Re: [ccp4bb] Rejected posting to CCP4BB@JISCMAIL.AC.UK
Dear Bill, as I pointed out in my previous post I have the impression that using wish (instead of bltwish) libblt provided in Debian stable now works fine with ccp4, including loggraph etc. So it might be too late for a complaint, although my statement is based only on a first impression rather than an extensive test. Tim On Wed, Apr 14, 2010 at 07:58:50PM -0700, William G. Scott wrote: Yo Hari: The symbolic link won't solve the problem. I managed to scrape together a blt for tcltk 8.5 on OSX, so it is possible. Most of the hacks in turn came from a different Linux distro (gentoo) so it should be possible on ubuntu. Here by the way is the patch: http://fink.cvs.sourceforge.net/viewvc/fink/dists/10.4/unstable/main/finkinfo/x11/blt-x86_64.patch debian and ubuntu distribute blt without the bltwish executable, and I apparently put a huge weed up their arse a couple of years ago by reporting this as a bug. They are totally inflexible. Like IT support people, the maintainers think you work for them rather than vice versa. It might be worth flooding them with complaints and bug reports. Bill On Apr 14, 2010, at 7:49 AM, hari jayaram hari...@gmail.com wrote: Hi Tim, Thanks a tonne Tim for the pointer on how bltwish is handled in debian. A symlink from /usr/bin/wish to /usr/bin/bltwish. Seems to at-least start ccp4i. Now to see if it will also plot my graphs Hari -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] To model or not to model...
Dear Katherine, I would like to add a futher point to this discussion: If you do model residues with poor or no electron density, then you should be sure of what you are doing and not just make more or less unguided guesses. This seems obvious but unfortunately it isn't, at least not to everybody. For example, I recently found a PDB entry of a glycoprotein that had an a-L-IdopNAc residue attached to an Asn side chain. Usually the first monosaccharide linked to an Asn is b-D-GlcpNAc -- there are only very few exceptions to that. Therefore, I had a look at the electron density and I saw that only part of the ring was resolved there; around the C5 atom of the sugar there was no electron density. b-D-GlcpNAc and a-L-IdopNAc only differ in the stereochemistry of the C5 atom, so it seems to me that the crystallographers arbitrarily placed the C6 and O6 somewhere and picked a wrong position, which led to wrong stereochemistry of the C5 atom. (This is in principle the same as adding an Ala CB atom somewhere near the CA atom and picking a position resulting in D-Ala instead of L-Ala.) In the example above it would have been better to leave out the unresolved atoms rather than creating an obviously wrong model, I think. The problem of non-crystallographers non knowing about occupancy or B-factors might be solved (in part) by better pointing out problematic parts of the structures, e.g. by highlighting them in visualization tools by default and not only when the user sets color mode to temperature or something like that. Cheers, Thomas
Re: [ccp4bb] To model or not to model...
On Apr 15, 2010, at 10:08, Thomas Lütteke wrote: Dear Katherine, I would like to add a futher point to this discussion: If you do model residues with poor or no electron density, then you should be sure of what you are doing and not just make more or less unguided guesses. This seems obvious but unfortunately it isn't, at least not to everybody. For example, I recently found a PDB entry of a glycoprotein that had an a-L-IdopNAc residue attached to an Asn side chain. Usually the first monosaccharide linked to an Asn is b-D- GlcpNAc -- there are only very few exceptions to that. Therefore, I had a look at the electron density and I saw that only part of the ring was resolved there; around the C5 atom of the sugar there was no electron density. b-D-GlcpNAc and a-L-IdopNAc only differ in the stereochemistry of the C5 atom, so it seems to me that the crystallographers arbitrarily placed the C6 and O6 somewhere and picked a wrong position, which led to wrong stereochemistry of the C5 atom. (This is in principle the same as adding an Ala CB atom somewhere near the CA atom and picking a position resulting in D-Ala instead of L-Ala.) In the example above it would have been better to leave out the unresolved atoms rather than creating an obviously wrong model, I think. Wouldn't it be even better to use to correct the restraints to keep the stereochemistry? If we relax the Cbeta stereochemistry, I bet we can end up with a few L- residues as well ... A. The problem of non-crystallographers non knowing about occupancy or B-factors might be solved (in part) by better pointing out problematic parts of the structures, e.g. by highlighting them in visualization tools by default and not only when the user sets color mode to temperature or something like that. Cheers, Thomas P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] Cryo Vs crystal size
