[ccp4bb] Refinement of partly occupied water molecules
Dear all, I have a question concerning the refinement of partly occupied water molecules: in some of my macromolecular crystal structures with resolutions between 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are detectable which show after assignment of a water molecule to these blobs and subsequent refinement with Phenix.refine a good-looking 2Fo-Fc electron density. However, there also occurs a small negative Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The negative Fo-Fc electron density disappears if the occupancy of the water molecule is automatically refined by Phenix.refine (occupancy manually set to a value below 100% followed by refinement) or manually set to 50% and fixed for this value (Fix occupancy option in phenix.refine). Therefore, I think these positions are partly occupied by water molecules, but I am not sure how I should handle it/how it is generally handled. Which one of the two options described above is the better one? I would be thankful for any advice and/or literature about this topic. Thank you for your help! Stefan
[ccp4bb] Post-doctoral position at Diamond Light Source
Post-doctoral position at Diamond Light Source, UK For those of you out there with an interest in methodological/software development, particularly in experimental phasing, a keenness and ability to work in a multidisciplinary team at a large scale facility and a desire to work and live in Oxfordshire, UK, I would like to draw your attention to a post-doctoral position available at Diamond Light Source: http://www.diamond.ac.uk/Home/Jobs/Current/DIA0849_SB.html Note this is a collaborative position in conjunction with our MX colleagues at the Soleil synchrotron located near Paris. Please contact myself (david.h...@diamond.ac.ukmailto:david.h...@diamond.ac.uk) or Graeme Winter (graeme.win...@diamond.ac.ukmailto:graeme.win...@diamond.ac.uk) for further details but please apply following the instructions at the link above. Best wishes, Dave and Graeme. -- I04 PBS MX Village Coordinator | Diamond Light Sourcehttp://www.diamond.ac.uk/Home.html t: 01235 778926 | e: david.h...@diamond.ac.ukmailto:david.h...@diamond.ac.uk w: http://www.diamond.ac.uk/mx-home -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
[ccp4bb] SUBSCRIPTION OF CCP4
Re: [ccp4bb] Refinement of partly occupied water molecules
You are desribing the reason many people limit their refinement to lower resolution! I think it is probably universally true that there are multiple conformations for sidechain/water networks at the surface, which we just dont model properly. If you are going to tackle the fine details you need to judge how the nearby side chains are fitted as well as the water. And of course then you can probably also see stuff present in the crystallisation media. The rewards are a lower R factor, and a better understanding of mobility I guess. Eleanor On 12 July 2013 09:08, Stefan Krimmer krim...@staff.uni-marburg.de wrote: Dear all, I have a question concerning the refinement of partly occupied water molecules: in some of my macromolecular crystal structures with resolutions between 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are detectable which show after assignment of a water molecule to these blobs and subsequent refinement with Phenix.refine a good-looking 2Fo-Fc electron density. However, there also occurs a small negative Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The negative Fo-Fc electron density disappears if the occupancy of the water molecule is automatically refined by Phenix.refine (occupancy manually set to a value below 100% followed by refinement) or manually set to 50% and fixed for this value (Fix occupancy option in phenix.refine). Therefore, I think these positions are partly occupied by water molecules, but I am not sure how I should handle it/how it is generally handled. Which one of the two options described above is the better one? I would be thankful for any advice and/or literature about this topic. Thank you for your help! Stefan
Re: [ccp4bb] NCS information from different crystal parameters?
Hard to comment without more information, but check how many molecules you expect per asymmetric unit. If more than one look at the self rotation function - it might help. And you can with difficulty do a cross rotation between two data sets, which sometimes suggests how crystal 1 is related to crystal 2. But if you have the Se sites for both crystals, it is probably easier to try to match them and get transformation matrices to use for cross-averaging that way. Is that your case? Eleanor. On 12 July 2013 03:33, Yuan SHANG shangyuan5...@gmail.com wrote: Dear all, Currently, I was stuck in a coiled-coil crystal. I have two Se-derivative crystals in similar crystallization conditions. And the cell parameters of these two are list below : Crystal A: P21, a=69,b=43,c=135, beta=100.7 Crystal B: P21, a=29,b=230,c=42, beta=92.2 Assuming the crystal packings in A and B are overall similar, there seems to be another P21 axis in crystal A, which may help to determine the NCS parameters during phasing as there are two moleculars in the ASU. Could anyone help to tell me how to derive the NCS parameters? Best regards, Yuan SHANG
Re: [ccp4bb] Methods to reduce the model bias and increase the SAD phase
