Re: [ccp4bb] Angle restraints
Hi Kavya, Which validation program did you use? How big is the deviation (in sigma values)? Is it the only outlier? What is your overall bond angle rmsZ? Using external restraints is a bit over the top here, especially if it is the only outlier. If your rmsZ is high (close to or over 1) then you may want to try tighter geometric restraints overall. In a normal distribution it is not surprising to find a 'true' outlier, so if your structure is large you need to worry less. Cheers, Robbie Sent from my Windows Phone From: Kavyashree Manjunath Sent: 2013-04-15 07:13 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Angle restraints Dear users, Validation of a structure showed a deviation in the angle between atoms NH1-CZ-NE and NH2-CZ-NE in the arginine residue. Several trials of modification of the orientation failed to solve this problem. I also confirmed by deleting the side chain and refining, it confirmed the presence of complete side chain. So I proceeded to use external restraints for these two angles in refmac5 (version 5.6.0117). The keyword was as follows - external angle first chain A residue 93 atom NE next chain A residue 93 atom CZ next chain A residue 93 atom NH1 value 120.3 sigma 0.5 external angle first chain A residue 93 atom NH2 next chain A residue 93 atom CZ next chain A residue 93 atom NE value 120.3 sigma 0.5 Still there is no change in the angle, it continues to have the same deviation. So kindly suggest whether there is any error in the keyword provided or other way to handle this problem. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Angle restraints
Sir, I used RCSB validation server. Deviations are as given below - Deviation Resi AT1 - AT2 - AT3BondDict Std Angle ValueDev -- 3.2ARG NE - CZ - NH1123.5 120.30.5 -3.4ARG NE - CZ - NH2116.9 120.30.5 Thus its more than 6 sigmas in both the cases. Its not an outlier. zANGL is 0.628 and Rms BondAngle is 1.4077. would you suggest me to ignore this deviation? But will it not be a problem during PDB submission? What is the reason I am getting a deviation like this? Should I reduce the weighing term further down? Thanking you Regards Kavya Hi Kavya, Which validation program did you use? How big is the deviation (in sigma values)? Is it the only outlier? What is your overall bond angle rmsZ? Using external restraints is a bit over the top here, especially if it is the only outlier. If your rmsZ is high (close to or over 1) then you may want to try tighter geometric restraints overall. In a normal distribution it is not surprising to find a 'true' outlier, so if your structure is large you need to worry less. Cheers, Robbie Sent from my Windows Phone From: Kavyashree Manjunath Sent: 2013-04-15 07:13 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Angle restraints Dear users, Validation of a structure showed a deviation in the angle between atoms NH1-CZ-NE and NH2-CZ-NE in the arginine residue. Several trials of modification of the orientation failed to solve this problem. I also confirmed by deleting the side chain and refining, it confirmed the presence of complete side chain. So I proceeded to use external restraints for these two angles in refmac5 (version 5.6.0117). The keyword was as follows - external angle first chain A residue 93 atom NE next chain A residue 93 atom CZ next chain A residue 93 atom NH1 value 120.3 sigma 0.5 external angle first chain A residue 93 atom NH2 next chain A residue 93 atom CZ next chain A residue 93 atom NE value 120.3 sigma 0.5 Still there is no change in the angle, it continues to have the same deviation. So kindly suggest whether there is any error in the keyword provided or other way to handle this problem. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Angle restraints
Dear Kavya, I would examine the electron density of your Arginine. It may be present in two alternative conformations and you may have fitted the Arginine in the middle, e.g. with the NH1 in one conformation and the NH2 in the other. The Arginine may also just be incorrectly fitted. Using Coot with Auto Fit Rotatmer and Real Space Refine Zone you might be able to correct it. Just using Regularize Zone on the Arginine might give hints how the Arginine got distorted. However, before running another round of refinement, it needs to be fitted back in electron density. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kavyashree Manjunath Sent: Monday, April 15, 2013 9:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Angle restraints Sir, I used RCSB validation server. Deviations are as given below - Deviation Resi AT1 - AT2 - AT3BondDict Std Angle ValueDev -- 3.2ARG NE - CZ - NH1123.5 120.30.5 -3.4ARG NE - CZ - NH2116.9 120.30.5 Thus its more than 6 sigmas in both the cases. Its not an outlier. zANGL is 0.628 and Rms BondAngle is 1.4077. would you suggest me to ignore this deviation? But will it not be a problem during PDB submission? What is the reason I am getting a deviation like this? Should I reduce the weighing term further down? Thanking you Regards Kavya Hi Kavya, Which validation program did you use? How big is the deviation (in sigma values)? Is it the only outlier? What is your overall bond angle rmsZ? Using external restraints is a bit over the top here, especially if it is the only outlier. If your rmsZ is high (close to or over 1) then you may want to try tighter geometric restraints overall. In a normal distribution it is not surprising to find a 'true' outlier, so if your structure is large you need to worry less. Cheers, Robbie Sent from my Windows Phone From: Kavyashree Manjunath Sent: 2013-04-15 07:13 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Angle restraints Dear users, Validation of a structure showed a deviation in the angle between atoms NH1-CZ-NE and NH2-CZ-NE in the arginine residue. Several trials of modification of the orientation failed to solve this problem. I also confirmed by deleting the side chain and refining, it confirmed the presence of complete side chain. So I proceeded to use external restraints for these two angles in refmac5 (version 5.6.0117). The keyword was as follows - external angle first chain A residue 93 atom NE next chain A residue 93 atom CZ next chain A residue 93 atom NH1 value 120.3 sigma 0.5 external angle first chain A residue 93 atom NH2 next chain A residue 93 atom CZ next chain A residue 93 atom NE value 120.3 sigma 0.5 Still there is no change in the angle, it continues to have the same deviation. So kindly suggest whether there is any error in the keyword provided or other way to handle this problem. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Angle restraints
Dear Kavya, First try Herman's suggestions. You can try changing the restraint weight but it will probably not solve the problem; it may hide it. If you cannot solve the problem and you did the best you can do, you can deposit the model with the outlier. The PDB does not reject models with (minor) issues. Future users will be warned by REMARK 500 if they want to use that specific arginine for further research. Cheers, Robbie Sent from my Windows Phone From: Kavyashree Manjunath Sent: 2013-04-15 09:35 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Angle restraints Sir, I used RCSB validation server. Deviations are as given below - Deviation Resi AT1 - AT2 - AT3BondDict Std Angle ValueDev -- 3.2ARG NE - CZ - NH1123.5 120.30.5 -3.4ARG NE - CZ - NH2116.9 120.30.5 Thus its more than 6 sigmas in both the cases. Its not an outlier. zANGL is 0.628 and Rms BondAngle is 1.4077. would you suggest me to ignore this deviation? But will it not be a problem during PDB submission? What is the reason I am getting a deviation like this? Should I reduce the weighing term further down? Thanking you Regards Kavya Hi Kavya, Which validation program did you use? How big is the deviation (in sigma values)? Is it the only outlier? What is your overall bond angle rmsZ? Using external restraints is a bit over the top here, especially if it is the only outlier. If your rmsZ is high (close to or over 1) then you may want to try tighter geometric restraints overall. In a normal distribution it is not surprising to find a 'true' outlier, so if your structure is large you need to worry less. Cheers, Robbie Sent from my Windows Phone From: Kavyashree Manjunath Sent: 2013-04-15 07:13 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Angle restraints Dear users, Validation of a structure showed a deviation in the angle between atoms NH1-CZ-NE and NH2-CZ-NE in the arginine residue. Several trials of modification of the orientation failed to solve this problem. I also confirmed by deleting the side chain and refining, it confirmed the presence of complete side chain. So I proceeded to use external restraints for these two angles in refmac5 (version 5.6.0117). The keyword was as follows - external angle first chain A residue 93 atom NE next chain A residue 93 atom CZ next chain A residue 93 atom NH1 value 120.3 sigma 0.5 external angle first chain A residue 93 atom NH2 next chain A residue 93 atom CZ next chain A residue 93 atom NE value 120.3 sigma 0.5 Still there is no change in the angle, it continues to have the same deviation. So kindly suggest whether there is any error in the keyword provided or other way to handle this problem. Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
[ccp4bb] salt or not?
Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina
Re: [ccp4bb] salt or not?
Dear Careina, I would be cautious of using dyes. Much better to 1) try in-situ diffraction if possible as this is least invasive or 2) pick a sensible cryo and just freeze the crystal(s). I would try to same something from the experiment for seed stock possibly sequencing. Dave David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com mailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms Sent: 15 April 2013 11:18 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] salt or not? Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies.
Re: [ccp4bb] salt or not?