Hi - My two cents: First, you say: I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? I think this assumption is confusing. If the crystals were grown in the same drop/condition, they have identical percentage solvent content. Thus, you do not want to look at dehydration, the 'percentage solvent content' is fine. What you want to look at is the mechanics of vitrification. Big crystals, are simply hard to freeze: because of their volume they cannot be vitrified as rapidly and uniformly as smaller crystals. I will not be surprised if there are papers that quantify that, but what I am saying here is only from experience and adding a 'logical' explanation to that experience. Thus, I would simply stay with the smaller crystals (I have a feeling that you 'small' crystals are 'big' for many other people) and be happy they diffract to 2.5 A (is that SR or RA?) A. On Apr 15, 2010, at 3:16, syed ibrahim wrote: Dear Jurgen and Ho Leung To add few more point regarding my question: 1. Crystal was first frozen in LN2 and then transfered to cryo stream (in presence of LN2 in vial) 2. Anealing did not help (both short time and long time) - perhaps the crystal dies. 3. Spots are clear to available resolution (is: 6-7A). In the high resolution region there is no spot but looks like smear in the whole area. 4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on 0.5mm loop. So the liquid around the crystal was very less. I deliberately avoided more solvent in the loop to help diffraction. Thanks Syed --- On Thu, 4/15/10, Jürgen Bosch jubo...@jhsph.edu wrote: From: Jürgen Bosch jubo...@jhsph.edu Subject: Re: [ccp4bb] Cryo Vs crystal size To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, April 15, 2010, 3:46 AM There are a couple of additional factors not taken into account here. 1. LN2 versus frozen in strem or propane etc 2. did you try to flash anneal the larger crystal 3. smeary diffraction from the big crystal or not ? 4. how much residual solvent was around your crystal when freezing ? In general smaller crystals are anyhow better in my hands. Jürgen On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote: Hi All I had two crystals grown in same well, one is small and other is 10 times bigger. I treated both crystal in same cryo and same time. The smaller one diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one to diffract high resolution. I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? Thank you Syed PS: Taken care of less solvent to be present in the loop - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
[ccp4bb] Stabilization of sensitive crystals - use of glutaraldehyde
Dear All, I have a question about the use of glutaraldehyde as a cross-linking agent. It is a practical question. The glutaraldehyde you can buy from Sigma- Aldrich contains a mixture of polymers according to the product information with an increasing number of polymers with increasing pH. Is it this mixture that is used for cross-linking crystals or do crystallographers use monomeric glutaraldehyde purified on charcoal? Best regards, Maria Håkansson __ Maria Håkansson, Ph.D. Senior Scientist, Max-lab, Lund University Phone: +46 (0) 76 8585 706 Fax: +46 (0) 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) SE-221 00 Lund, Sweden Web address: www.maxlab.lu.se Email: maria.hakans...@maxlab.lu.se __
Re: [ccp4bb] Cryo Vs crystal size
and don't forget to check diffraction without freezing. Mark On 15 April 2010 10:37, Anastassis Perrakis a.perra...@nki.nl wrote: Hi - My two cents: First, you say: I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? I think this assumption is confusing. If the crystals were grown in the same drop/condition, they have identical percentage solvent content. Thus, you do not want to look at dehydration, the 'percentage solvent content' is fine. What you want to look at is the mechanics of vitrification. Big crystals, are simply hard to freeze: because of their volume they cannot be vitrified as rapidly and uniformly as smaller crystals. I will not be surprised if there are papers that quantify that, but what I am saying here is only from experience and adding a 'logical' explanation to that experience. Thus, I would simply stay with the smaller crystals (I have a feeling that you 'small' crystals are 'big' for many other people) and be happy they diffract to 2.5 A (is that SR or RA?) A. On Apr 15, 2010, at 3:16, syed ibrahim wrote: Dear Jurgen and Ho Leung To add few more point regarding my question: 1. Crystal was first frozen in LN2 and then transfered to cryo stream (in presence of LN2 in vial) 2. Anealing did not help (both short time and long time) - perhaps the crystal dies. 3. Spots are clear to available resolution (is: 6-7A). In the high resolution region there is no spot but looks like smear in the whole area. 4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on 0.5mm loop. So the liquid around the crystal was very less. I deliberately avoided more solvent in the loop to help diffraction. Thanks Syed --- On *Thu, 4/15/10, Jürgen Bosch jubo...@jhsph.edu* wrote: From: Jürgen Bosch jubo...@jhsph.edu Subject: Re: [ccp4bb] Cryo Vs crystal size To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, April 15, 2010, 3:46 AM There are a couple of additional factors not taken into account here. 1. LN2 versus frozen in strem or propane etc 2. did you try to flash anneal the larger crystal 3. smeary diffraction from the big crystal or not ? 4. how much residual solvent was around your crystal when freezing ? In general smaller crystals are anyhow better in my hands. Jürgen On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote: Hi All I had two crystals grown in same well, one is small and other is 10 times bigger. I treated both crystal in same cryo and same time. The smaller one diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one to diffract high resolution. I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? Thank you Syed PS: Taken care of less solvent to be present in the loop - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ *P** **please don't print this e-mail unless you really need to* Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 -- Mark J van Raaij http://webspersoais.usc.es/mark.vanraaij http://www.ibmb.csic.es
Re: [ccp4bb] Phasing statistics
