[ccp4bb] Protein/DNA microcrystals
Hello All, I am trying to crystallise a heterodimeric transcription factor complex bound to DNA. At the moment I have a construct which crystallises in many different conditions, all containing a PEG (3350, 4K, 6K, 8K) as precipitant along with various buffers and/or salts. When fine screening (in hanging drops) around these conditions I commonly get a shower of microcrystals at 20% (or higher) PEG. At lower PEG concentrations the crystals become more round and eventually the drops progress to phase separation (around 15% PEG). Lowering the protein concentration in the drops results in similar numbers of crystals forming, which end up smaller in size. I have tried seeding with the microcrystals from higher to lower PEG concentrations which results in crystal melting/phase separation. Also, the best crystals seem to grow on the glass slide and when I try to move them or pick them up, they are quite soft and hard to handle. Any advice on how to optimise conditions for growth of larger crystals and also tips for handling these soft crystals would be great. Thanks, Scott
Re: [ccp4bb] Protein/DNA microcrystals
Hi Scott, I'd try and set up drops at 20% PEG, then back off by dilution to 15% PEG at different time points (15 min, 30 min 1 hour...) Since equilibration of PEG in hanging drops is much slower than this, you actually need to dilute the drops. You can also consider microbatch. Flip -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Scott Berry Sent: Monday, August 11, 2008 07:57 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein/DNA microcrystals Hello All, I am trying to crystallise a heterodimeric transcription factor complex bound to DNA. At the moment I have a construct which crystallises in many different conditions, all containing a PEG (3350, 4K, 6K, 8K) as precipitant along with various buffers and/or salts. When fine screening (in hanging drops) around these conditions I commonly get a shower of microcrystals at 20% (or higher) PEG. At lower PEG concentrations the crystals become more round and eventually the drops progress to phase separation (around 15% PEG). Lowering the protein concentration in the drops results in similar numbers of crystals forming, which end up smaller in size. I have tried seeding with the microcrystals from higher to lower PEG concentrations which results in crystal melting/phase separation. Also, the best crystals seem to grow on the glass slide and when I try to move them or pick them up, they are quite soft and hard to handle. Any advice on how to optimise conditions for growth of larger crystals and also tips for handling these soft crystals would be great. Thanks, Scott
Re: [ccp4bb] problem running DISTANG
It is not accepting your input pdb; can you give more details? eg the command script.. eleanor Xie Jiabao wrote: Hello, I am trying to run the CCP4 program DISTANG from the command line (in order to do a translational grid search of a molecule over the asymmetric unit). But the program aborts with the following error message when I type DISTANG at the command line. ### ### CCP4 6.0: DISTANGversion 6.0 : 06/09/05## ### Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. !--SUMMARY_END--/FONT/B unknown-format file is being opened on unit 1 for INPUT. *** RWBROOK error: point code unitfunction ***1 -41MMDB_F_Open *** file : XYZIN *** reason : cannot open a file *** continue running, may crash ... BFONT COLOR=#FF!--SUMMARY_BEGIN-- DISTANG: XYZOPEN: Error opening logical name XYZIN Times: User: 0.0s System:0.0s Elapsed: 0:00 /pre /html !--SUMMARY_END--/FONT/B I am using CCP4i ver 6.0.1 installed on a machine running SuSe linux 10.1. Any help will be appreciated. Thanks in advance, Xie
Re: [ccp4bb] merohedral twinning problem
