Re: [ccp4bb] smeared spot in diffraction
Hi all, yesterday i have tried annealing, no significant improvement. Thank you so much for all those suggestions. Summary: 1. check diffraction on RT. 2. dehydrate crystal, many methods 3. try annealing 4. grow crystal in presence of cryo 5. try smaller crystal, sometimes small crystal is better 6. change loop. but my crystal is not thin plate and needle. I will try one by one next week, when machine is available to me, and let you know the result. Thanks again. Fengxia
[ccp4bb] Vacancies: Scientific Database Curator (2 Posts) - PDBe
The Protein Data Bank in Europe (PDBe http://www.ebi.ac.uk/pdbe/) is looking for two Scientific Database Curators to work as part of the PDBe Annotation Team on the annotation of preliminary data submissions to the PDB/EMBD. Details of the job and application are given below. Please get in touch with me if you need any further information. regards- Jawahar Swaminathan, Ph.D. Coordinator - PDBe Annotation Depositions, PDBe, European Bioinformatics Institute, Cambridge CB10 1SD --- http://www.embl.de/aboutus/jobs/jobs_embl_ebi_hinxton/2010/database_curators/w_10_007_ebi/ Grade: 5 or 6 depending on qualifications and experience EMBL site: EMBL-EBI Hinxton Commencing date: As soon as possible after closing date Job description: The Protein Data Bank in Europe (PDBe) Team (www.ebi.ac.uk/pdbe) is part of the worldwide Protein Data Bank organisation (wwPDB; www.wwpdb.org), which maintains the global archive of structural data on biomacromolecules. The Team also maintains a number of databases that support deposition and advanced search services for structural biologists and the wider scientific community. The Team consists of an international and inter-disciplinary mix of professionals (structural biologists, software engineers, and database engineers). The high-quality curation of PDB entries is essential in establishing the PDBe as a world-leading source of protein-structure information. To maintain and extend this position, the PDBe team is looking for an expert structural biologist for a demanding role in database curation. The work involves annotating preliminary PDB submissions and extracting biological information relevant to a given entry. In addition, curators draw on their own area of expertise in contributing to the development of methods and procedures. Qualifications and experience: Applicants should possess a PhD in some area of structural biology or chemistry as well as have a broad knowledge in molecular biology. An in-depth knowledge of biochemistry and/or protein structure analysis would be highly advantageous. The ideal candidate will be computer literate and be comfortable working with Linux/Unix, Emacs, molecular graphics software and also have some scripting experience. Excellent English, communication and interpersonal skills are essential. Contract: An initial contract of 2 years will be offered to the successful candidate. This can be renewed, depending on circumstances at the time of the review. Closing date: 27 February 2010 EMBL is an inclusive, equal opportunity employer offering attractive conditions and benefits appropriate to an international research organisation. Please note that EMBL does not return CVs or attached documents to applicants. *To apply, please send a CV (including names and addresses of referees) and covering letter, by email, quoting ref. no. W/10/007/EBI in the subject line, to: applicati...@ebi.ac.uk * General enquiries may be sent to applicati...@ebi.ac.uk or to the following address: EMBL-EBI, Personnel, Wellcome Trust Genome Campus, Hinxton, CB10 1SD. *Important Information to Applicants:* EMBL does not charge a fee at any stage of the recruitment process (application, interview meeting, processing, training or any other fees). EMBL does not concern itself with information on bank accounts. ---
[ccp4bb] coot: fit ligand
Dear all, we would like to ask coot to fit the Val-Lys dipeptide of a thermolysin structure. We read in a PDB-file with the VK-fragment and run the Other Modelling Tools-Find Ligands tool. Unfortunately coot rips the pair apart and only places the valine into the density, the lysine is simply omitted. Is there a way to kindly as coot to not separate the two when trying to find a suitable home for them? Ta, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Refining against images instead of only reflections
-Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of James Holton Sent: 21 January 2010 08:39 To: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Refining against images instead of only reflections It is interesting and relevant here I think that if you measure background-subtracted spot intensities you actually are measuring the AVERAGE electron density. Yes, the arithmetic average of all the unit cells in the crystal. It does not matter how any of the vibrations are correlated, it is still just the average (as long as you subtract the background). The diffuse scatter does NOT tell you about the deviations from this average; it tells you how the deviations are correlated from unit cell to unit cell. James, as I've pointed out before this is completely inconsistent with both established DS theory and many experiments performed over the years. If you're simulation is producing this result, then the