What else in in the conditions? Calcium Sulphate/Phosphate is poorly soluble, so if there is any sulphate or phosphate in your condition I would be suspicious. The age of the plate is also a bad sign, as evaporation over an extended time can lead to salt crystals. Check the well solution for crystals, if there are any then it's almost certainly salt. Softness and lack of birefringence are cautiously good signs, however the only way to know for sure is to stick them in an x-ray beam, which is always worth while for a crystal. Good luck Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms [careinaedgo...@yahoo.com] Sent: 15 April 2013 11:18 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] salt or not? Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina
Re: [ccp4bb] Angle restraints
Sir, Thank you Sir. It worked. I used alternate conformations which differ by 180 deg around CZ-NE bond. Now there is no angle deviation error. Thank you all for useful suggestions. With Regards Kavya what you may have is two alternate conformations - i.e. your current one and one with the CD-NE bond rotated 180 degrees. Your density looks like an average between these two (to me at least and adaict from your image). Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
[ccp4bb] AW: [ccp4bb] cell disruptor / homogenizer - final summary
P.S.: The C5 is what we had in Bochum. Also not perfect, but I guess theres no perfect machine .. Von: Clemens Steegborn [mailto:clemens.steegb...@ruhr-uni-bochum.de] Gesendet: Donnerstag, 28. März 2013 13:58 An: 'Clemens Steegborn'; 'CCP4BB@JISCMAIL.AC.UK' Betreff: AW: [ccp4bb] cell disruptor / homogenizer - final summary Since my previous summary on cell disruption equipment elicited a pretty clear response, I want to give a final update: Several methods and machines were mentioned (see below), but the Avestin Emulsiflex C5 is now the clear winner, with five happy users and no really negative comment. Thanks again to everyone for this helpful feedback! Happy Holidays Clemens Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Clemens Steegborn Gesendet: Dienstag, 26. März 2013 19:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] AW: [ccp4bb] cell disruptor / homogenizer - summary Dear colleagues, To summarize the feedback on cell disruptors: One person found the Emulsiflex performance worth the maintenance required, there were no comments at all on Fluidizer and TS 0.75! As alternatives, French press (Glenn Milles), beadbeaters (Biospec), Panda homegenizer (GEA Niro Soavi), and nitrogen cavitation in a pressure bomb (http://www.ncbi.nlm.nih.gov/pubmed/21041386) were suggested. Thanks for the comments and suggestions. Best Clemens Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Clemens Steegborn Gesendet: Montag, 25. März 2013 10:17 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] cell disruptor / homogenizer Dear colleagues, We are currently looking for new cell disruptor/homogenizer equipment, mainly for E.coli work. We currently have a Branson sonifier and a Microfluidics Fluidizer the latter one keeps causing trouble, and we think about a replacement. We previously also used an Avestin C-5 Emulsiflex, required quite some maintenance, and I inquired about a Constant Systems TS 0.75 and got mixed responses from users (not to mention their outrageous pricing). My question: What is your experience with Fluidizer, Emulsiflex, TS 0.75? Is there any other great (low maintenance, affordable especially concerning follow-up costs: repairs, replacement parts) equipment I missed? Thanks in advance for your comments Best regards Clemens
Re: [ccp4bb] salt or not?
Dear Careina, altough your crystals does't take up the Izit dye it sounds promising. The uptake of Izit depends on the solvent channels of the protein molecule - sometimes the dye just can't enter the molecule. Concerning the Calciumchloride - if the concentration is not too high and without other ingredients which could cause less solube salt there is the possiblity that you have got protein crystals. I once had a condition whith 34 % MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting crystals To speak from my experience I think 1 month after setting up the trays the drops should not be dried out. If the wells are sealed properly new crytals can appear even after 1 year. You should definately check the diffraction then you will know for sure. Cheers, Ulrike
[ccp4bb] Off-topic: PDB statistics
Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Postdoctoral position - ESRF, Grenoble