Sorry - I had not twigged that this was a SAD discussion. In this case DM is as you say gonna save you from bimodal ambiguities - as we all know that is why the FOMs get so much better during DM - but that seems fine to me as it is pretty much new information coming in from the solvent flattening, histogram matching, probably some averaging. So that improvement is not 'artifactual' but in fact part of the experimental detail of just how the structure was solved. I guess FOMs are really only useful in the Crick and Blow centroid case. Still think any and all info on the experiment(s) is useful to see - if only for people to plan their own experiments building on past experience. Best wishes and regards Martyn Martyn Symmons Cambridge - Original Message From: Ian Tickle ianj...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 14 April, 2010 15:42:51 Subject: Re: [ccp4bb] Phasing statistics I have to say that I don't share James' enthusiasm for the FOM as a useful statistic, even for experimental phases, and particularly not in the SAD (and SIR) cases, which after all is what this thread is supposed to be about. This is even without delving further into the murky issues of wildly inflated FOM estimates from DM that he and others have raised. My point is that the definition of FOM is that it's the *expected* cos(phase error), as opposed to *actual* cos(phase error) and the 'expected' bit makes all the difference! It's essentially the difference between precision and accuracy, i.e. the FOM tells you how precise the phase estimate is and not at all how accurate it is: for the latter you need to calculate the errors relative to the phases of the final model, as James suggested. The FOM is roughly related (anti-correlated to be precise) to the variance: this is clear if we take the case of small phase deviations, then cos(x) expands to (1-x^2/2) and the variance is x^2, where x is the deviation from the mean (NOT the same thing as the error!). So just like the variance it measures the 'peakedness' of the distribution: a FOM=1 corresponds to variance=0, i.e. an infinitely sharp distribution (delta function). Now a particular problem arises in SAD (and SIR) because then in general we always get a bimodal distribution: i.e. 2 separate peaks. If these peaks happen to separated by 180 deg then the FOM is 0 (cos(90)=0). A large separation is most likely to occur when the anomalous contribution is large, when of course you expect the phase estimates to be optimal, for example see Fig 2(a) in Wang et al., Acta Cryst. (2007). D63, 751–758. Conversely if the anomalous contribution is small, so the phase estimates are poor, the separation between the 2 estimates is small and the FOM is close to 1! So we apparently have a situation where the *better* are the phases, the *lower* is the FOM! What the Figure in the Wang paper ignores of course are the experimental errors which will tend to broaden the distribution and lower the FOM in the case where the anomalous contribution is small relative to the errors. Even so it means that the FOM is not a good measure of phase quality. Much better IMO is the phasing power (mean heavy-atom amplitude / mean P-weighted lack of closure error). This essentially measures the degree to which the phase ambiguity is capable of being successfully resolved by DM methods. One other thing puzzles me: why do many programs that purport to calculate average phase differences (relative to model phases say) use statistics such as, well, average |phase difference| when we already have a perfectly good measure in the FOM!, i.e. average cos(phase difference)? Then you would be able to directly compare the FOMs from experimental phases with those from model phases. Even better still would be the average log likelihood: if the phase probability is exp(A cos(delta_phi)) then the log likelihood is simply A cos(delta_phi) and you just average that, i.e. it's essentially the averaged FOM-weighted cos(phase difference), so that the average is weighted according to the reliability of the phase estimates. A poor phase estimate is likely to be associated with a small F which is not going to contribute much to the map anyway, so it makes no sense to give poor phases the same weight as good phases in the average. Cheers -- Ian On Tue, Apr 13, 2010 at 6:47 PM, James Holton jmhol...@lbl.gov wrote: Probably the only phasing stat that I pay any attention to these days is the Figure of Merit (FOM). This is because, the _definition_ of FOM is that it is the cosine of the phase error (or at least your best estimate of it). FOM=1 is perfect phases and FOM=0 is random phases, and a reasonable cutoff value for FOM is 0.5 (see Lunin Woolfson, Acta D, 1993). Yes, there are ways to get various programs to report very inaccurate values for FOM (such as running DM for thousands of cycles), and yes, there are often legitimate reasons to run these programs in this
Re: [ccp4bb] xorg.conf set-up for Ubuntu with NVIDIA 980 xgl and Crystal Eyes
Ensure that these lines are somewhere in your xorg.conf file: # - Section "Module" Load "glx" EndSection Section "Device" Identifier "Configured Video Device" Driver "nvidia" Option "Stereo" "3" EndSection Section "Extensions" Option "Composite" "Disable" EndSection # -- The stereo and composite options settings are required for stereo function. Cheers. Claudia Scotti wrote: Dear All, I've moved from RedHat to Ubuntu and I have thus lost the X11 config file for 3D stereo vision. I've tried to reset it up following the instructions, but the only result I get is the following: - the stereo emitter is on, - the glasses switch on when I open them (replaced batteries as well), - the monitor splits the image as expected, but: - the emitter is not flickering and the crystal eyes do not become active. Anybody has solved this problem before, please? I couldn't find it in previous threads... Any help would be very much appreciated. Claudia Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Via Ferrata, 1 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 Hotmail: Trusted email with Microsofts powerful SPAM protection. Sign up now.