obvious conclusion is that you're not simulating what you claim to be. I don't know of a single experimental result that supports your claim. In order for it to be true the total background (i.e. the sum of the detector noise, air scatter, scattering from the cryobuffer, Compton scattering from the crystal and of course the diffuse scattering itself) would have to be a linear function (or more precisely planar since the detector co-ords are obviously 2-D) of the detector co-ordinates in the region of the Bragg spots, since that is the background model that is used for background subtraction. Whilst it may be true that detector noise and non-crystalline scattering can be accurately modeled by a linear background model (at least in the local region of each Bragg spot), this cannot possibly be generally true of the DS component, and since getting at the DS component is the whole purpose of the experiment, it is crucial that this be modeled accurately. Of course your claim may well be true if there's no DS, but we're talking specifically about cases where there is observable DS (otherwise what's the point of your simulation?). The reason it can't be true that the DS is a linear function is that there's a wealth of simulation work and experimental data that demonstrate that it's not true (not to mention simple manual observation of the images!). The simulations cannot easily be dismissed as unrealistic because in many cases they give an accurate fit to the experimental data. As an example see here: http://journals.iucr.org/a/issues/2008/01/00/sc5007/sc5007.pdf . Looking at the various simulations here (Figs 3 5) it's obvious that the DS is very non-linear at the Bragg positions (and more importantly it's also non-linear between the Bragg positions). Note that the simulated calculated patterns here contain no Bragg peaks since as noted in the Figure legends, the average structure (or the average density) has been subtracted in the calculation, i.e. the simulations are showing only the DS component. I fail to see how any kind of background subtraction model could cope with the DS and give the right answer for the Bragg intensity in these kind of cases. Even from the observed patterns it's plain that the DS is non-linear, and therefore a linear background correction couldn't possibly correct the raw integrated intensity for the DS component. Well-established theory says that the total coherent scattered intensity is proportional to (~=) the time-average of the squared modulus of the structure factor of the crystal: I(coherent) ~= |Fc|^2 If we make the assumption that the deviations of the contributions to the structure factor from different unit cells are uncorrelated, we can show that the Bragg intensity is the squared modulus of the time and lattice-averaged SF sampled at the reciprocal lattice points: I(Bragg)~= |F|^2 The time/lattice-averaged SF is the FT of the average density, and therefore I(Bragg) indeed corresponds to the average density. The diffuse intensity is the difference between these: I(diffuse) = I(coherent) - I(Bragg) ~= |F|^2 - |F|^2 The assumption above implies that we're assuming that there's no 'acoustic' component of the DS, since this arises from correlations between different unit cells. However this doesn't mean that there *is* no acoustic component, it simply means that we are ignoring it: for one thing we have no alternative since the acoustic and Bragg scattering are practically inseparable; for another, correlations between different unit cells are purely an artifact of the crystallisation process, so have no biological significance, hence we're usually not interested in them anyway. The diffuse scatter does NOT tell you about the deviations from this average; it tells you how the deviations are correlated from unit cell to unit cell. This is completely wrong, the previous equation can be rewritten as:
[ccp4bb] SCAM email
Dear All, The email about the network sales company which appears as if sent from me on 22 Jan is a scam and was certainly not sent by me. I hope that there will be no more of these but I guess you will need to be aware that they might appear again in some form. Best wishes, John Campbell _ We want to hear all your funny, exciting and crazy Hotmail stories. Tell us now http://clk.atdmt.com/UKM/go/195013117/direct/01/
Re: [ccp4bb] SCAM email
Dear John Campbell (not you, the real John Campbell whom I know and who would never do such a thing as to send SPAM, the other John Campbell), Thank you very much for your kind offer. May I suggest that you fly to Haiti immediately and offer these commodities there at once, for free? They are in need of autocar, computer, TV, mobile telephone and others (others include water, food, blankets, tents, medical supplies, medicines and so forth). Once this has been done, I will happily connect to your web site for these unforeseen harvests you are promising. And I won't even honour you with my signature on this reply. John Campbell wrote: Dear All, The email about the network sales company which appears as if sent from me on 22 Jan is a scam and was certainly not sent by me. I hope that there will be no more of these but I guess you will need to be aware that they might appear again in some form. Best wishes, John Campbell [and Hi to the real John Campbell, signed Fred.]