Se the twinning rules in http://www.ccp4.ac.uk/dist/html/twinning.html P3 pace group number space group point group possible twin operators 143 P3 PG3 -h,-k,l; k,h,-l; -k,-h,-l 144 P31 PG3 -h,-k,l; k,h,-l; -k,-h,-l 145 P32 PG3 -h,-k,l; k,h,-l; -k,-h,-l 146 H3 PG3 k,h,-l ie you need to test -h,-k,l (TWIN -1 0 0 0 -1 0 0 0 1) k,h,-l )TWIN 0 1 0 1 0 0 0 0 -1) -k,-h,-l (TWIN 0 -1 0 -1 0 0 0 0 -1) Eleanor pointless or sfcheck will suggest which is the most likely to be real. Eleanor Kristof Van Hecke wrote: Dear, Sorry for the off-topic question. I'm facing a (probably) merohedral twinning problem, regarding a small molecule. Using Xprep, I get a Hexagonal P-lattice with cell: 18.014 18.014 22.048 90.00 90.00 120.00 Mean |E*E-1| = 0.902 [expected .968 centrosym and .736 non-centrosym] However, based on the systematic absence exceptions, the probable (apparent) SG's are: P6(3)/m (Laue '6/m') P6(3) (Laue '6') P6(3)22 (Laue '622') 61/65 62=31 63-c- --c N60 50 36 2471 1420 N I3s 19 19 0 420 161 I186.6 223.1 4.6 30.015.5 I/s 2.3 2.6 0.3 1.6 1.2 I know there is a twin law to transform the (apparent) Laue group '6/m' to the (true) Laue group '-3' (TWIN law -1 0 0 0 -1 0 0 0 1) and merging the data in a trigonal SG, but this is not solving the structure at all... Has anyone noticed a similar case that could be of any help please..? Many thanks Kristof Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
Re: [ccp4bb] interface
Not sure if this is relevant, however as an addendum: a couple of years ago I looked at some data from a colleague using AREAIMOL and learned that while it's not intuitively obvious, it is possible that the calculated accessible surface area buried on one subunit may differ from that buried on the other subunit in the same interaction i.e. more surface area may be buried on one subunit than the other. I think that this effect is simply an artefact of how the accessible surface is defined, but it does mean that in some cases the simple division by 2 of the total calculated buried area may not be accurate for the individual subunits. In the example that I looked at the differences could be quite significant - in the most extreme case the split was 60/40 (although in others it was much smaller). I suppose this is really just a curiosity, but it does add more weight to the argument for reporting the total change in buried area due to interface formation. Best wishes Peter Steven Darnell wrote: Phil, I had a follow up conversation regarding this very topic. Here is an excerpt: The following is from Chothia and Janin (1975) Nature, 256:705-708, one of the early articles regarding buried surface area and protein interfaces: The surface area buried in the complex is then defined as the accessible surface area of one subunit plus that of the other subunit minus that of the complex. I believe that definition has not changed in 30 years. While I will agree that dividing by 2 approximates the physical area of the interface, this does not represent the total amount of surface area that is no longer accessible to solvent. In terms of desolvating the interface for binding, the latter is more appropriate. As you point out, PISA appears to be reporting the area of the interface, not the total surface area occluded from solvent. Confusing indeed. Regards, Steve Darnell Phil Jeffrey said the following on 8/8/08 10:03 AM: Which brings up something about PISA. If I run PISA on pdb entry 2IE3, which I'm familiar with, I get the following numbers from PISA and CCP4's AREAIMOL (surface areas in Angstrom^2) for the A:C interface. PISA for 2IE3 Automatic A:C interface selection 907.9 (a crystal packing interface is larger than this, but this surface is the A:C interface) AreaIMol with some editing of 2IE3 to separate the chains Chain A25,604.4 Chain C11,847.4 Total 37,451.8 Chain AC 35,576.6 Difference 1,875.2 Difference/2 937.6 For buried S.A. I agree with Steve Darnell's definition. However PISA appears to be reporting half that value, or what it calls interface area. Potentially confusing. Phil Jeffrey Princeton -- ___ Peter J Briggs, [EMAIL PROTECTED] Tel: +44 1925 603826 CCP4, [EMAIL PROTECTED] Fax: +44 1925 603825 http://www.ccp4.ac.uk/ Daresbury Laboratory, Daresbury, Warrington WA4 4AD
Re: [ccp4bb] interface