I check ha sites by 1) looking at the shelxe plots ( these are displayed in the CCP4 GUI task - prob also in phenix? ) If there is good contrast for one hand rather than the other the sites are likely to be correct - if not - hmmm; there might be a reason but they could be wrong.. Then I check the sites by giving the strongest ones to phaser_ep and seeing if it suggests more which agree with SHELX ones.. Bias is not usually a problem if the initial build is correct but if the Rfactors are not coming down that may not be the case. Can you see sidechains - espec MET near your Se sites? Eleanor On 12 July 2013 04:12, Yuan SHANG shangyuan5...@gmail.com wrote: Dear All, I got a Se-crystal diffracted to 3.3A. The protein is predicted to be a coiled coil. After I got the heavy atom positions from a shelxd based programs from AutoRickShaw, I run phaser and phenix.autobuild to get a initial phase shown in the attachment. I fit a standard helix into the density maps. After that, I did a two step iteration to improve my phases similar to the methods mentioned in Roversi's paper With phases: how two wrongs can sometimes make a right. Step 1: Run DM with current model, heavy atom positions, the initial density maps to get a new DM map. Step 2: In the new DM map, some new density appears. Fit new helixes in this new map. The iteration ends when I found no obvious helix densities. However, when I run refinement, I still got very bad R/Rfree values. It seemed I got a lot of model bias during the process above. Now, I have some questions that puzzled me a lot. 1. Is there any programs that could help to compare and check the heavy atom positions in shelxd solutions? Especially in cases when the solutions are not obvious. SitCOM seemed very good, is there a 32-bit distribution of this programs? Or any others have similar functions? 2. How to reduce the model bias and phase errors in my situations? any suggestions? 3. Should I collect a MAD data instead of SAD data? How much would that could help? Best regards Many thanks! Yuan
[ccp4bb] CCP4 Support 32 bit OSX - request for feedback
Dear CCP4 Users, CCP4 Core Team is in final stages for 6.4.0 Release. Every release and subsequent support takes a considerable effort, which scales linearly with the number of supported platforms. This makes us to periodically revise the scope of support we can offer to community. Since at least 4 years ago, Apple stopped manufacturing and support of 32-bit architectures, both on hardware and software side. This means that further support of 32-bit Macs by CCP4 will be completely unjustified in some future, and may be uneconomical already today. Yet, before taking any radical actions, we would like to estimate the number of actual 32-bit Mac OSX users of CCP4. I therefore solicit a feedback from everybody who: a) has a 32-bit Mac machine running CCP4 AND b) uses this machine for CCP4ing routinely AND c) has no plans to acquire a 64-bit equivalent in 6 months time. It would help us greatly to estimate the demand, if all such users send content-less e-mails to mail-to:eugene.krissi...@stfc.ac.uk Subject: 32-bit Mac: YES [system] where [system] is your Mac OSX release, such as 10.4, 10.5 or 10.6. PLEASE DO NOT CC THIS TO THE WHOLE CCP4BB BY MERE REPLYING TO THIS E-MAIL! I appreciate your time and willingness to help. Many thanks, Eugene Krissinel. Begin forwarded message: Date: 10 July 2013 16:35:44 GMT+01:00 To: hari jayaram hari...@gmail.com Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] CCP4 package manager not available for 32 bit OSX? Sadly not, we are dropping support for 32-bit Macs. You can still install 32-bit CCP4 6.3.0 from dmg. Sorry -- Eugene On 10 Jul 2013, at 16:04, hari jayaram wrote: Hi , I am trying to use the CCP4 package manager on my 32 bit OSX 10.6.8 running laptop and it complains /bin/sh: Volume/setup/.pm/Setup.app/Contents/MacOS/Setup: Bad CPU type in executable. Is there a 32 bit package manager available... Thanks Hari Screen shot 2013-07-10 at 10.57.53 AM.png -- Scanned by iCritical.
[ccp4bb] Fwd: [ccp4bb] CCP4 Support 32 bit OSX - request for feedback
PS Please consult http://support.apple.com/kb/ht3696 for how to determine your CPU type. Many thanks -- Eugene Begin forwarded message: From: Eugene Krissinel eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk Date: 12 July 2013 12:47:26 GMT+01:00 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] CCP4 Support 32 bit OSX - request for feedback Reply-To: Eugene Krissinel eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk Dear CCP4 Users, CCP4 Core Team is in final stages for 6.4.0 Release. Every release and subsequent support takes a considerable effort, which scales linearly with the number of supported platforms. This makes us to periodically revise the scope of support we can offer to community. Since at least 4 years ago, Apple stopped manufacturing and support of 32-bit architectures, both on hardware and software side. This means that further support of 32-bit Macs by CCP4 will be completely unjustified in some future, and may be uneconomical already today. Yet, before taking any radical actions, we would like to estimate the number of actual 32-bit Mac OSX users of CCP4. I therefore solicit a feedback from everybody who: a) has a 32-bit Mac machine running CCP4 AND b) uses this machine for CCP4ing routinely AND c) has no plans to acquire a 64-bit equivalent in 6 months time. It would help us greatly to estimate the demand, if all such users send content-less e-mails to mail-to:eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk Subject: 32-bit Mac: YES [system] where [system] is your Mac OSX release, such as 10.4, 10.5 or 10.6. PLEASE DO NOT CC THIS TO THE WHOLE CCP4BB BY MERE REPLYING TO THIS E-MAIL! I appreciate your time and willingness to help. Many thanks, Eugene Krissinel. Begin forwarded message: Date: 10 July 2013 16:35:44 GMT+01:00 To: hari jayaram hari...@gmail.commailto:hari...@gmail.com Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] CCP4 package manager not available for 32 bit OSX? Sadly not, we are dropping support for 32-bit Macs. You can still install 32-bit CCP4 6.3.0 from dmg. Sorry -- Eugene On 10 Jul 2013, at 16:04, hari jayaram wrote: Hi , I am trying to use the CCP4 package manager on my 32 bit OSX 10.6.8 running laptop and it complains /bin/sh: Volume/setup/.pm/Setup.app/Contents/MacOS/Setup: Bad CPU type in executable. Is there a 32 bit package manager available... Thanks Hari Screen shot 2013-07-10 at 10.57.53 AM.png -- Scanned by iCritical. -- Scanned by iCritical.