Re: [ccp4bb] Acidic protein purification
Megha, If you want to prepare your sample at a pH below the pI, you will want to use a cation column. Enzymes at pH's below their pI have an overall positive charge. Cation exchange is a column which has a negative charge, thus it will bind to positively charged proteins. I would suggest looking at GE healthcare's chromatography section. They have pretty good books (or chapters rather) that describe all kinds of chromatography and will guide you in the right direction. http://www4.gelifesciences.com/aptrix/upp01077.nsf/Content/protein_purification~ion_exchange# The have detailed information about pH stability of each resin type as well. Kelly *** Kelly Daughtry PhD Candidate Department of Physiology and Biophysics Boston University School of Medicine 590 Commonwealth Ave R 390 Boston MA, 02215 (P) 617-358-5548 *** On Fri, Jan 8, 2010 at 10:01 PM, megha goyal mgbio...@gmail.com wrote: Dear all, our protein is stable in acidic pH at a pI of 6. we do not have any tag in it and it is expressed in inclusion body form. which ion exchange chromatography will suit it cation or anion exchange and do i use a strong or a weak ion excahnge resin. thanks and regards.
[ccp4bb] question on the effect of a fire alarm on crystallization
Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
If your crystals exploded, the alarm would provide warning to other residents of the building. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu On Fri, 2010-01-22 at 09:13 -0500, Jacqueline Vitali wrote: Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
I do not know the effect myself, but the idea of vibration fee has been tossed around a while. I believe that no vibration reduces the amount of nucleation that one might get, thus a vibration-free environment is detrimental when screening for hits. If you already have a method to grow crystals, then I can be convinced that a vibration-free environment might be helpful. Some of my best crystals have grown after subjecting the trays to physical or thermal shock. Since those observations, I never work about vibrations or temperature control. I think about it, but I do not worry about it. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacqueline Vitali Sent: Friday, January 22, 2010 8:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] question on the effect of a fire alarm on crystallization Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
On nucleation I'm not sure if there is any effect - I suspect you've got far more trouble from Brownian motion. For growth low frequency vibration (picking up the tray and putting it down) has much more influence than high-frequency (e.g. fans or motors). The liquid acts to damp most of the high-frequency effects. I would not worry about the effect of the fire alarm, just the process of putting it in! Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, January 22, 2010 10:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] question on the effect of a fire alarm on crystallization I do not know the effect myself, but the idea of vibration fee has been tossed around a while. I believe that no vibration reduces the amount of nucleation that one might get, thus a vibration-free environment is detrimental when screening for hits. If you already have a method to grow crystals, then I can be convinced that a vibration-free environment might be helpful. Some of my best crystals have grown after subjecting the trays to physical or thermal shock. Since those observations, I never work about vibrations or temperature control. I think about it, but I do not worry about it. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacqueline Vitali Sent: Friday, January 22, 2010 8:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] question on the effect of a fire alarm on crystallization Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
When working on tobacco Rubisco in Los Angeles, we got the best crystals of the activated form of the protein after small earthquakes (4-5 on the richter scale). However, as people in the lab. noticed, this earthquake parameter is somewhat difficult to control. Handling the linbro plates sometimes caused massive nucleation: I would look at a plate with only clear drops and by the time I would put the plate back in the incubator there was so much nucleation that the plate was of no use any more. When the nucleation of your protein is sensitive to vibrations, I would worry more about handling than about the fire alarm. Best, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacqueline Vitali Sent: Friday, January 22, 2010 3:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] question on the effect of a fire alarm on crystallization Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
