[ccp4bb] 3-year BBSRC-funded Postdoc Position - structural studies on an inter-MT bridge complex - correction!
A postdoctoral research post is available within my research group in the Department of Biochemistry at the University of Leicester. The position is funded by the Biotechnology and Biological Sciences Research Council and is available from 1st September 2014 for three years. The aim of the project is to assemble a high-resolution model of the clathrin/TACC3/ch-TOG (CTC) complex, capitalizing on our recent paper in which we identified the binding regions (Hood et al. J. Cell Biol. 2013). During mitosis, the CTC complex forms inter-microtubule bridges that stabilize the mitotic spindle. These are dynamic bridges, whose assembly is dependent on the activity of Aurora-A kinase. The overall architecture of the complex is unknown, and there are currently no structures of the individual sub-complexes. The postdoctoral researcher will investigate the structure and dynamics of the CTC complex using X-ray crystallography, protein-protein interaction studies and low-resolution methods such as SAXS and TIRF microscopy. They will work closely with a researcher who will focus on NMR-based techniques. The two researchers will coordinate their efforts to piece the structures together into a model of the core MT-binding module of the complex. Our work will shed light on the regulation, assembly, dynamics and MT-binding properties of this complex. My research group is located in the state-of-the-art Henry Wellcome Building with access to excellent facilities for biochemical studies, structural biology, cell culture, microscopy and computer analysis. This project is related to our ongoing work on microtubule binding proteins (Richards et al. PNAS 2014) and the protein kinases that regulate mitosis (Richards et al. Mol. Cell. 2009, Dodson et al. Sci. Signal 2013). The successful applicant will have a PhD in a relevant subject, and have a proven track record in protein expression and purification, structural biology and/or related biophysical methods. Informal enquiries are welcome and should be made by email to richard.bayl...@le.ac.ukmailto:richard.bayl...@le.ac.uk The deadline for applications is 28th July. For detailed information on the post, and to submit your application, consult the University job vacancies page - reference MBP01113. http://www2.le.ac.uk/offices/jobs/opportunities/jobsearch === Prof. Richard Bayliss Professor of Molecular Medicine Department of Biochemistry Henry Wellcome Building University of Leicester Lancaster Road, Leicester LE1 9HN Tel: 0116 2297100 Web: http://www2.le.ac.uk/departments/biochemistry/staff/richard-bayliss/research Twitter: @baylisslab [cid:8B11F347-BA95-4B16-89CA-F8FA127437A2@le.ac.uk]
Re: [ccp4bb] Solvent channels
Sorry for the delayed response - The situation of 'normal' drug-lead molecule, no restriction of solvent channel access, no other hindrance to mobility, and rapid on-rates and/or/with a low Kd driving/maintaining the concentration gradient might be considered almost optimal. But let us assume that it is indeed typical, and put it in perspective: One of the remarks made previously was that even seconds can suffice (for ions). Say 5 sec. The time factor between that and the 'typical' 30 min soaking then is 360, while the factor to 10 hrs (movie time) is only 20, that is, 10 hrs being an 18x more typical soak time than 5 seconds ;-). But seriously now, why would I beat the dead 5 sec horse dead again? Because of the cautionary tales of failed 'typical' 5-sec ligand soaks where beating proteolysis by 'flash-soaking' was apparently the motivation to ignore prior odds: http://www.ruppweb.org/cvs/br/rupp_2001_NSB_questions_BotA.pdf http://www.nature.com/nsmb/journal/v16/n7/full/nsmb0709-795.html 8 years this model stayed in the literature, frequently cited and presumably used... Its little small-molecule friend did not live as long: http://pubs.acs.org/doi/pdf/10.1021/ja025109g While advertising again the perils of too short soaking and subsequent pressure for optimistic interpretation, I think that Dale's assessment of faster diffusion vs slower binding in the lysozyme-methylene blue case is correct. Maybe growing a clear crystal first in a counter diffusion tube so that it fills the entire tube, and then sticking it into the blue dye and documenting the dye diffusion in solution vs in crystal might work. Could be a summer student project... Soak boldly and stay off the twilight list, BR
Re: [ccp4bb] Buried surface area calculation..