Re: [ccp4bb] frosted crystals during storage in pucks
Ditto - I was always impressed with the contraption in the Garman lab which, if I remember correctly, is made of a thick block of wood and some plumbing pipes. It is designed to hold empty open Dewars inverted so they could dry. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Senior Scientist, 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! -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ginell, Stephan L. Sent: Friday, July 12, 2013 12:45 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] frosted crystals during storage in pucks My experience with xtals frosting in LN2 either in a dewar, while freezing, or in pucks, has been because the LN2 was contaminated with ice crystals The fog you see above your dewar when freezing xtals is frozen water vapor...it will fall and collect in the LN2 and also deposit on the xtals. Dewars filled with recycled LN2 get contaminated with ice. Dewars dried upside down allow the cold gas to flow out and warm moist air to flow in and the water to condense inside the dewar (basic physics). To dry shipping dewars keep up right while warming. Steve Sent from my iPad On Jul 11, 2013, at 5:25 PM, Nathaniel Clark nathanielcl...@gmail.commailto:nathanielcl...@gmail.com wrote: At our last synchrotron trip, the beamline staff suggested that the problem was due to moisture accumulation in the dry shipper. They recommended storing them inverted (for a few weeks, if I recall), and/or putting a supply of dry air in the dewer. Haven't tried it yet! Nat On Thu, Jul 11, 2013 at 5:08 PM, Rain Field rainfiel...@163.commailto:rainfiel...@163.com wrote: Hi All, We found if the crystals are storaged in pucks for 3-4 days in shipping dewar (with liquid nitrogen), they are almost frosted. Although I can wash them with liquid nitrogen, but it's not convenient to do that for each crystals. I doubt it's because the humid air in North West America. Does anyone has an idea how to avoid this? Thank you!
Re: [ccp4bb] Methods to reduce the model bias and increase the SAD phase
Dear Yuan. 1. Is there any programs that could help to compare and check the heavy atom positions in shelxd solutions? Especially in cases when the solutions are not obvious. SitCOM seemed very good, is there a 32-bit distribution of this programs? Or any others have similar functions? If you use shelxd to search the heavy atom, you can get the value of CC All/Weak in shelxd.log. CC All should be larger than 35 if you want to solve the structure by SAD. 2. How to reduce the model bias and phase errors in my situations? any suggestions? Try TLS refinement in refmac or phenix.refine after model building. 3. Should I collect a MAD data instead of SAD data? How much would that could help? I don't think the MAD data will be better than SAD data. Maybe you can try to collect data with small oscillation angle (e.g.0.5deg) 。 If you need more help, please send me the log file outputted by HKL2000. Best regards Many thanks! Yuan Wei
Re: [ccp4bb] 5D data storage
It seems like every couple of years I see an article about a new high density optical format. Then they don't make it to the marketplace. I think it has something to do with resistance from the music movie business over piracy concerns. But anyway, I have learned not to get too excited until a product is actually available for purchase. On 07/11/13 18:47, Scott Classen wrote: I stumbled across this interesting abstract today, and though I'd rekindle the perennial data storage debate on ccp4bb. Apparently these researchers have figured out a way to store 360TB of data on a disc (not sure of the actual dimensions). The memory crystal should have a thermal stability of 1000ºC and the data should remain readable forever. Here is the news release: http://www.southampton.ac.uk/mediacentre/news/2013/jul/13_131.shtml and a PDF abstract from the recent Conference on Lasers and Electro-Optics (CLEO’13) in San Jose: http://www.orc.soton.ac.uk/fileadmin/downloads/5D_Data_Storage_by_Ultrafast_Laser_Nanostructuring_in_Glass.pdf -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Refinement of partly occupied water molecules
On Fri, Jul 12, 2013 at 1:08 AM, Stefan Krimmer krim...@staff.uni-marburg.de wrote: in some of my macromolecular crystal structures with resolutions between 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are detectable which show after assignment of a water molecule to these blobs and subsequent refinement with Phenix.refine a good-looking 2Fo-Fc electron density. However, there also occurs a small negative Fo-Fc electron density detectable inside the 2Fo-Fc density blob. The negative Fo-Fc electron density disappears if the occupancy of the water molecule is automatically refined by Phenix.refine (occupancy manually set to a value below 100% followed by refinement) or manually set to 50% and fixed for this value (Fix occupancy option in phenix.refine). Therefore, I think these positions are partly occupied by water molecules, but I am not sure how I should handle it/how it is generally handled. Which one of the two options described above is the better one? I would be thankful for any advice and/or literature about this topic. When I had to deal with this in the past, I followed this advice (from Thomas Schneider): http://www.embl-hamburg.de/~tschneider/shelxl/shelxl_faq/shelxlfaq.html#Q16 This is especially true at the resolutions you're working with; even with subatomic resolution data I believe that the observation in the FAQ (that refining the occupancies doesn't improve R-factors and may even make them worse) will be true in most cases - and regardless of program used, btw. (I can't remember if I ever tried comparing the outcomes myself, though.) -Nat
