[ccp4bb] AW: [ccp4bb] off-topic;Crystal cannot dissolved in buffer
Hi Xiao, I have observed this phenomenon with two proteins I worked with: rubber-like crystals which you can bend without breaking. As others mentioned: if you have this rubber-like crystals it must be protein, since salt crystals behave very differently. Also I could not think of any reason why salt crystals would refuse to dissolve. Fresh crystals did not suffer from this phenomenon, so I suspect it is due to some (slow) crosslinking. I did not try myself, but you could consider adding reducing agents (glutathion, TCEP, DTT) to your crystallization setup to see if this helps. Also try the freshest crystals you have for Xray diffraction since in my hands this “rubber-transition” led to a dramatic reduction in resolution of the data. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Xiao Xiao Gesendet: Freitag, 26. September 2014 01:53 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] off-topic;Crystal cannot dissolved in buffer Hi everyone, Sorry for an off-topic question. I got a problem with crystal dissolving. Basically I got crystals of my protein in various conditions, most conditions contain PEGs but different salts. These crystals has very similar shape, so it should not be salt. Now I am trying to dissolve the crystal to make sure it is my protein, by SDS-PAGE and N-term sequencing. I washed the crystals in its original crystallization buffer few times then transfered them into regular buffer (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal didn't dissolve. I then tried to heat it then add SDS loading buffer to run a gel, I did see very small amount of protein on the gel, at the correct position, but it's not enough for N-term sequencing. Is it normal for a protein crystal? And does anyone have any suggestion for dissolving such crystals?
[ccp4bb] Postdoctoral fellowship on structural biology of eukaryotic complexes in Pavia (Italy)
The Department of Biology and Biotechnology of the University of Pavia (Italy) is announcing a new postdoctoral programme on a few selected strategic topics. With an above-average salary, successful postdoctoral fellows will potentially be considered for tenure-track positions at the end of the fellowships which are for two years with possible extension to third year. One of the topics will be on stuctural and biophysical studies of eukaryotic multiprotein complexes and membrane proteins. Expertise in protein expression using insect and/or mammalian cells is essential. Proven experience with expression and purification of membrane protein targets will be a plus. The language of the application will be English, however knowledge of (or commitment to learn) Italian language is highly recommended. The fellowships will be assigned on a competitive basis by an external advisory panel and successful candidates will expectedly have a strong CV. Applicants should have a PhD in Molecular Biology, Crystallography, Biophysics or a related area, and relevant publications, and must be under 35 years old. The Department of Biology and Biotechnology of the Univerisity of Pavia (Italy) has a strong division in structural biology with two highy integrated and well-equipped laboratories led by Dr. Federico Forneris and Prof. Andrea Mattevi, as illustrated at www.unipv.it/biocry. Pavia is a lovely university town in the north of Italy with very good life quality. Interested candidates should request details about application procedure, detailed topics and deadlines to federico.forne...@unipv.it or andrea.matt...@unipv.it no later than October 10th. Best, F. Federico Forneris, PhD --- The Armenise-Harvard Laboratory of Structural Biology Department of Biology and Biotechnology University of Pavia Via Ferrata, 9 27100 Pavia - Italy http://fornerislab.unipv.it http://www.unipv.it/biocry/index.php?page=investigators#forneris
[ccp4bb] New! Interactive structure factor tutorial...
Hi folks! Nearly two decades ago I wrote the interactive structure factor tutorial, to help people understand diffraction beyond the simple explanation of Bragg's law. As such it has been one of the longest lived interactive teaching tools on the web. However time and technology have caught up with it, and it is increasingly difficult to run, requiring plugins, security exceptions, and even obsolete browsers. So new I've completely rewritten it using modern web technologies. Try it here: http://www.ysbl.york.ac.uk/~cowtan/sfapplet2/sfintro.html It should work on any version of Firefox or Chrome since about the mid Permian, and I expect Safari and Opera as well. It's even useable on tablets (although a big screen helps). Internet Explorer will probably work since version 9. Show me a userbase and willingness to help with testing and I might be able to get it running on IE 7 or 8. Problem reports very welcome. Once it's stable this version will replace the existing version. Kevin -- EMAIL DISCLAIMER: http://www.york.ac.uk/docs/disclaimer/email.htm
[ccp4bb] How to generate cif file?
Dear all, In my protein structure I found a modified Tyrosine that is covalently bound to a nucleotide by a phosphodiester bound. I am having problem to generate the library description for such a modification. Would someone have any ideas on how to generate such a cif file? Thanks in advance. Regards, Gaelle
Re: [ccp4bb] How to generate cif file?