I guess the crystals will be safe but freaked out. I think handling the crystal trays is a process which would much more influence the crystal growth. Do you ask because you have frequent fire drills or your colleagues have a affinity for arson? Sorry for the jokes, could not resist. Cheers, Djordje Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
Hi , Maybe the lower frequency of the earthquake vibrations or the handling helped your crystals nucleate. I would assume that even the resonance frequency of the linbro plates are low, and may cause disturbances in them. However, fire alarm systems usually are of a much higher frequency (shrill whines and stuff ), and so they may not affect the linbro plates. Ganesh On Fri, 22 Jan 2010 17:08:41 +0100, herman.schreu...@sanofi-aventis.com wrote: When working on tobacco Rubisco in Los Angeles, we got the best crystals of the activated form of the protein after small earthquakes (4-5 on the richter scale). However, as people in the lab. noticed, this earthquake parameter is somewhat difficult to control. Handling the linbro plates sometimes caused massive nucleation: I would look at a plate with only clear drops and by the time I would put the plate back in the incubator there was so much nucleation that the plate was of no use any more. When the nucleation of your protein is sensitive to vibrations, I would worry more about handling than about the fire alarm. Best, Herman - FROM: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] ON BEHALF OF Jacqueline Vitali SENT: Friday, January 22, 2010 3:14 PM TO: CCP4BB@JISCMAIL.AC.UK SUBJECT: [ccp4bb] question on the effect of a fire alarm on crystallization Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali -- What is true for E. coli is also true for an elephant - Jacques Monod.
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
We are going to be a pretty unpopular bunch if this thread concludes that we need to set off fire alarms in order to nucleate our crystals. :) Sean
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
Hi Jim, On Fri, Jan 22, 2010 at 09:11:00AM -0600, Jim Pflugrath wrote: I do not know the effect myself, but the idea of vibration fee has been tossed around a while. What? I'll have to pay for vibration now as well? Are you collecting that personally? Or is it just another scheme to save some failing banks? Maybe I'm lucky and that only applies to US citizens ... I'll ask my tax adviser ... Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
If Eddie were a bit more self-promoting, he might have pointed out this paper: http://journals.iucr.org/d/issues/1997/06/00/gr0718/gr0718.pdf Snell et al. (1997). Acta Cryst. D 53, 747-755. Where they found that crystals growing in space could tell when the astronauts were exercising (low frequency vibrations). But I admit they did not test the effects of fire alarms. -James Holton MAD Scientist Edward Snell wrote: On nucleation I’m not sure if there is any effect – I suspect you’ve got far more trouble from Brownian motion. For growth low frequency vibration (picking up the tray and putting it down) has much more influence than high-frequency (e.g. fans or motors). The liquid acts to damp most of the high-frequency effects. I would not worry about the effect of the fire alarm, just the process of putting it in! Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Jim Pflugrath *Sent:* Friday, January 22, 2010 10:11 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] question on the effect of a fire alarm on crystallization I do not know the effect myself, but the idea of vibration fee has been tossed around a while. I believe that no vibration reduces the amount of nucleation that one might get, thus a vibration-free environment is detrimental when screening for hits. If you already have a method to grow crystals, then I can be convinced that a vibration-free environment might be helpful. Some of my best crystals have grown after subjecting the trays to physical or thermal shock. Since those observations, I never work about vibrations or temperature control. I think about it, but I do not worry about it. *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Jacqueline Vitali *Sent:* Friday, January 22, 2010 8:14 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] question on the effect of a fire alarm on crystallization Dear all, Does anyone know the effect of a fire alarm placed in the same room where one grows crystals? (1) On trays placed inside styrofoam boxes (2) On trays placed inside an incubator which has not been tested as vibration free. Thank you. Jackie Vitali