[ccp4bb] Harmful effect of X-ray
Dear All, Can anyone please tell me regarding the harmful effects of X-ray , we are using for protein crystallography, on human being and what are the precautions we should take. Thanks
[ccp4bb] Position opening at RCSB PDB/Rutgers University- BIOCHEMICAL INFORMATION ANNOTATION SPECIALIST
The RCSB Protein Data Bank (http://www.pdb.org) is a publicly accessible information portal for researchers and students interested in structural biology. At its center is the PDB archive – the sole international repository for the 3-dimensional structure data of biological macromolecules. These structures hold significant promise for the pharmaceutical and biotechnology industries in the search for new drugs and in efforts to understand the mysteries of human disease. The primary mission of the RCSB PDB is to provide accurate, well-annotated data in the most timely and efficient way possible to facilitate new discoveries and scientific advances. The RCSB PDB processes, stores, and disseminates these important data, and develops the software tools needed to assist users in depositing and accessing structural information. The RCSB Protein Data Bank at Rutgers University in Piscataway, NJ has one opening for a Biochemical Information Annotation Specialist to curate and standardize macromolecular structures for distribution in the PDB archive. The annotation specialists validate, annotate, and release structural entries in PDB archive. The annotation specialists also communicate daily with members of the deposition community. The position is an academic position with state benefit. The salary is compatible with faculty level. A background in macromolecular crystallography or small molecule crystallography is a strong advantage. Biological chemistry (PhD, MS) is required. Experience with linux computer systems, biological databases is preferred. The successful candidate should be self-motivated, pay close attention to details, possess strong written and oral communication skills, and meet deadlines. This position offers the opportunity to participate in an exciting project with significant impact on the scientific community. Please send resume (pdf preferred) to Dr. Jasmine Young at pdbj...@rcsb.rutgers.edu. -- Jasmine Young, Ph.D. RCSB Protein Data Bank Assistant Research Professor Lead biocurator Center for Integrative Proteomics Research Rutgers The State University of New Jersey 174 Frelinghuysen Rd Piscataway, NJ 08854-8087 Email: jas...@rcsb.rutgers.edu Phone: (848)-445-0103 ext 4920 Fax:(732)-445-4320
Re: [ccp4bb] Harmful effect of X-ray
Is this a joke or are you serious??? Hopefully you haven't actually started shooting people with highly focused X-rays... Jon On 07/12/2013 10:14 AM, diptimayee mishra wrote: Dear All, Can anyone please tell me regarding the harmful effects of X-ray , we are using for protein crystallography, on human being and what are the precautions we should take. Thanks -- Jonathan P. Schuermann, Ph. D. Beamline Scientist, NE-CAT Argonne National Laboratory, 436E 9700 S. Cass Ave. Argonne, IL 60439 Email: schue...@anl.gov Tel: (630) 252-0682
Re: [ccp4bb] Harmful effect of X-ray
Most modern textbooks have sections on the proper protections and measures to take, although the information may be dated. See chapter 6 in Volume III of the International Tables for X-ray Crystallography. With the modern equipment and regulatory measures in most countries, you really have to work hard to be exposed at dangerous levels (which can lead to skin lesions and burns). However, you can get yourself exposed if you intentionally circumvent the safety measures and interlocks. In my experience, X-ray exposure in crystallography labs is very low and not dangerous. Our radiation safety people find our labs to be very clean with respect to scattered radiation around the sample compared to medical X-ray labs. Talk to your institute's safety people for advice. For in-house equipment, you are most at risk of exposure during aligning the equipment. If you talk to the old crystallographers (or their students who are now +50 year old), you might hear stories of aligning collimators and cameras by the tingle on your eye as you look into the beam. By the time protein crystallography came around (50s-60s), phosphors and film were used for alignment so the danger comes mostly from scattered radiation and poor shielding. In all the years I have worked with X-rays without protection (I only wore a lab coat to prevent film developer from staining my clothes), neither I nor my colleagues have ever had X-ray exposures above background as determined by film badges and ring badges. In fact, we once exposed a film badge intentionally to see if anyone cared. We caught hell for doing that. For synchrotron sources, chances of being exposed as a general user are now nil unless you really work hard to subvert the safety measures (which will get you kicked out). Hope this helps, 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 Jul 12, 2013, at 11:14 AM, diptimayee mishra wrote: Dear All, Can anyone please tell me regarding the harmful effects of X-ray , we are using for protein crystallography, on human being and what are the precautions we should take. Thanks
[ccp4bb] How to disable auto-renaming of files in ccp4i refmac menu?