Post-Doctoral Fellow (m/f) for the Macromolecular Crystallography /in-crystallo/ spectroscopy facility : Grenoble, France You will play a major role in the operation and development of the ESRF /in-crystallo/ spectroscopy Facility (ID29S) dedicated to the application of optical spectroscopy in the context of Macromolecular Crystallography (MX). The interpretation of electron density maps resulting from MX experiments can often benefit from the use of such complementary techniques. The successful candidate will support Raman spectroscopy and X-ray diffraction experiments on both ID29S and the MX beamline ID29 and will be responsible for developing new instruments suitable for various on-line spectroscopy experiments on the MX beamline ID30A currently under construction. Additionally the successful candidate will carry out research, using a combination of X-ray crystallography and Raman spectroscopy, aimed at studying the effect of radiation damage on DNA molecules. For more information about ID29S please consult: http://www.esrf.eu/UsersAndScience/Experiments/MX/Cryobench/. You should hold a Ph.D. in optical spectroscopy as applied to biological macromolecules, protein crystallography or a related subject. Experience in the use of optical spectroscopy as applied in Structural Biology is highly desirable as is knowledge of the operation of synchrotron radiation beamlines. You should be capable of integrating into a multidisciplinary and international research team. The working language of the ESRF is English. For further information on employment terms and conditions, please refer to http://www.esrf.fr/Jobs/Conditions. The ESRF is an equal opportunity employer and encourages applications from disabled persons. Contract of 18 months, renewable for a further 6 to 18 month-period. Only candidates holding a Ph.D. obtained less than 3 years ago are eligible for Post-doctoral positions. If you are interested in this position, please apply on-line at this address: http://www.esrf.fr/Jobs. Ref. PDID29-2 -- Deadline for returning application forms: 30th April 2013
Re: [ccp4bb] Off-topic: PDB statistics
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Raji, the numbers you have accumulated might already be accurate, it is more a question of how precise your numbers should be - and this might become a long discussion without resulting in a single answer: Take into account that the PDB query relies on the entries in the PDB headers. And take into account that it might not be clear e.g. what is a MR solution: If I solved a structure with a new ligand and get the phases from refining the new data against the protein without ligand, you might call this MR, although personally I think of the search for the correct placement in the unit cell when I speak of MR, so in this case it is not really MR. Other people will have different opinion about this and so forth. Best, Tim On 04/15/2013 03:48 PM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRbAkAUxlJ7aRr7hoRAujtAKCVnnbGN2Y/15+ZGydTXciFWqGe0wCeM9FL bc9b/l3Hkqz2uLHIJhrTSz4= =hm6F -END PGP SIGNATURE-
Re: [ccp4bb] Off-topic: PDB statistics
Structures of a protein-ligand complex are often determined without the need of running a molecular replacement program: a difference Fourier is generated to locate the ligand, usually after a few rounds of rigid bodies and / or atom positional refinement.. They may account for many of the 19,000 unaccounted structures. There is not a full consensus on what Molecular Replacement mean. Some consider a structure determined by simple Fourier difference maps as a case of Molecular Replacement where the solution is identity (or close to identity). Others (like me) think the terms of solved by Molecular Replacement should be reserved to cases where the non-isomorphism between the original structure and the one to solve is beyond the radius of convergence of refinement and reaching the solution requires the use of a Molecular Replacement program (precisely). The point is that it's unclear whether you can truly get the statistics you want if there is no universal definition for the terms used. Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji Edayathumangalam Sent: Monday, April 15, 2013 9:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic: PDB statistics Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Off-topic: PDB statistics
From my own db program: Number of entries in histogram: 711 Total number of instances : 78467 0 48249 0.6149 MOLECULAR REPLACEMENT 1 8557 0.1091 NULL 2 5632 0.0718 SAD 3 5128 0.0654 MAD 4 3600 0.0459 FOURIER SYNTHESIS 5 1762 0.0225 OTHER 6 1171 0.0149 MIR 7 511 0.0065 SIRAS 8 505 0.0064 DIFFERENCE FOURIER 9 392 0.0050 MIRAS 10 229 0.0029 AB INITIO 11 226 0.0029 MR 12 151 0.0019 RIGID BODY REFINEMENT 13 146 0.0019 ISOMORPHOUS REPLACEMENT 14 110 0.0014 AB INITIO PHASING 15 109 0.0014 MULTIPLE ISOMORPHOUS 1683 0.0011 N/A 1775 0.0010 SIR 1870 0.0009 RIGID BODY 1964 0.0008 DIRECT METHODS 2050 0.0006 RE-REFINEMENT USING 2137 0.0005 DIFFERENCE FOURIER PLUS 2236 0.0005 ISOMORPHOUS 2334 0.0004 REFINEMENT 2430 0.0004 MOLREP 2526 0.0003 SE-MET MAD PHASING 2625 0.0003 RIGID-BODY REFINEMENT 2724 0.0003 ISOMORPHOUS METHOD etc It's a very heterogeneous field, that REMARK 3 field, and the ones above are the most dominant entries (note the 8,557 that are NULL that are in fact crystal structures). At least in some versions of ADIT the guidance that RCSB gives about this field is very weak, which accounts for the variation. I'm interested in what ab initio phasing really means, but I've been too lazy to mine the actual entries for details. Phil Jeffrey Princeton On 4/15/13 9:48 AM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Off-topic: PDB statistics
