It is completely label free, and appears to be able to detect very low affinity interactions (high microM for small molecules). We'd be happy to send detailed protocols if you are interested. http://www.pnas.org/content/106/51/21984.full Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
Re: [ccp4bb] Trying to digest PISA results
I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer.
Re: [ccp4bb] Trying to digest PISA results
Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing jinghu...@mednet.ucla.edu wrote: Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to digest PISA results
Ideally, the more nonspecific the protease, the better. (That would be a separate protein engineering project, or chemical catalyst project..) For now, we found that Pronase works well enough. Cheers, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 3:44 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing jinghu...@mednet.ucla.edu wrote: Excellent point indeed. We always include (at least one, preferably multiple) control proteins that are proteolysed equally across to make sure that the observed target stabilization is not due to fortuitous protease inhibition. What protease did you use for your nucleotide binding case? The protease sometimes matters. For example, thermolysin mainly only digests proteins that are unfolded, whereas pronase, which is a mixture of various proteases, can digest both folded and unfolded proteins. Best, Jing -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 12:18 PM To: Huang, Jing Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g -- Jing Huang, Ph.D. Associate Professor UCLA Department of Molecular Medical Pharmacology 310-825-4329 jinghuang at mednet dot ucla dot edu http://labs.pharmacology.ucla.edu/huanglab/ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, September 05, 2011 10:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Trying to digest PISA results NMR...take that! JPK 2011/9/5 Andreas Förster docandr...@gmail.com: AUC ! Andreas On 05/09/2011 6:00, Jacob Keller wrote: mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergenjubo...@jhsph.edu wrote: Hi Jacob, you forgot cross-linking to stabilize a weak complex and verify that it exists. Jürgen On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure or failure to maintain confidentiality may subject you to federal and state penalties. If you are not the intended recipient, please immediately notify us by return email, and delete this message from your computer. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** IMPORTANT WARNING: This email (and any attachments) is only intended for the use of the person or entity to which it is addressed, and may contain information that is privileged and confidential. You, the recipient, are obligated to maintain it in a safe, secure and confidential manner. Unauthorized redisclosure
Re: [ccp4bb] How to help crystal grow bigger
vary the precipitant/protein ratio Agree with Enrico. Reducing the concentration of your protein sample to make it form less nuclei, then it might grow bigger. Best R, tiantian On Mon, Sep 5, 2011 at 7:48 PM, Enrico Stura est...@cea.fr wrote: dear SnowDeer, The first step to see if bigger crystals can be grown is to use bigger protein drops and vary the precipitant/protein ratio at the beginning of the vapour diffusion experiment. You can work out for yourself why this should give you all the informations that you need in subsequent experiments. Enrico. On Mon, 05 Sep 2011 10:06:22 +0200, SnowDeer huanxu...@gmail.com wrote: Dear All: Recently I am working on a protein which can already grow nice pyramid-like crystals after the condition was optimized, while the crystals are too small to be picked up. The crystals grew quite fast and densely, so I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put the plate under 16 degree to slow down the evaporation, while the crystals were still the same. I also tried macro or micro seeding with or without the paraffin oil. Macroseeding would give a larger crystal (not very nice) with many small crystals in the drop even I washed the seeds carefully. For microseeding, the same small crystals grew. I don't have many experiences in crystallography, so I have no idea how to make it grow bigger... Any suggestion is most welcome. Thanks. SnowDeer -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/**institutes/institute-of-** biology-and-technology-saclay-**ibitec-s/unites-de-recherche/** department-of-molecular-**engineering-of-proteins-** simopro/molecular-toxinology-**and-biotechnology-laboratory-** ltmb/crystallogenesis-e.-sturahttp://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/**protein/mirror/stura/index2.**htmlhttp://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71 -- Shanghai Institute of Materia Medica, Chinese Academy of Sciences Address: Room 101, 646 Songtao Road, Zhangjiang Hi-Tech Park, Shanghai, 201203