I dearly love ccp4i, but there is one aspect of the interface that drives me to distraction. When I type (or paste) an input file name (let's say input.mtz) into the refmac menu, ccp4i changes various output file names automatically according to some scheme that doesn't at all match my work flow. How can I disable this? I imagine it is simply a matter of commenting out a couple of lines in a script but I haven't been able to figure out which lines those are. Better yet - if it must rename, is there a way I can give it a new rule for naming output files? My workflow wants all output files from the same refinement run to have the same base name. E.g. RUN_N.pdb RUN_N.mtz and RUN_N.tls regardless of what the input file names might be (typically dataset.mtz and RUNsomething-coot-N.pdb). Is there still time to request this as a feature for the next release? Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Membrane Protein expression and purification
Hi Krish, You may need to do some construct engineering to get your protein to express well to the plasma membrane. These steps involve creating many expression construct variants including N and C-terminal truncations, fusion partners at the N-or C-termini, or even signal peptides at the N-terminus to properly target your protein to the plasma membrane. Pay attention to post-translational modifications as well. You may also want to look into protein trafficking signals, which can be found in a nice paper by Duvernay et al 2006, which is focussed on GPCRs but applies to other membrane proteins as well. Additionally, you may want to try other expression systems like BV, mammalian, or even e.coli with all of these variants. Of course, if you just want to try more detergents you can purchase a detergent screen from (sorry for the self-plug) Emerald Bio to test a larger amount of detergents than you probably have thus far. Expressing membrane proteins can be a daunting task, but with enough hard work you can be successful. Best of luck-Tim CraigEmerald Bio Date: Fri, 12 Jul 2013 00:29:28 -0400 From: krishna@gmail.com Subject: [ccp4bb] Membrane Protein expression and purification To: CCP4BB@JISCMAIL.AC.UK Dear All, First of all sorry for bringing the non-ccp4 post. As our CCP4 community is filled with experts in various fields of structural biology: I'd like to get some help/suggestions from the membrane proteins expert community. I'm trying to express my membrane protein in Pichia pastoris (SMD1163H). I see my protein getting expressed but the yield is very low. I tried optimizing the media, temperature and additive like DMSO but none of them improved the yield. Apart from that, the main problem is my protein is not completely extractable from the Pichia membranes. Tried DM, DDM (2 to 4%), beta-OG, LDAO,CHAPS and C12E8 (2 hrs to over night in 4 C and 1hr at RT). Only I could get little bit of my protein in DDM. If I spin my protein even at 30K to 50K g after extraction most of it is ending up in the pellet. I know every protein behaves different but I'd like to know - Did anyone observe the same behavior with Pichia membrane protein expression? I'm even trying with the interaction partner (chaperone like) to express it in happy form! I'd like to get some suggestions regarding the expression in Pichia or any other system that might help. Any tips on the solubility part of membranes would be really helpful. Thank you. Krishna
Re: [ccp4bb] Membrane Protein expression and purification
Hi Krish, Seconding Tim's comments, you may need to switch construct or expression host. I've seen many times when a membrane protein seems to be in the yeast membrane, but seems unextractable or most likely, improperly folded. Since you're already working with yeast, I would recommend trying S.cerevisiae. It's worked for many integral membrane proteins, and smells a whole lot better than Pichia. http://csmp.ucsf.edu/pdf/Pub14_PMID20946832.pdf My 2 cents. Good luck, John On Fri, Jul 12, 2013 at 1:34 PM, Timothy Craig tlmcra...@hotmail.comwrote: Hi Krish, You may need to do some construct engineering to get your protein to express well to the plasma membrane. These steps involve creating many expression construct variants including N and C-terminal truncations, fusion partners at the N-or C-termini, or even signal peptides at the N-terminus to properly target your protein to the plasma membrane. Pay attention to post-translational modifications as well. You may also want to look into protein trafficking signals, which can be found in a nice paper by Duvernay et al 2006, which is focussed on GPCRs but applies to other membrane proteins as well. Additionally, you may want to try other expression systems like BV, mammalian, or even e.coli with all of these variants. Of course, if you just want to try more detergents you can purchase a detergent screen from (sorry for the self-plug) Emerald Bio to test a larger amount of detergents than you probably have thus far. Expressing membrane proteins can be a daunting task, but with enough hard work you can be successful. Best of luck -Tim Craig Emerald Bio -- Date: Fri, 12 Jul 2013 00:29:28 -0400 From: krishna@gmail.com Subject: [ccp4bb] Membrane Protein expression and purification To: CCP4BB@JISCMAIL.AC.UK Dear All, First of all sorry for bringing the non-ccp4 post. As our CCP4 community is filled with experts in various fields of structural biology: I'd like to get some help/suggestions from the membrane proteins expert community. I'm trying to express my membrane protein in Pichia pastoris (SMD1163H). I see my protein getting expressed but the yield is very low. I tried optimizing the media, temperature and additive like DMSO but none of them improved the yield. Apart from that, the main problem is my protein is not completely extractable from the Pichia membranes. Tried DM, DDM (2 to 4%), beta-OG, LDAO,CHAPS and C12E8 (2 hrs to over night in 4 C and 1hr at RT). Only I could get little bit of my protein in DDM. If I spin my protein even at 30K to 50K g after extraction most of it is ending up in the pellet. I know every protein behaves different but I'd like to know - Did anyone observe the same behavior with Pichia membrane protein expression? I'm even trying with the interaction partner (chaperone like) to express it in happy form! I'd like to get some suggestions regarding the expression in Pichia or any other system that might help. Any tips on the solubility part of membranes would be really helpful. Thank you. Krishna