[ccp4bb] Proportion of MR in the PDB
Does anyone have a good estimate of the proportion of molecular replacement structures in the PDB? I tried using the search tools at RCSB and PDBe. The PDBe gives 55.1% but apparently only 3411 structures have phase determination information recorded, so this does not look to be terribly comprehensive. Thanks Nick Keep
Re: [ccp4bb] Cryo Vs crystal size
You've gotten some helpful replies already. I have found the following reference to be helpful in understanding some of the physics behind damage incurred during the crystal cooling process and a general strategy to help avoid it. It expands upon what's already been said - that larger crystals are more prone to distress during cooling. This and other papers from the same group contain useful information and advice. A General Method for Hyperquenching Protein Crystals Matthew Warkentin and Robert E. Thorne Struct Funct Genomics. 2007 December ; 8(4): 141–144. doi:10.1007/s10969-007-9029-0. Best, -Andy On 4/15/2010 4:48 AM, Mark J. van Raaij wrote: and don't forget to check diffraction without freezing. Mark On 15 April 2010 10:37, Anastassis Perrakis a.perra...@nki.nl mailto:a.perra...@nki.nl wrote: Hi - My two cents: First, you say: I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? I think this assumption is confusing. If the crystals were grown in the same drop/condition, they have identical percentage solvent content. Thus, you do not want to look at dehydration, the 'percentage solvent content' is fine. What you want to look at is the mechanics of vitrification. Big crystals, are simply hard to freeze: because of their volume they cannot be vitrified as rapidly and uniformly as smaller crystals. I will not be surprised if there are papers that quantify that, but what I am saying here is only from experience and adding a 'logical' explanation to that experience. Thus, I would simply stay with the smaller crystals (I have a feeling that you 'small' crystals are 'big' for many other people) and be happy they diffract to 2.5 A (is that SR or RA?) A. On Apr 15, 2010, at 3:16, syed ibrahim wrote: Dear Jurgen and Ho Leung To add few more point regarding my question: 1. Crystal was first frozen in LN2 and then transfered to cryo stream (in presence of LN2 in vial) 2. Anealing did not help (both short time and long time) - perhaps the crystal dies. 3. Spots are clear to available resolution (is: 6-7A). In the high resolution region there is no spot but looks like smear in the whole area. 4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on 0.5mm loop. So the liquid around the crystal was very less. I deliberately avoided more solvent in the loop to help diffraction. Thanks Syed --- On *Thu, 4/15/10, Jürgen Bosch /jubo...@jhsph.edu mailto:jubo...@jhsph.edu/* wrote: From: Jürgen Bosch jubo...@jhsph.edu mailto:jubo...@jhsph.edu Subject: Re: [ccp4bb] Cryo Vs crystal size To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Date: Thursday, April 15, 2010, 3:46 AM There are a couple of additional factors not taken into account here. 1. LN2 versus frozen in strem or propane etc 2. did you try to flash anneal the larger crystal 3. smeary diffraction from the big crystal or not ? 4. how much residual solvent was around your crystal when freezing ? In general smaller crystals are anyhow better in my hands. Jürgen On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote: Hi All I had two crystals grown in same well, one is small and other is 10 times bigger. I treated both crystal in same cryo and same time. The smaller one diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one to diffract high resolution. I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? Thank you Syed PS: Taken care of less solvent to be present in the loop - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ http://web.me.com/bosch_lab/ *P** **please don't print this e-mail unless you really need to* Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 -- Mark J van Raaij http://webspersoais.usc.es/mark.vanraaij http://www.ibmb.csic.es
[ccp4bb] PhD fellowship University of Copenhagen: Nicotinic acetylcholine alpha4beta2 receptor model systems