This will in general be true in an asymmetric interface (between two different molecules) if you look at the accessible surface, defined by the locus of a water molecule rolling over the surface. The surface measured by that method is larger on a convex surface than on a concave one. Phil On 11 Aug 2008, at 11:06, P.J.Briggs wrote: Not sure if this is relevant, however as an addendum: a couple of years ago I looked at some data from a colleague using AREAIMOL and learned that while it's not intuitively obvious, it is possible that the calculated accessible surface area buried on one subunit may differ from that buried on the other subunit in the same interaction i.e. more surface area may be buried on one subunit than the other. I think that this effect is simply an artefact of how the accessible surface is defined, but it does mean that in some cases the simple division by 2 of the total calculated buried area may not be accurate for the individual subunits. In the example that I looked at the differences could be quite significant - in the most extreme case the split was 60/40 (although in others it was much smaller). I suppose this is really just a curiosity, but it does add more weight to the argument for reporting the total change in buried area due to interface formation. Best wishes Peter Steven Darnell wrote: Phil, I had a follow up conversation regarding this very topic. Here is an excerpt: The following is from Chothia and Janin (1975) Nature, 256:705-708, one of the early articles regarding buried surface area and protein interfaces: The surface area buried in the complex is then defined as the accessible surface area of one subunit plus that of the other subunit minus that of the complex. I believe that definition has not changed in 30 years. While I will agree that dividing by 2 approximates the physical area of the interface, this does not represent the total amount of surface area that is no longer accessible to solvent. In terms of desolvating the interface for binding, the latter is more appropriate. As you point out, PISA appears to be reporting the area of the interface, not the total surface area occluded from solvent. Confusing indeed. Regards, Steve Darnell Phil Jeffrey said the following on 8/8/08 10:03 AM: Which brings up something about PISA. If I run PISA on pdb entry 2IE3, which I'm familiar with, I get the following numbers from PISA and CCP4's AREAIMOL (surface areas in Angstrom^2) for the A:C interface. PISA for 2IE3 Automatic A:C interface selection 907.9 (a crystal packing interface is larger than this, but this surface is the A:C interface) AreaIMol with some editing of 2IE3 to separate the chains Chain A25,604.4 Chain C11,847.4 Total 37,451.8 Chain AC 35,576.6 Difference 1,875.2 Difference/2 937.6 For buried S.A. I agree with Steve Darnell's definition. However PISA appears to be reporting half that value, or what it calls interface area. Potentially confusing. Phil Jeffrey Princeton -- ___ Peter J Briggs, [EMAIL PROTECTED] Tel: +44 1925 603826 CCP4, [EMAIL PROTECTED] Fax: +44 1925 603825 http://www.ccp4.ac.uk/ Daresbury Laboratory, Daresbury, Warrington WA4 4AD
[ccp4bb] Workshop Announcement: Biological physics at large facilities: From molecules to cells
*Workshop Announcement: Biological physics at large facilities: From molecules to cells* Jointly organised by ESRF-ILL Grenoble, 19 - 23 October 2008 The workshop will bring together the Biology and Physics communities for a discussion on the state-of-the-art and future perspectives of physics instrumentation and methods for the study of biological structures and dynamics at neutron sources and synchrotron radiation facilities. The impact of using physics methods in the study of biological systems is growing rapidly with unique opportunities offered by new instrumentation and methodology at large facilities worldwide. Both ESRF and ILL are planning considerable upgrades, with biophysical methods at the forefront. An integrated approach is essential to understand the physics of biological processes. The workshop will actively input into the facility upgrade plans and encourage networks of connections between complementary physical techniques. Please go to the webpage for details: http://www.ill.eu/news-events/workshops-events/biological-physics-at-large-facilities/home/ *Invited speakers