Dear Gajana, you can use . PISA ((Krissinel and Henrick, 2007) (EMBL server) and NACCESS (Hubbard and Thornton, 1993). (not for windows i think). Hope to help Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Gajanan Arbade [gajanan_pbi...@diat.ac.in] Envoyé : mardi 1 juillet 2014 20:49 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Buried surface area calculation.. Hello, Am working with some DNA binding proteins. If I want to calculate the buried surface area, which software tools should i use? Suggest me some windows based programs to solve my purpose. Thank you Regards, Gajanan __ Gajanan K Arbade Research Scholar Defence Institute of Advanced Technology (DIAT) Pune Maharashtra, India Pin Code-411025 Contact No. Lab.+ 91-20-24304377 Mob: 08698198016
Re: [ccp4bb] Solvent channels
Hi Reza, I can recommend the work of Marc-Olivier Coppens and Kourosh Malek and the references therein for an interesting journey through solvent channels. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics 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 Reza Khayat Sent: 27 June 2014 12:01 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
[ccp4bb] How to transfer non-frozen crystals with less disturbance?
Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
You can cut a small piece of sponge and put that into the reservoir; this prevents the reservoir buffer from splashing up into the drop. The sitting drops should be reasonably safe, but the 10 uL hanging drops are big; they'll be vulnerable to falling off if the tray is jarred. Good luck! Pat On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Dear Meisam, i don't know exactly what you need to do after the transfert, but if you want collect without frozen, maybe you can try to use capillar. Maybe can you mount your crystall in capillar before the travel ? It's simple suggestion, i never did that. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Meisam Nosrati [meisam.nosr...@gmail.com] Envoyé : mercredi 2 juillet 2014 22:17 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] How to transfer non-frozen crystals with less disturbance? Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
You can prevent them from falling off by also removing 5 microliter or so of mother liquor from the drop and repositioning it back over the reservoir On Wed, Jul 2, 2014 at 3:38 PM, Patrick Loll pat.l...@drexel.edu wrote: You can cut a small piece of sponge and put that into the reservoir; this prevents the reservoir buffer from splashing up into the drop. The sitting drops should be reasonably safe, but the 10 uL hanging drops are big; they'll be vulnerable to falling off if the tray is jarred. Good luck! Pat On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Dear Meisam I second what Nicolas had suggested: to mount your crystals in glass capillaries. I used to do this when travelling to the ESRF from the UK, keeping the crystals in a plastic case (alternatively, you may prefer using the Granada Box from Hampton Res. for this). One important consideration is to add a few uL of your well solution into the capillary tube (close to the mounted crystal) to avoid crystal damage/dessication. I will be happy to give you more details of this method -offline- if you like. With best regards, Victor On 2 July 2014 21:51, FOOS Nicolas nicolas.f...@synchrotron-soleil.fr wrote: Dear Meisam, i don't know exactly what you need to do after the transfert, but if you want collect without frozen, maybe you can try to use capillar. Maybe can you mount your crystall in capillar before the travel ? It's simple suggestion, i never did that. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Meisam Nosrati [meisam.nosr...@gmail.com] Envoyé : mercredi 2 juillet 2014 22:17 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] How to transfer non-frozen crystals with less disturbance? Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Another thing I always did when traveling with trays was pack them surrounded by ‘blue Ice’ packets (or the equivalent) equilibrated at the same temperature at which the trays have grown. It prevents the trays from moving around in the transport box (I used a small plastic Coleman cooler with a handle), and also provides a buffer against any temperature changes during transport. -pam On Jul 2, 2014, at 4:07 PM, Gloria Borgstahl gborgst...@gmail.commailto:gborgst...@gmail.com wrote: You can prevent them from falling off by also removing 5 microliter or so of mother liquor from the drop and repositioning it back over the reservoir On Wed, Jul 2, 2014 at 3:38 PM, Patrick Loll pat.l...@drexel.edumailto:pat.l...@drexel.edu wrote: You can cut a small piece of sponge and put that into the reservoir; this prevents the reservoir buffer from splashing up into the drop. The sitting drops should be reasonably safe, but the 10 uL hanging drops are big; they'll be vulnerable to falling off if the tray is jarred. Good luck! Pat On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edumailto:pat.l...@drexelmed.edu --- Pamela J. Focia, Ph.D. Research Assistant Professor Structural Biology Facility Manager Robert H. Lurie Comprehensive Cancer Center in the Departments of: Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, and Molecular Biosciences, Northwestern University 303 E. Chicago Ave., S-215, Chicago 60611 o (312)503-0848 c (312)286-3274 f (312)503-5349 fo...@northwestern.edumailto:fo...@northwestern.edu