General announcement and how to apply: http://www.farma.ku.dk/index.php?id=8206 Project description: http://www.farma.ku.dk/index.php/Project-6/8214/0/ Deadline for applications: Monday 3 May 2010 at 12 o'clock noon About the group: http://www.farma.ku.dk/BR/ Please contact me if you wish further information. Best regards Michael Gajhede -- Professor Michael Gajhede Institute of Medicinal Chemistry University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Ø Denmark Phone: +45 35306407 Email: m...@farma.ku.dk.dk
[ccp4bb] Gordon Research Conference on Diffraction Methods
The biannual Gordon Conference on Diffraction Methods in Structural Biology will be held at Bates College in Lewiston, Maine from 18-23rd July. The program covers all aspects of macromolecular structure solution from crystallization to structure solution and refinement and includes sessions on complementary techniques and the potential of free electron lasers. Many of the leading methods developers will be present and the format of the meeting, with free afternoons, provides ample opportunities for informal discussions. Further details, including a full program and details on the application procedure can be obtained from: http://www.grc.org/programs.aspx?year=2010program=diffrac We hope to see you there ! Andrew Leslie (Chair) Ana Gonzalez (Vice Chair) Outline Program: Sunday pm: Macromolecular Structures, Pushing the boundaries Discussion leader: Michael Rossmann 1. ABC Transporters: Doug Rees 2. The Spliceosome : Kyoshi Nagai Monday am: Crystallisation and the diffraction experiment Discussion leader: Zbyszek Dauter 1. Nanolitre crystallisation: Seth Harris 2. Twinning and disorder: Todd Yeates 3. Simulating the diffraction experiment: James Holton 4. Microcrystallography: Gwyndaf Evans Monday pm: Radiation damage and data collection strategies Discussion leader: Vivian Stojanoff 1. Radiation damage, experimental: Elspeth Garman 2. Microbeams: Yanhui Zou 3. Testing data collection strategies with the Pilatus: Marcus Mueller Tues am: Synchrotron developments and automated data processing Discussion leader: Thomas Schneider 1. Synchrotrons, latest developments: Sean Mc Sweeney 2. Synchrotrons, latest developments: Janet Smith 3. Data processing with Xia2/CHEF: Graeme Winter 4. Data processing with AUTOPROC: Clemens Vonrhein Tues pm: Complementary techniques Discussion leader: Ana Gonzales 1. Electron tomography: Sriram Subramaniam 2. SAXS: Hiro Tsuruta 3. Spectroscopy: Carrie Wilmot Weds am: Structure solution and refinement Discussion leader: Paul Adams 1. SAD Phasing: Randy Read 2. Molecular replacement with BALBES: Garib Murshudov 3. PHENIX: Tom Terwilliger 4. TLS: Ethan Merritt Weds pm: Challenging problems/Membrane Proteins Discussion leader: TBC 1. Using cubic lipidic phase: Martin Caffrey 2. Membrane Proteins: Stephen White 3. The Bacterial Ribosome: Venki Ramakrishnan Thurs am: Selected Posters and Map improvement and Model building Discussion leader: Tassos Perrakis 1. COOT: Paul Emsley 2. Modelling nucleic acids: Johan Hattne Thursday pm: Future Methods, FELs, Diffraction imaging Discussion leader: Ed Lattman 1. Anton Barty 2. Serial Crystallography: John Spence and Petra Fromme
[ccp4bb] Summary: Phasing statistics
Dear Colleagues, I am very grateful to everyone who contributed to the discussion regarding phasing statistics that I initiated. I certainly found it very informative. Below is a summary of the technical responses that I regarding this problem. 1) Use some of the statistics that SHELXD and SHELXE do provide (e.g. CC/CCfree for SHELXD, CCfree and connectivity for SHELXE). These could be compared to statistics produced for well determined structures (e.g. see Debreczeni et al. 2003 Acta Cryst. D., D59, 688-696). 2) Take the results from SHELX and put them into SHARP to generate the statistics. 3) Take the results from SHELX and put them into phaser_er, CRANK, or MLPHARE (perhaps with more difficulty) to generate the statistics. Thanks to Rick Lewis, Boaz Shaanan, Ed Lowe and Eleanor Dodson for suggestions. Cheers, Nic Harmer [For anyone interested, I took approaches 1 and 2. I got a good figures for phasing power from SHARP (somehow I failed to find the Rcullis, never mind), quoted the FOM at the end of SHELX, and the values for CC/CCfree from SHELXD, and the map contrast in the original and inverted hands from SHELXE. These all looked quite convincing, so hopefully my referees will be happy.]