[ccp4bb] Postdoc position available at UT Southwestern Medical Center at Dallas
We are looking for one or two highly motivated and multi-talented postdoctoral researcher(s) to join a collaborative project studying the structure and mechanism of circadian clocks (See Huang et al. http://www.sciencemag.org/content/337/6091/189.abstract 2012 Science). The overall goal of the project is to understand how various multi-domain clock proteins interact with each other; and how these interactions modulate the clock-mediated transcription activation. Person(s) working on this project will have a unique opportunity to gain some unusual insights into the molecular clockworks governing our sleep-wake cycles. Candidates should have a Ph.D. degree in a relevant field and must be proficient in molecular cloning, protein expression, purification, crystallization, and X-ray crystal structure determination. Additional experiences in eukaryotic protein expression systems, cell culture, various biochemical and biophysical techniques to study protein-protein interactions are also highly desirable. To apply, please email CV and contact info of two or more referees to zh...@chop.swmed.edu. All the Best, Hong Zhang, Ph.D. Associate Professor Department of Biophysics and Department of Biochemistry UT Southwestern Medical Center at Dallas 4323 Harry Hines Blvd. Dallas, Texas 75390-8816 Phone: 214-645-6372 Fax: 214-645-5948 http://profiles.utsouthwestern.edu/profile/23445/hong-zhang.html
Re: [ccp4bb] Harmful effect of X-ray
My first reaction to this question was well, it depends. The effect of X-rays depends on (among other things) the energy (wavelength) and the intensity (and of course the dose). But I decided not to write about this until... In response to Michael's note below, I want to point out that instruments constantly improve: I just re-wrote our safety plan because we have to renew our radiation license for our new in-house instrument. On this plan, they ask you to write a worst-case scenario. For an in-house sealed tube instrument, producing CuKa radiation, we calculated (with the help of the friendly manufacturer, thank you) that if you had a direct exposure to the beam, for example because your hand would be in the wrong place at the wrong time, you would burn a 7mm deep hole in your hand. Interestingly and surprisingly, when compared to our old rotating anode generator, the estimated worst case scenario for the old setup was an order of magnitude less severe (less than 1 mm deep burn). The constant improvement of instruments also makes them just a little more dangerous. The old days of 'tingle' are long gone and you *really* need to watch yourself, even on in-house instruments. Indeed, if you do not tinker with the safety system and have shielding in place at all time, as you say, the exposure is never above background. So: obey all the rules and worry very little. At the synchrotron, if I recall my safety training correctly, the worst case scenario (which is very nearly impossible to accomplish, even if you tried, which of course you should not), is instant death. So yes, that gets you kicked out, but not the way you imagined. There are many less severe variations, but probably all are much more severe than the in-house case. There is a reason for all the safety systems. The original question (what happens and how bad is it), is not that easy to answer. Apart from burns, there are of course the known effects of DNA damage etc. Since we share a building with people who call their profession radiation health physics, I was reminded with an interesting discussion that there are people who study how you can use radiation for your benefit - use enough to kill bad things, but not so much to badly harm the good things - and these people make a living that way, quite successfully. In the end, it is hard to know what anything does to your body and it is best to stay on the safe side. Remember ALARA and observe it. It is possibly the most sensible safety rule I have seen in my life. Hopefully now nobody will be tempted to see if it tingles when you stick your finger in the beam. Mark -Original Message- From: R. M. Garavito rmgarav...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Jul 12, 2013 10:14 am Subject: Re: [ccp4bb] Harmful effect of X-ray Most modern textbooks have sections on the proper protections and measures to take, although the information may be dated. See chapter 6 in Volume III of the International Tables for X-ray Crystallography. With the modern equipment and regulatory measures in most countries, you really have to work hard to be exposed at dangerous levels (which can lead to skin lesions and burns). However, you can get yourself exposed if you intentionally circumvent the safety measures and interlocks. In my experience, X-ray exposure in crystallography labs is very low and not dangerous. Our radiation safety people find our labs to be very clean with respect to scattered radiation around the sample compared to medical X-ray labs. Talk to your institute's safety people for advice. For in-house equipment, you are most at risk of exposure during aligning the equipment. If you talk to the old crystallographers (or their students who are now +50 year old), you might hear stories of aligning collimators and cameras by the tingle on your eye as you look into the beam. By the time protein crystallography came around (50s-60s), phosphors and film were used for alignment so the danger comes mostly from scattered radiation and poor shielding. In all the years I have worked with X-rays without protection (I only wore a lab coat to prevent film developer from staining my clothes), neither I nor my colleagues have ever had X-ray exposures above background as determined by film badges and ring badges. In fact, we once exposed a film badge intentionally to see if anyone cared. We caught hell for doing that. For synchrotron sources, chances of being exposed as a general user are now nil unless you really work hard to subvert the safety measures (which will get you kicked out). Hope this helps, 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) 353-9334
Re: [ccp4bb] Membrane Protein expression and purification