HI Gaelle, If both the nucleotide already exists in the PDB (you can check on LigandExpo), you can just create a LINK description with Jligand in CCP4. See this tutorial: http://www.ysbl.york.ac.uk/mxstat/JLigand/tutorial_link.html If you have the cif file with the LINK description, you can just load it in Refmac. You also need to add a LINK record to you PDB file. Jligand will give you an example record. HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gaelle PESCE Sent: Friday, September 26, 2014 12:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to generate cif file? Dear all, In my protein structure I found a modified Tyrosine that is covalently bound to a nucleotide by a phosphodiester bound. I am having problem to generate the library description for such a modification. Would someone have any ideas on how to generate such a cif file? Thanks in advance. Regards, Gaelle
Re: [ccp4bb] off-topic;Crystal cannot dissolved in buffer
Xiao, You could be the victim of the dreaded PEG cross-linking effect. One of the unfortunate by-products of keeping PEG stock solutions in water is that they will form peroxides and aldehydes. They will slowly cross-link the surface of some crystals. However, it is dependent on the nature of your protein's composition of surface residues, so not every protein crystal does this. I had one case where PEG4000 grown crystals would be resistant to dissolving and would easily bend (just like Herman related); the thinner rods would spring back straight. After placing the crystals into buffer known to dissolve them, I poked the crystals hard and the insides squeezed out like toothpaste, leaving an empty sack behind. The bottom-line is that fresh crystals diffracted better than old crystals because of this cross-linking. Suggestions: 1) make your PEG stocks up fresh or store them in the freezer as aliquots. 2) Remove oxidized PEGs from your stocks: see Ray et al. Biochemistry 1991, 30, 6866-6875 and Jurnak, J. Cryst. Growth, 76, 577-582, 1986. 3) Check to see if freshly grown crystals behave better. Best of luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Sep 25, 2014, at 7:53 PM, Xiao Xiao victor41...@gmail.com wrote: Hi everyone, Sorry for an off-topic question. I got a problem with crystal dissolving. Basically I got crystals of my protein in various conditions, most conditions contain PEGs but different salts. These crystals has very similar shape, so it should not be salt. Now I am trying to dissolve the crystal to make sure it is my protein, by SDS-PAGE and N-term sequencing. I washed the crystals in its original crystallization buffer few times then transfered them into regular buffer (500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal didn't dissolve. I then tried to heat it then add SDS loading buffer to run a gel, I did see very small amount of protein on the gel, at the correct position, but it's not enough for N-term sequencing. Is it normal for a protein crystal? And does anyone have any suggestion for dissolving such crystals?
Re: [ccp4bb] how to measure the angle between two aromatic ring plane in PYMOL?
On Thu, 25 Sep 2014, Wang, Bing wrote: Hi everyone, I want to tell an angel between two aromatic planes which comes from two different molecules and cross each other. First, I tried the easier ways. I presume these two aromatic rings are in the perfect planes, that is why I tried plane_wizard.py and draw_plane_cgo.py which work very well. However after I got two plates by plane_wizard or draw_plane, I don't know how to measure the angle between these two plates. I also don't know whether I could get the angle I wanted from these two ways. I tried vector_angly.py which cann't loaded into pymol properly. If it could, how can i do to get the angel from these two plates I drew. Solutions? Since I am in the beginner state, please show me details which i could follow step by step. Second, If these two aromatic rings are not in the perfect planes, how can i find the best fit planes? And then find the angle between the best fit planes? I tried svdplos.py and makeCGOplates.py which are downloaded on line. unfortunately both of these can't be loaded into pymol properly. Solutions? Thanks! Bing Wang Wang, I am sure the other replies will sort your problem in PyMOL itself. But I thought it might be useful for you to consider checking any calculation of the angle works properly by using the well-established CCP4 tool GEOMCALC: http://www.ccp4.ac.uk/html/geomcalc.html I (and others) use the tool to calculate butterfly bend angles in flavin rings (this involves finding the angle between two mean-plane rings). If you want any help on running it then contact me off list and I would be happy to help. Good luck, Oliver PS. I really like the phrase tell an angel. Paul Emsley's coot refers to geometry only optimization as send in the bond angels. | Dr Oliver Smart | | Global Phasing Ltd., Cambridge UK | | http://www.globalphasing.com/people/osmart/ |
[ccp4bb] is small dialysis tubes reusable??
Hi all, I use Slide-A-Lyzer MINI Dialysis Devices from Thermo for dialysis of small protein volumes. Are they reusable? if so how can we store them?? Thanks and Regards, Manjula R Research Scholar Department of Biophysics National Institute of Mental Health and Neurosciences Bengaluru-29, Karnataka INDIA E-mail: manjula@gmail.com Mobile no:+91-9538553356 http://www.nimhans.kar.nic.in/ https://www.nimhans.kar.nic.in/
Re: [ccp4bb] is small dialysis tubes reusable??