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Hi Meisam - We do this frequently, using pieces of spongy foam to pad the inside of a sturdy styrofoam shipping box, which keeps the temperature from fluctuating too much. This is then carried in a reusable grocery bag by one person whose sole responsibility it is to ensure it isn’t subjected to any jarring movements during the trip. We haven’t used anything quite so large as your 10+ μl drops, but this setup works fine with 24-well plates of ~2-4 μl hanging drops for the 1.5-hour drive to our friendly neighborhood synchrotron. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On Jul 2, 2014, at 4:17 PM, Meisam Nosrati meisam.nosr...@gmail.commailto:meisam.nosr...@gmail.com wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Somebody really needs to hurry up and invent a molecular beam transporter you know like the one on Star Trek. Then we could just send our crystals wherever we want it that way. Sorry rough day in the lab. But if we could do that, we probably wouldn't need to solve structures anymore anyway. I have used the sponge in the reservoir method to carry trays on an airplane and it seemed to work. Laurie On Jul 2, 2014 5:18 PM, Pamela J Focia fo...@northwestern.edu wrote: Another thing I always did when traveling with trays was pack them surrounded by ‘blue Ice’ packets (or the equivalent) equilibrated at the same temperature at which the trays have grown. It prevents the trays from moving around in the transport box (I used a small plastic Coleman cooler with a handle), and also provides a buffer against any temperature changes during transport. -pam On Jul 2, 2014, at 4:07 PM, Gloria Borgstahl gborgst...@gmail.com wrote: You can prevent them from falling off by also removing 5 microliter or so of mother liquor from the drop and repositioning it back over the reservoir On Wed, Jul 2, 2014 at 3:38 PM, Patrick Loll pat.l...@drexel.edu wrote: You can cut a small piece of sponge and put that into the reservoir; this prevents the reservoir buffer from splashing up into the drop. The sitting drops should be reasonably safe, but the 10 uL hanging drops are big; they'll be vulnerable to falling off if the tray is jarred. Good luck! Pat On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu --- Pamela J. Focia, Ph.D. Research Assistant Professor Structural Biology Facility Manager Robert H. Lurie Comprehensive Cancer Center in the Departments of: Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, and Molecular Biosciences, Northwestern University 303 E. Chicago Ave., S-215, Chicago 60611 o (312)503-0848 c (312)286-3274 f (312)503-5349 fo...@northwestern.edu
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Hi Meisam, Here is what I have done before: 1. transfer the whole drop to a capillary and close both sides with plasticine. Notice that I am talking about the whole drop, not individual crystals. It is a lot simpler this way and involves less crystal manipulation. Make sure you remove any skin if present. 2. Transfer the well solution to an eppendorf tube. Don't make a new one, take the equilibrated solution from you tray. 3. Pack everything inside a styrofoam box with blue ice equilibrated in you incubator, as someone suggested. 4. When you get to the facility, reassemble your drop. Hopefully your crystals will survive the transfer (test before). Cheers, Mario Sanches On Wed, Jul 2, 2014 at 1:17 PM, Meisam Nosrati meisam.nosr...@gmail.com wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati -- Mario Sanches http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?
Of course, if you can plan ahead, you can grow your crystals in shipping friendly plates. See the In-Situ plate at Mitegen for details. http://www.mitegen.com/mic_catalog.php?c=insituplates you also might try to add agarose to the drop to fix the crystal in place, but that could be tough with a very large drop (20uL). Good luck! Bryan On Jul 2, 2014, at 5:33 PM, Sampson, Jared wrote: Hi Meisam - We do this frequently, using pieces of spongy foam to pad the inside of a sturdy styrofoam shipping box, which keeps the temperature from fluctuating too much. This is then carried in a reusable grocery bag by one person whose sole responsibility it is to ensure it isn’t subjected to any jarring movements during the trip. We haven’t used anything quite so large as your 10+ μl drops, but this setup works fine with 24-well plates of ~2-4 μl hanging drops for the 1.5-hour drive to our friendly neighborhood synchrotron. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On Jul 2, 2014, at 4:17 PM, Meisam Nosrati meisam.nosr...@gmail.commailto:meisam.nosr...@gmail.com wrote: Dear CCP4ers I need to transfer some crystals mainly in sitting drops to the site of data collection without freezing them. I do not know what is the best solution to secure the boxes in their place to minimize the disturbance. I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have one or two hanging drop boxes as well with 10 microliter size drops. If you have any experience about this matter, I greatly appreciate, if you share it with me. Thanks Meisam Nosrati This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. = -- 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.
[ccp4bb] Rename chain ID of water molecules
Dear CCP4BB members, I want to keep the chain ID of water molecules the same as their interacting protein molecule. For example, I have two protein molecules in the structure —— named chain A and chain B; then I want all the water molecules interacting with protein A (or B) have the same chain name A (or B). Any tool in CCP4 can do this? I remembered that when we deposited our coordinates to PDB, the server can do this automatically. Thank you. Kind regards, Wenhe