Hi Tim, Indeed, I engineered several constructs, both N- and C-terminal truncations, to express this protein in E.coli, yeast and also in BV. Nothing much success. I'm going to try mammalian cell lines. I have some constructs with signal peptides and right now I'm trying them. Hope I get something out of it! Thank you very much for all your suggestions. Krish On Fri, Jul 12, 2013 at 2:34 PM, Timothy Craig tlmcra...@hotmail.comwrote: Hi Krish, You may need to do some construct engineering to get your protein to express well to the plasma membrane. These steps involve creating many expression construct variants including N and C-terminal truncations, fusion partners at the N-or C-termini, or even signal peptides at the N-terminus to properly target your protein to the plasma membrane. Pay attention to post-translational modifications as well. You may also want to look into protein trafficking signals, which can be found in a nice paper by Duvernay et al 2006, which is focussed on GPCRs but applies to other membrane proteins as well. Additionally, you may want to try other expression systems like BV, mammalian, or even e.coli with all of these variants. Of course, if you just want to try more detergents you can purchase a detergent screen from (sorry for the self-plug) Emerald Bio to test a larger amount of detergents than you probably have thus far. Expressing membrane proteins can be a daunting task, but with enough hard work you can be successful. Best of luck -Tim Craig Emerald Bio -- Date: Fri, 12 Jul 2013 00:29:28 -0400 From: krishna@gmail.com Subject: [ccp4bb] Membrane Protein expression and purification To: CCP4BB@JISCMAIL.AC.UK Dear All, First of all sorry for bringing the non-ccp4 post. As our CCP4 community is filled with experts in various fields of structural biology: I'd like to get some help/suggestions from the membrane proteins expert community. I'm trying to express my membrane protein in Pichia pastoris (SMD1163H). I see my protein getting expressed but the yield is very low. I tried optimizing the media, temperature and additive like DMSO but none of them improved the yield. Apart from that, the main problem is my protein is not completely extractable from the Pichia membranes. Tried DM, DDM (2 to 4%), beta-OG, LDAO,CHAPS and C12E8 (2 hrs to over night in 4 C and 1hr at RT). Only I could get little bit of my protein in DDM. If I spin my protein even at 30K to 50K g after extraction most of it is ending up in the pellet. I know every protein behaves different but I'd like to know - Did anyone observe the same behavior with Pichia membrane protein expression? I'm even trying with the interaction partner (chaperone like) to express it in happy form! I'd like to get some suggestions regarding the expression in Pichia or any other system that might help. Any tips on the solubility part of membranes would be really helpful. Thank you. Krishna
Re: [ccp4bb] Membrane Protein expression and purification
Hi John, Thank you very much for all the valuable suggestions and the helpful link. I think it's worth trying S.cerevisiae both for expression and also protecting the environment from that strong Pichia odour. Krish On Fri, Jul 12, 2013 at 3:17 PM, John Lee kw...@msg.ucsf.edu wrote: Hi Krish, Seconding Tim's comments, you may need to switch construct or expression host. I've seen many times when a membrane protein seems to be in the yeast membrane, but seems unextractable or most likely, improperly folded. Since you're already working with yeast, I would recommend trying S.cerevisiae. It's worked for many integral membrane proteins, and smells a whole lot better than Pichia. http://csmp.ucsf.edu/pdf/Pub14_PMID20946832.pdf My 2 cents. Good luck, John On Fri, Jul 12, 2013 at 1:34 PM, Timothy Craig tlmcra...@hotmail.comwrote: Hi Krish, You may need to do some construct engineering to get your protein to express well to the plasma membrane. These steps involve creating many expression construct variants including N and C-terminal truncations, fusion partners at the N-or C-termini, or even signal peptides at the N-terminus to properly target your protein to the plasma membrane. Pay attention to post-translational modifications as well. You may also want to look into protein trafficking signals, which can be found in a nice paper by Duvernay et al 2006, which is focussed on GPCRs but applies to other membrane proteins as well. Additionally, you may want to try other expression systems like BV, mammalian, or even e.coli with all of these variants. Of course, if you just want to try more detergents you can purchase a detergent screen from (sorry for the self-plug) Emerald Bio to test a larger amount of detergents than you probably have thus far. Expressing membrane proteins can be a daunting task, but with enough hard work you can be successful. Best of luck -Tim Craig Emerald Bio -- Date: Fri, 12 Jul 2013 00:29:28 -0400 From: krishna@gmail.com Subject: [ccp4bb] Membrane Protein expression and purification To: CCP4BB@JISCMAIL.AC.UK Dear All, First of all sorry for bringing the non-ccp4 post. As our CCP4 community is filled with experts in various fields of structural biology: I'd like to get some help/suggestions from the membrane proteins expert community. I'm trying to express my membrane protein in Pichia pastoris (SMD1163H). I see my protein getting expressed but the yield is very low. I tried optimizing the media, temperature and additive like DMSO but none of them improved the yield. Apart from that, the main problem is my protein is not completely extractable from the Pichia membranes. Tried DM, DDM (2 to 4%), beta-OG, LDAO,CHAPS and C12E8 (2 hrs to over night in 4 C and 1hr at RT). Only I could get little bit of my protein in DDM. If I spin my protein even at 30K to 50K g after extraction most of it is ending up in the pellet. I know every protein behaves different but I'd like to know - Did anyone observe the same behavior with Pichia membrane protein expression? I'm even trying with the interaction partner (chaperone like) to express it in happy form! I'd like to get some suggestions regarding the expression in Pichia or any other system that might help. Any tips on the solubility part of membranes would be really helpful. Thank you. Krishna
Re: [ccp4bb] Harmful effect of X-ray