[ccp4bb] Crystallography training
Dear All, Attached is information about a new X-ray crystallography training program being introduced at Pennsylvania State University. Deadline for registering is May 1st 2010. Kindly pass it along to anybody that may be interested in such an opportunity. Thank you Neela attachment: AD 4-15-10 Xray.gif
[ccp4bb] X-Ray films
Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
[ccp4bb] crystallography movies
Both the original movie and the made-for-TV movies of The Andromeda Strain had short references to crystallography, the first in how the strain was organized, the second in how a message from the future was encoded in buckyball nanotechnology crystal. Perhaps this is why they are my favorite sci-fi movies James Phillips Duke University
Re: [ccp4bb] X-Ray films
Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at 12:16 PM, harry powell ha...@mrc-lmb.cam.ac.ukwrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] bacillus protein expression
Hi Amit- We used a well-expressing soluble protein fusion (SUMO) at the N-term of a couple of B. anthracis proteins that we were trying to produce in E. coli, which improved the soluble yield of both targets tremendously. The disadvantage of a fusion is of course you produce a non-native protein- upon cleaving away the fusion (e.g. with thrombin or TEV), you will probably retain one or more exogenous residues. We picked SUMO because after cleaving with Ulp1 (SUMO protease) you produce a native protein with no additional residues from the fusion. If you want more details about this and things we tried to boost expression, please feel free to contact me off-list. Best- Brad On Wed, Apr 14, 2010 at 3:30 PM, Amit Kumar amit23...@gmail.com wrote: Hi all, Sorry for a non-ccp4 query. I will highly appreciate your comments on the expression: I am trying to express HIS tagged ~60 kDa cytosolic protein from Bacillus in E.coli pET expression system. I tried codon+ strain, time of expression and various temperatures ( 18, 25, 30, 37C) for the expression. Neither full-length nor truncated form of the protein was found to be expressing (also checked by western blot). What other parameters should I explore to get the expression in Escherichia coli? Thank you very much for your comments. Amit K.
[ccp4bb] composite omit map in CCP4
Hi Folks, Is there an easy way to generate a composite omit map using CCP4? I know this has been discussed a few times before. But has anybody written some new programs recently? Regards, Weikai
Re: [ccp4bb] X-Ray films
The documentary Naturally Obsessed: The Making of a Scientist is definitely a must-see film. It captures the story of life and science in a crystallography lab, that of Dr. Larry Shapiro at Columbia University, and follows the graduate students journey of fortune and misfortune that crystallographers know so well. Not to sound too sappy about it, but it is almost like a coming of age story for graduate students Check it out: http://naturallyobsessed.com/ -Chayne Piscitelli From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Brad Bennett [bradbennet...@gmail.com] Sent: Thursday, April 15, 2010 9:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-Ray films Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at 12:16 PM, harry powell ha...@mrc-lmb.cam.ac.ukmailto:ha...@mrc-lmb.cam.ac.uk wrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
[ccp4bb] Summary of replies: bacillus protein expression
Hi all, I am highly thankful to all of you for very useful replies. The following are the list of the replies to the query. Query: Hi all, Sorry for a non-ccp4 query. I will highly appreciate your comments on the expression: I am trying to express HIS tagged ~60 kDa cytosolic protein from Bacillus in E.coli pET expression system. I tried codon+ strain, time of expression and various temperatures ( 18, 25, 30, 37C) for the expression. Neither full-length nor truncated form of the protein was found to be expressing (also checked by western blot). What other parameters should I explore to get the expression in Escherichia coli? Thank you very much for your comments. Amit K. Replies to above query: reply-1 Did you tried to check on the insoluble fraction to see if your protein is there? If the protein is insoluble tried the following: -Different E.coli strains -Less rich medium (LB instead of 2XYT) -Modify protein (shorter construct based on proteolysis, 2D structure prediction) - Most probably you will have to use a different vector using larger tags like GST, MBP, SUMO etc. - If all fails, you will need to go to other expression systems (Yeast, Pichia) reply-2 Looks like my very recent experience. In the expression profile, my protein started expressing after 20 hrs after low conc IPTG induction. I had to continue the expression for 36-40 hrs. reply-3 Are your codons optimized? I would look at the first 50 aa's to see if its particularly rich in rare codons, if so mutate them to more prefered e. coli codons. An N-His tag also helps with this since after about 50 aa's the ribosome doesn't tend to stall as much. The protein may also be toxic, so using a toxic resistance strain of e. coli is also good. Finally and maybe most simply I would get some fresh competent cells. If they aren't made properly they can have started to express the Lac inacativator protein which will absolutely kill your inducability. reply-4 You could try other tags, as His tags are not always the best. In addition, you didn't mention which side of the protein you tagged- it can make a difference whether it is N- or C-terminal. One last thing is to get the gene synthesized to codon optimize and RNA optimize. In this case I don't think codon optimization is a big factor, but your RNA could be forming interesting secondary structures which may inhibit translation in E. coli. reply-5 1. look up Bacillus megaterium expression system (commercial, pretty cheap relatively easy). It's easier to use than some of the integration-based B. subtilis systems. Or you could ask around for a suitable B.sut. shuttle vector as an alternative. 2. Bacillus codon set is heavily biased towards AT-richness and even in the codon+ may not be expressed well - there may also be mRNA issues (stability). It's not hard to make synthetic genes these days - if you're desperate to continue using E. coli for this work you can make a synthetic codon-optimized version specific to the host. reply-6 We used a well-expressing soluble protein fusion (SUMO) at the N-term of a couple of B. anthracis proteins that we were trying to produce in E. coli, which improved the soluble yield of both targets tremendously. The disadvantage of a fusion is of course you produce a non-native protein- upon cleaving away the fusion (e.g. with thrombin or TEV), you will probably retain one or more exogenous residues. We picked SUMO because after cleaving with Ulp1 (SUMO protease) you produce a native protein with no additional residues from the fusion. If you want more details about this and things we tried to boost expression, please feel free to contact me off-list.