Hi Manjula, I imagine that they could be reusable, if you stored them in a buffer with some sort of preservative like sodium azide (NB Toxic) or maybe a bacteriostatic agent like EDTA or something. However, when you use them, a small amount of protein is going to remain in the cassette after each use, so I would only use each cassette for a specific protein to prevent cross-contamination. Additionally, I'd worry about protein from old preps falling apart/denaturing and then contaminating future fresh preps of protein. I guess it depends at what step during purification you are using them. HTH, Dave [image: David Briggs on about.me] David Briggs about.me/david_briggs http://about.me/david_briggs On 26 September 2014 15:51, Manjula Ramu manjula@gmail.com wrote: Hi all, I use Slide-A-Lyzer MINI Dialysis Devices from Thermo for dialysis of small protein volumes. Are they reusable? if so how can we store them?? Thanks and Regards, Manjula R Research Scholar Department of Biophysics National Institute of Mental Health and Neurosciences Bengaluru-29, Karnataka INDIA E-mail: manjula@gmail.com Mobile no:+91-9538553356 http://www.nimhans.kar.nic.in/ https://www.nimhans.kar.nic.in/
Re: [ccp4bb] calculation of active site area
Fpocket and CAVER are good. Yarrow On Friday, September 19, 2014, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Please tell me the names of good servers / tools which calculate the size and surface area of the active site pocket of a protein.. -- Regards Faisal School of Life Sciences JNU
[ccp4bb] Off Topic: The 72nd Annual Pittsburgh Diffraction Conference
3rd Announcement The 72nd Annual Pittsburgh Diffraction Conference October 26 - 28, 2014 Georgia Hotel Conference Center Athens, GA, Organized by John Rose and B.C. Wang Deadlines (more information below) Hotel reservations: Deadline September 29, 2014 Conference Registration:Deadline October 15, 2014 Sidhu Award:Deadline October 1, 2014 Abstract Deadline: Deadline October 15, 2014 Reception: Sunday October 26 7:00 pm Opening Remarks:Monday October 27 8:45 am Banquet:Monday October 27 7:00 pm Scientific Sessions I Scientific Sessions begin at 9:00 am and 1:30 pm Oct 27 AM Session: 100 years of X-ray Diffraction Chair John Rose (University of Georgia) Brian Matthews (University of Oregon) Bi-Cheng Wang (University of Georgia) Charles Campana (Bruker-AXS) Aina Cohen(Stanford Synchrotron Radiation Laboratory) Larry DeLucas (University of Alabama, Birmingham) Oct 27 PM Session: Structure Elucidation, Refinement and Interpretation through Powder Diffraction Crystallography Chair Charles Lake (Indiana University of Pennsylvania) Brian Toby (APS, Argonne National Laboratory) Bob von Dreele (APS, Argonne National Laboratory) Ashfia Huq (SNS,Oak Ridge National Laboratory) Angus Wilkinson (Georgia Institute of Technology) Clarina dela Cruz (Oak Ridge National Laboratory) Scott Speakman (PANalytical) October 27: Poster Session Posters on all topics related to X-ray diffraction are invited. The C.S. Yoo Poster award ($400) will be given for the best poster presented by a student in the areas of macromolecular crystallography, small molecule crystallography and diffraction based material science). Please see the PDC website (pdc14.bmb.uga.edu) for information about abstract preparation. Scientific Sessions II Scientific Sessions begin at 9:00 am and 1:30 pm Oct 28 AM Session: Crystallographic Education Chair Joe Ng (University of Alabama, Huntsville) Cora Lind-Kovacs (University of Toledo) Joe NG (University of Alabama, Huntsville) Leighton Coates (Oak Ridge National Laboratory) Larry DeLucas (University of Alabama, Birmingham) Bill Duax (Hauptman-Woodward Institute) Claudia Rawn (University of Tennessee) Oct 28 PM Session: Advances in Neutron Crystallography Chair Paul Langan (Oak Ridge National Laboratory) Irene Weber (Georgia State University) Don Ronning (University of Toledo) Andrey Kovalevsky (Oak Ridge National Laboratory) Daniel Unruh (Texas Tech University) Mayank Aggarwal (Oak Ridge National Laboratory) Conference Workshop Sunday October 26 9:00 - 5:00 Georgia Hotel Conference Center Powder Diffraction Structure Solution with GSAS-II Robert B. Von Dreele and Brian Toby X-ray Science Division, Advanced Photon Source, Argonne National Laboratory The new GSAS-II package is believed to be the only general-purpose crystallographic analysis package to be started in the current century. It is also the only package written in a modern scientist-friendly language, such as Python. GSAS-II performs structural analysis from x-ray and neutron diffraction data, which may be single-crystal and/or powder diffraction data. It is hoped that by the time of the PDC, work will have begun on extending GSAS-II to include TOF data from the neutron spallation source. Beyond structural analysis, GSAS-II can be used for powder diffraction data reduction, texture and stress analysis as well as pattern indexing and structure solution. This workshop will highlight these latter capabilities of GSAS-II: indexing and structure solution from powder diffraction, although all features of the package will be introduced. Participants should bring a