Thanks for the sobering analysis. I would not have expected that. Fortunately you have to be very persistent to override all the interlocks on an in-house system. I do know on our Agilent instrument that the backscatter from a crystal at full power with the doors open is only 3 times background--learned that from the installer during commissioning. With the doors closed you can't measure any stray radiation. Roger Rowlett On Jul 12, 2013 3:57 PM, Mark van der Woerd mjvdwo...@netscape.net wrote: My first reaction to this question was well, it depends. The effect of X-rays depends on (among other things) the energy (wavelength) and the intensity (and of course the dose). But I decided not to write about this until... In response to Michael's note below, I want to point out that instruments constantly improve: I just re-wrote our safety plan because we have to renew our radiation license for our new in-house instrument. On this plan, they ask you to write a worst-case scenario. For an in-house sealed tube instrument, producing CuKa radiation, we calculated (with the help of the friendly manufacturer, thank you) that if you had a direct exposure to the beam, for example because your hand would be in the wrong place at the wrong time, you would burn a 7mm deep hole in your hand. Interestingly and surprisingly, when compared to our old rotating anode generator, the estimated worst case scenario for the old setup was an order of magnitude less severe (less than 1 mm deep burn). The constant improvement of instruments also makes them just a little more dangerous. The old days of 'tingle' are long gone and you *really* need to watch yourself, even on in-house instruments. Indeed, if you do not tinker with the safety system and have shielding in place at all time, as you say, the exposure is never above background. So: obey all the rules and worry very little. At the synchrotron, if I recall my safety training correctly, the worst case scenario (which is very nearly impossible to accomplish, even if you tried, which of course you should not), is instant death. So yes, that gets you kicked out, but not the way you imagined. There are many less severe variations, but probably all are much more severe than the in-house case. There is a reason for all the safety systems. The original question (what happens and how bad is it), is not that easy to answer. Apart from burns, there are of course the known effects of DNA damage etc. Since we share a building with people who call their profession radiation health physics, I was reminded with an interesting discussion that there are people who study how you can use radiation for your benefit - use enough to kill bad things, but not so much to badly harm the good things - and these people make a living that way, quite successfully. In the end, it is hard to know what anything does to your body and it is best to stay on the safe side. Remember ALARA and observe it. It is possibly the most sensible safety rule I have seen in my life. Hopefully now nobody will be tempted to see if it tingles when you stick your finger in the beam. Mark -Original Message- From: R. M. Garavito rmgarav...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Jul 12, 2013 10:14 am Subject: Re: [ccp4bb] Harmful effect of X-ray Most modern textbooks have sections on the proper protections and measures to take, although the information may be dated. See chapter 6 in Volume III of the International Tables for X-ray Crystallography. With the modern equipment and regulatory measures in most countries, you really have to work hard to be exposed at dangerous levels (which can lead to skin lesions and burns). However, you can get yourself exposed if you intentionally circumvent the safety measures and interlocks. In my experience, X-ray exposure in crystallography labs is very low and not dangerous. Our radiation safety people find our labs to be very clean with respect to scattered radiation around the sample compared to medical X-ray labs. Talk to your institute's safety people for advice. For in-house equipment, you are most at risk of exposure during aligning the equipment. If you talk to the old crystallographers (or their students who are now +50 year old), you might hear stories of aligning collimators and cameras by the tingle on your eye as you look into the beam. By the time protein crystallography came around (50s-60s), phosphors and film were used for alignment so the danger comes mostly from scattered radiation and poor shielding. In all the years I have worked with X-rays without protection (I only wore a lab coat to prevent film developer from staining my clothes), neither I nor my colleagues have ever had X-ray exposures above background as determined by film badges and ring badges. In fact, we once exposed a film badge intentionally to see if anyone cared. We caught hell for doing that.
Re: [ccp4bb] Harmful effect of X-ray
On Jul 12, 2013, at 12:57 PM, Mark van der Woerd mjvdwo...@netscape.net wrote: I just re-wrote our safety plan because we have to renew our radiation license for our new in-house instrument. On this plan, they ask you to write a worst-case scenario. For an in-house sealed tube instrument, producing CuKa radiation, we calculated (with the help of the friendly manufacturer, thank you) that if you had a direct exposure to the beam, for example because your hand would be in the wrong place at the wrong time, you would burn a 7mm deep hole in your hand. Interestingly and surprisingly, when compared to our old rotating anode generator, the estimated worst case scenario for the old setup was an order of magnitude less severe (less than 1 mm deep burn). The constant improvement of instruments also makes them just a little more dangerous. The old days of 'tingle' are long gone and you *really* need to watch yourself, even on in-house instruments. Indeed, if you do not tinker with the safety system and have shielding in place at all time, as you say, the exposure is never above background. So: obey all the rules and worry very little. Although I've never knowingly stuck any bodily appendage into an X-ray beam, I recently had the delightful experience of having a skin cancer hacked off my hand. Even though it is hard to know if the cause was an X-ray beam, I've met other crystallographers who have had similarly located lesions, leading me to believe that this might be more common than we tend to acknowledge to ourselves or each other. So I would modify the last quoted sentence to Obey all the rules and worry anyway. Ionizing radiation, especially when collimated, it a significant hazard, ipso facto.
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