Re: [ccp4bb] X-Ray films
Wrath of Khan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli pisci...@ohsu.edu wrote: The documentary Naturally Obsessed: The Making of a Scientist is definitely a must-see film. It captures the story of life and science in a crystallography lab, that of Dr. Larry Shapiro at Columbia University, and follows the graduate students journey of fortune and misfortune that crystallographers know so well. Not to sound too sappy about it, but it is almost like a coming of age story for graduate students Check it out: http://naturallyobsessed.com/ -Chayne Piscitelli From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Brad Bennett [bradbennet...@gmail.com] Sent: Thursday, April 15, 2010 9:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-Ray films Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at 12:16 PM, harry powell ha...@mrc-lmb.cam.ac.uk wrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH -- --- --- Brock Schuman, Graduate Student Department of Biochemistry Microbiology University of Victoria PO Box 3055 STN CSC Victoria, BC, V8W 3P6 CANADA tel: 250-721-8945 FAX:250-721-8855
Re: [ccp4bb] crystallography movies
Well, the original movie was reasonably well done. But the made for TV version is deeply rewarding, for all the wrong reasons, being so bad, it's good. SPOILER ALERT: Read no further if you want to truly enjoy the the silliest four minutes in the history of made-for-television movies, specifically those minutes during which: that thumb is cut off and tossed up four stories or so to a thumb-awaiting Benjamin Bratt, who then has to grope his way to the thumb-dependent fail-safe ATM machine. It's a good thing that Bratt lost his eyesight (to a broken steam pipe!) only after he had already caught the cut-off thumb. Thanks for the memory! I've saved that four minutes on a dvd, and I'll be sure to watch it again soon. On Thu, Apr 15, 2010 at 12:36 PM, james.phill...@duke.edu wrote: Both the original movie and the made-for-TV movies of The Andromeda Strain had short references to crystallography, the first in how the strain was organized, the second in how a message from the future was encoded in buckyball nanotechnology crystal. Perhaps this is why they are my favorite sci-fi movies James Phillips Duke University -- Gary A. Ratner gary.a.rat...@gmail.com
Re: [ccp4bb] X-Ray films
Terminator 3 - IIRC, a synchrotron strips the fluid-metal exoskeleton off of TX - that counts, right? -- Delivered via an Android. On Apr 15, 2010 9:07 PM, Brock Schuman bro...@gmail.com wrote: Wrath of Khan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli pisci...@ohsu.edu wrote: The documentary N... -- --- --- Brock Schuman, Graduate Student Department of Biochemistry Microbiology University of Victoria PO Box 3055 STN CSC Victoria, BC, V8W 3P6 CANADA tel: 250-721-8945 FAX:250-721-8855
[ccp4bb] Postdoctoral position in protein crystallography at Emory University
POSTDOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY AT EMORY UNIVERSITY A postdoctoral position is available in the Department of Biochemistry at Emory University (Atlanta, GA). This position has been funded for two years to support an NIH R01 grant aimed at understanding the structural and mechanistic basis for the evolution of novel function within the steroid hormone receptors. For publications resulting from this work see: Ortlund et al, Science. 2007 Sep 14;317(5844). Bridgham et al, Nature. 2009 Sep 24;461(7263):515-9. The successful candidate will have a very strong background in X-ray crystallography, molecular biology and biochemistry, along with good organizational, communication and interpersonal skills. Emory University maintains a state-of-the-art in house X-ray diffraction system and has regular synchrotron access. Please send (email) cover letter, CV and the names and address of three references to: Eric Ortlund, Ph.D. Assistant Professor Department of Biochemistry Emory University School of Medicine 1510 Clifton Road, NE, Room G235 Atlanta, GA 30322 Tel 404-727-5014 Fax 404-727-2738 eric.ortl...@emory.edu
Re: [ccp4bb] X-Ray films