include:* */ Martin Caffrey, Limerick, EI Leslie Leiserowitz, Rehovot, IS/* */Thierry Charitat, Strasbourg, F Jian Lu, Manchester, UK/* */Valeria De Marco, Amsterdam, NLRichard Ortega, Gradignan, F/* */Donald Engelman, Newhaven, USA Tassos Perrakis, Amsterdam, NL/* */Pieter Glatzel, Grenoble, F /**/Maikel Rheinstädter, Columbia-Missouri, USA/**//* */Thomas Hauss, Berlin, D/**//**/Tim Salditt, Göttingen, D/**//* */Malcolm Howells, Grenoble, F Tanya Smith, Leipzig, D/**//* */John Katsaras, Chalk River, CAMotomu Tanaka, Munich, D/* */Jeremy Lakey, Newcastle, UK Ada Yonath, Rehovot, IS/* */ /* *//* *Abstracts for Oral or Poster presentations : Deadline 12 September 2008.**//* */A few bursaries are available for financial help to attend the workshop/* -- Matthew Bowler Macromolecular Crystallography Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE = Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://www.esrf.fr/UsersAndScience/Experiments/MX/About_our_beamlines/ID14-2/ =
Re: [ccp4bb] Protein/DNA microcrystals
This is just one option out of many, but what often works miracles in cases like this is to subtly alter the DNA oligo - add or remove 1-2 nt from the ends, or to methylate/phosphorylate/dephosphorylate etc. This is quite cheap for short oligos and therefore can be very cost effective. Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Scott Berry Sent: Monday, August 11, 2008 1:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein/DNA microcrystals Hello All, I am trying to crystallise a heterodimeric transcription factor complex bound to DNA. At the moment I have a construct which crystallises in many different conditions, all containing a PEG (3350, 4K, 6K, 8K) as precipitant along with various buffers and/or salts. When fine screening (in hanging drops) around these conditions I commonly get a shower of microcrystals at 20% (or higher) PEG. At lower PEG concentrations the crystals become more round and eventually the drops progress to phase separation (around 15% PEG). Lowering the protein concentration in the drops results in similar numbers of crystals forming, which end up smaller in size. I have tried seeding with the microcrystals from higher to lower PEG concentrations which results in crystal melting/phase separation. Also, the best crystals seem to grow on the glass slide and when I try to move them or pick them up, they are quite soft and hard to handle. Any advice on how to optimise conditions for growth of larger crystals and also tips for handling these soft crystals would be great. Thanks, Scott
[ccp4bb] Rwork and Rfree in Sfcheck
Hello all, I have a complex structure with resolution 2.7A The total molecular weight is 80KD, and about 10% is disordered. I refined this structure to R and Rfree 25.5% and 29.7% with CNS. I also tried refmac5, but the R and Rfree have no difference with CNS. I use sfcheck of ccp4 to check this structure. But sfcheck give the R-factor for all relections 29%. and Rfree 33%. Why sfcheck and CNS give different R and Rfree? Which R and Rfree is true? Best Lisa
Re: [ccp4bb] Rwork and Rfree in Sfcheck
On Monday 11 August 2008 16:54, Lisa Wang wrote: Hello all, I have a complex structure with resolution 2.7A The total molecular weight is 80KD, and about 10% is disordered. I refined this structure to R and Rfree 25.5% and 29.7% with CNS. I also tried refmac5, but the R and Rfree have no difference with CNS. I use sfcheck of ccp4 to check this structure. But sfcheck give the R-factor for all relections 29%. and Rfree 33%. Why sfcheck and CNS give different R and Rfree? sfcheck usually does not have enough information to reproduce your full refinement. In particular, it does not reproduce the solvent model, TLS refinement, anomalous terms, variant scattering factors, etc. So it cannot reproduce your original Fcalc values. Given that it has different Fcalc, it is no wonder that it also has different R values. I suggest you ignore them. -- Ethan A MerrittCourier Deliveries: 1959 NE Pacific Dept of Biochemistry Regular Mail: Mailstop 357742 Health Sciences Building University of Washington - Seattle WA 98195-7742