laptop (Windows, Mac or Linux) with the GSAS-II software installed. To download GSAS-II see subversion.xray.aps.anl.gov/trac/pyGSAS. Contact the authors in advance for help with software installation. Limited assistance will also be available before the workshop start time. Registration $65.00 (includes lunch) Space is limited to 30 participants so register now Important Deadlines Conference Registrations – Deadline October 15, 2014 Everyone must register for the meeting including invited speakers. $150 Conference registration for professionals (non students) $ 50 Conference registration for students $ 65 GSAS-II Workshop registration only Reservations can be made via the conference web page pdc14.bmb.uga.edu (REGISTRATIONS) Hotel reservations – Deadline September 29, 2014 The PDS has reserved a block of 40 rooms at the UGA Hotel Conference Center at the conference rate of $104.00 per night. Hotel reservations may be made via HOUSING link on the PDC web page or by
[ccp4bb] R/Rfree gap with pseudotranslational symmetry
Hi all, I'm having an issue during structure refinement where I can't seem to get my Rfree to drop below about 0.30 (whereas Rwork is around 0.24) and was wondering if anyone had any thoughts on the matter. I have a 2.3Å dataset that I processed the data in P31 and got an MR solution in phaser with RFZ=29.3, TFZ=14.9. There is 6-fold NCS present in the structure and it appears there is pseudotranslational symmetry with P62 as well. I have run Zanuda and P31 seems to be the correct space group. Also, Xtriage didn't detect any sort of twinning. Before refinement in phenix the R/Rfree gap is rather small, however even after one round of refinement I am finding that this gap increases to almost 0.06. I have a feeling that the high symmetry present has something to with this R/Rfree gap but was hoping some of you may have some helpful suggestions for how to deal with it. Thanks, Kim
Re: [ccp4bb] R/Rfree gap with pseudotranslational symmetry
On Fri, Sep 26, 2014 at 8:17 PM, Kimberly Stanek kas...@virginia.edu wrote: Before refinement in phenix the R/Rfree gap is rather small, however even after one round of refinement I am finding that this gap increases to almost 0.06. I have a feeling that the high symmetry present has something to with this R/Rfree gap but was hoping some of you may have some helpful suggestions for how to deal with it. It's normal for the R/R-free gap to increase during the first round of refinement in molecular replacement - in fact, unless you are solving a near-identical crystal form and keeping the original R-free flags, this is almost guaranteed to happen. MR will use all reflections and the limited refinement Phaser does uses very coarse parameterization (rigid-body and group B-factor), so the R-free will usually be quite low and sometimes even lower than R-work. Restrained refinement will immediately start to open the gap, but if it's working properly, it won't keep expanding throughout refinement. At this resolution a gap in the range of 0.02-0.04 would be normal - less than this is unusual. My guess is you just need to change the relative weights of the X-ray target and geometry restraints so that the latter are stronger. Also, use NCS restraints if you aren't already. -Nat
Re: [ccp4bb] R/Rfree gap with pseudotranslational symmetry
Well - R / Rfree gaps are not really the thing to worry about after a MR solution. Both seem to have decreased sensibly, so now you need to worry about the map quality - can you see things to rebuild? water structure ? etc. It is possible the NC translation will affect the R factors. NC translation can make whole classes of reflections very weak, and of course weak reflections will have high R factors.. But I dont understand how an NC translation can make P31 SG be pseudo P62? ? Eleanor On 26 September 2014 21:56, Nat Echols nathaniel.ech...@gmail.com wrote: On Fri, Sep 26, 2014 at 8:17 PM, Kimberly Stanek kas...@virginia.edu wrote: Before refinement in phenix the R/Rfree gap is rather small, however even after one round of refinement I am finding that this gap increases to almost 0.06. I have a feeling that the high symmetry present has something to with this R/Rfree gap but was hoping some of you may have some helpful suggestions for how to deal with it. It's normal for the R/R-free gap to increase during the first round of refinement in molecular replacement - in fact, unless you are solving a near-identical crystal form and keeping the original R-free flags, this is almost guaranteed to happen. MR will use all reflections and the limited refinement Phaser does uses very coarse parameterization (rigid-body and group B-factor), so the R-free will usually be quite low and sometimes even lower than R-work. Restrained refinement will immediately start to open the gap, but if it's working properly, it won't keep expanding throughout refinement. At this resolution a gap in the range of 0.02-0.04 would be normal - less than this is unusual. My guess is you just need to change the relative weights of the X-ray target and geometry restraints so that the latter are stronger. Also, use NCS restraints if you aren't already. -Nat