The Race for the Double Helix (2004) http://www.amazon.com/Race-Double-Helix-VHS-Pigott-Smith/dp/6303247911/ref=sr_1_1?ie=UTF8s=dvdqid=1271365899sr=1-1 When crystallography was on FILMS not CCD's:) Eva Kirchner wrote: favourite movies involving real science The Dark Crystal (1982)! (Crystallography is voodoo, voodoo is magic, magic is fantasy, therefore fantasy is science, and this movie involves science. Ha!) It tells you what awful consequences arise if you break a crystal. ;-) Am 15.04.2010 um 22:07 schrieb Brock Schuman: Wrath of Khan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli pisci...@ohsu.edu wrote: The documentary Naturally Obsessed: The Making of a Scientist is definitely a must-see film. It captures the story of life and science in a crystallography lab, that of Dr. Larry Shapiro at Columbia University, and follows the graduate students journey of fortune and misfortune that crystallographers know so well. Not to sound too sappy about it, but it is almost like a coming of age story for graduate students Check it out: http://naturallyobsessed.com/ -Chayne Piscitelli From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Brad Bennett [bradbennet...@gmail.com] Sent: Thursday, April 15, 2010 9:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] X-Ray films Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at 12:16 PM, harry powell ha...@mrc-lmb.cam.ac.uk wrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH -- --- --- Brock Schuman, Graduate Student Department of Biochemistry Microbiology University of Victoria PO Box 3055 STN CSC Victoria, BC, V8W 3P6 CANADA tel: 250-721-8945 FAX:250-721-8855 -- Dr. Eva Kirchner Unité de Virologie Structurale Département de Virologie Institut Pasteur 25, rue du Dr Roux 75015 Paris France Phone: +33 (0)1 45 68 82 87 Email: eva.kirch...@pasteur.fr
Re: [ccp4bb] X-Ray films
I'm not sure there was actually any science in the movie, but 'The Incredible Hulk' had some opening scenes shot at the beautiful ALS synchrotron... and I just want to point out that I only saw that movie because my kids made me! Took a lot of junk food to make it through that one -Robyn -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of harry powell Sent: Thursday, April 15, 2010 9:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-Ray films Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] X-Ray films
Mike Carson claims that the spinning DNA in the computer monitor in Jurassic Park was the product of the Ribbons software package: http://www.cbse.uab.edu/carson/ James On Apr 15, 2010, at 9:16 AM, harry powell wrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
[ccp4bb] THANKS Cryo Shipping!!
Thanks everyone for letting me know its canister. I had a different idea of a canister. I really appreciate all your replies.Instead of replying individually, I am sending this common email. Thanks again, Ivan
Re: [ccp4bb] X-Ray films
(What is in the Hollywood district other than tourists looking for Hollywood?) I have a silly anecdote that does not answer Harry's question: It must have been 1995 or 1996, the Beckman sales representative appeared at my desk on a Friday afternoon without an appointment, but carrying a bribe. The second Jurassic Park movie was going into production, and the movie company had asked her to order a room full of laboratory equipment so that the wet lab scenes would look credible to a scientist viewer. As always, once production starts, everything has to happen NOW, so the Beckman sales rep needed to borrow my VWR catalog over the weekend. That's what the bribe was for. For completeness, here's my financial involvement disclosure: I accepted the bribe, which was a pastry, and I ate it. Despite the 10 HBO channels in my apartment, I never saw the entire Jurassic Park 2 movie, so I don't know if the wet lab scenes were retained. That's completely off-topic, Dan harry powell wrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] X-Ray films
Did I see a PyMol screenshot (or something very like it) at the beginning of Avatar? James Stroud wrote: Mike Carson claims that the spinning DNA in the computer monitor in Jurassic Park was the product of the Ribbons software package: http://www.cbse.uab.edu/carson/
Re: [ccp4bb] X-Ray films
Right you are, Chayne, I think it should be shown to every Protein crystallographer graduate student on their first day in the lab. ivan On Thu, Apr 15, 2010 at 11:42 AM, Chayne Piscitelli pisci...@ohsu.eduwrote: The documentary Naturally Obsessed: The Making of a Scientist is definitely a must-see film. It captures the story of life and science in a crystallography lab, that of Dr. Larry Shapiro at Columbia University, and follows the graduate students journey of fortune and misfortune that crystallographers know so well. Not to sound too sappy about it, but it is almost like a coming of age story for graduate students Check it out: http://naturallyobsessed.com/ -Chayne Piscitelli -- *From:* CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Brad Bennett [bradbennet...@gmail.com] *Sent:* Thursday, April 15, 2010 9:43 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] X-Ray films Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at 12:16 PM, harry powell ha...@mrc-lmb.cam.ac.ukwrote: Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