[ccp4bb] Course on Neutrons in Structural Biology
Graduate Course - Neutron Scattering techniques in Structural Biology Oak Ridge TN, June 7 – June 11, 2010 Dear Colleague, The completion of the Neutron Spallation Source (SNS) and the cold source at the High Flux Isotope Reactor (HFIR) at the Oak Ridge National Laboratory (ORNL) opens a new era for the application of neutron scattering techniques in structural biology with i) increased capacity and capability of neutron instrumentation and ii) broader range of biological questions addressed by neutron scattering techniques that cannot be answered by other techniques alone. The Graduate Course in Neutron in Biology aims at educating and enabling a new generation of researcher to fully exploit the latest instrumentation and software development at the SNS and HFIR facilities. Attendees will participate in an intensive course focusing on neutron techniques used in structural biology. The course is designed for graduate students with knowledge of protein function and structure, new or with limited experience of neutron sciences. Course Objectives: 1. Educate graduate students in neutron scattering techniques, instrumentation and data collection, analysis and interpretation by offering courses taught by neutron scattering scientist. 2. Expose participants to cutting-edge research in structural biology by presenting seminars from national and international scientists to detail how neutron scattering integrate in their research program. 3. Build interactions between graduate participants and their university groups and ORNL neutron scattering experts to develop new research projects. Information is available on the course web page: http://neutrons.ornl.gov/conf/gcnb2010/ Travel and accommodation grants are available. The application package consisting of 1) Information form, 2) CV, 3) Applicant motivation letter (1/2 to 1 page), 4) Principal Investigator letter of support (1/2 to 1 page), 5) Justification for level of travel support requested should be sent electronically to meille...@ornl.govmailto:meille...@ornl.gov before March 26, 2010. The number of participants is limited to 15. Attendance to the course is free of charge. Participants have the possibility to register as non degree students at NCSU for 2 credit-hours (BCH 590E). Best Regards, Flora Flora Meilleur Assistant Professor Molecular Structural Biochemistry N C State University Neutron Scattering Sciences Division Oak Ridge National Laboratory Phone: 865-241-2897
Re: [ccp4bb] question on the effect of a fire alarm on crystallization
Clemens Vonrhein wrote: Hi Jim, On Fri, Jan 22, 2010 at 09:11:00AM -0600, Jim Pflugrath wrote: I do not know the effect myself, but the idea of vibration fee has been tossed around a while. What? I'll have to pay for vibration now as well? Are you collecting that personally? Or is it just another scheme to save some failing banks? Maybe I'm lucky and that only applies to US citizens ... I'll ask my tax adviser ... Clemens Only if you exercise! Subbu
Re: [ccp4bb] coot: fit ligand
Good old Val-Lys. I've noticed in the recent overhaul of the PDB they adopted the convention that any peptide less than three amino acids in length is now HETATOMs instead of ATOMs. This may be screwing up Coot's ability to link the dipeptide. I suggest you edit the PDB file to change these keywords and see if that helps. On the other hand, I can't imagine what Coot could do to improve on the fantastic Val-Lys model in 8TLN. Surly that model is without flaw! ;-) Dale Tronrud Tim Gruene wrote: Dear all, we would like to ask coot to fit the Val-Lys dipeptide of a thermolysin structure. We read in a PDB-file with the VK-fragment and run the Other Modelling Tools-Find Ligands tool. Unfortunately coot rips the pair apart and only places the valine into the density, the lysine is simply omitted. Is there a way to kindly as coot to not separate the two when trying to find a suitable home for them? Ta, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