Thanks to everyone who responded. I appreciate your pointing out the caveats of such statistics mining expeditions. I remember (with no accuracy or precision) sitting in a lecture by Wayne Hendrickson years ago and watching him cringe at the notion of considering the simple case of difference Fourier (say, isomorphous protein and protein+ligand structures) as Molecular Replacement. Like you point out, Thierry, I believe he referred to these cases as Molecular Substitutions. And like you said, there's no consensus with the nomenclature. Phil, thanks much for the stats. Somewhat reassuring that they are somewhat similar to what I got from the PDB. Thanks to everyone! Raji On Mon, Apr 15, 2013 at 11:46 AM, Phil Jeffrey pjeff...@princeton.eduwrote: From my own db program: Number of entries in histogram: 711 Total number of instances : 78467 0 48249 0.6149 MOLECULAR REPLACEMENT 1 8557 0.1091 NULL 2 5632 0.0718 SAD 3 5128 0.0654 MAD 4 3600 0.0459 FOURIER SYNTHESIS 5 1762 0.0225 OTHER 6 1171 0.0149 MIR 7 511 0.0065 SIRAS 8 505 0.0064 DIFFERENCE FOURIER 9 392 0.0050 MIRAS 10 229 0.0029 AB INITIO 11 226 0.0029 MR 12 151 0.0019 RIGID BODY REFINEMENT 13 146 0.0019 ISOMORPHOUS REPLACEMENT 14 110 0.0014 AB INITIO PHASING 15 109 0.0014 MULTIPLE ISOMORPHOUS 1683 0.0011 N/A 1775 0.0010 SIR 1870 0.0009 RIGID BODY 1964 0.0008 DIRECT METHODS 2050 0.0006 RE-REFINEMENT USING 2137 0.0005 DIFFERENCE FOURIER PLUS 2236 0.0005 ISOMORPHOUS 2334 0.0004 REFINEMENT 2430 0.0004 MOLREP 2526 0.0003 SE-MET MAD PHASING 2625 0.0003 RIGID-BODY REFINEMENT 2724 0.0003 ISOMORPHOUS METHOD etc It's a very heterogeneous field, that REMARK 3 field, and the ones above are the most dominant entries (note the 8,557 that are NULL that are in fact crystal structures). At least in some versions of ADIT the guidance that RCSB gives about this field is very weak, which accounts for the variation. I'm interested in what ab initio phasing really means, but I've been too lazy to mine the actual entries for details. Phil Jeffrey Princeton On 4/15/13 9:48 AM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Puzzling Structure
Just to round up this topic, a bug report was submitted to PDBe concerning entry 2wlv and the PDB has how responded acknowledging the problem. An updated entry will be available in one week. As pointed out by Savvas, it is very likely the CRYST1 record was manually edited prior to deposition resulting in the wrong spacegroup being parsed in by AutoDep and subsequent processing moving the waters around in the wrong space group. Since there is no logical reason for the authors to do this, it was probably inadvertent. I imagine somebody accidentally deleted a space between P 21 21 2 and 18 and tried to fix it by adding it back, between 1 and 8. /Michel -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. Berry Sent: April-14-13 9:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling Structure Robbie Joosten wrote: Hi Martyn, I think the question is where the error was made - seeing the uploaded file would clear this up. But it seems unlikely to me that the depositor saw a huge R factor discrepancy at the end of refinement and just blithely uploaded it. So scenario 3 :- PDB : we cannot reproduce your R factor with our programs Depositor : that's your problem mate - it was fine when it left me...up to you to sort it... Which seems a sort of reasonable attitude to me. Not quite, the depositor has to give, i.e. type, the space group (example depositions: https://www.ebi.ac.uk/pdbe- xdep/autodep/AutoDep?param=QovCsvhNv06Mpnr%2BvIkqqfuqeeBd8leFN AVymZgS%2Fe7mULyfrCaTMN8jsyaGZUTyUDQyN3gMF3o%3D). Don't ask me why, because it is clearly a source of error. In my experience with RCSB autodep2, assuming the information was in the uploaded pdb file, this information is already pre-filled and the depositor just glances over to see it is correct. A missing or extra (1) might not be noticed. So perhaps it is a parsing error, perhaps related to the different ways the space group is represented on the CRYST1 card, and the different stringency of different programs in interpreting the CRYST1 card. But the validation report is included with the approval request letter, and major discrepancies are noted in the text of the validation letter in case the depositor is too busy to actually read the validation report. So if the depositor read more than the first line or two of the letter he should have known there was a problem. Then there is the 2-week default release policy: No major issues were raised during data processing. A summary of some of the key annotations in your entry is shown below. Please verify that these are correct. If we do not hear from you within two weeks, we will consider this entry to have been approved by you. The entry will then be released according to the release/hold instructions you have provided. If this 2-week default release is the rule even when there are issues, the depositor may have put it aside to find the problem when time was available, and waited too long and let the default release kick in. eab
Re: [ccp4bb] Puzzling Structure
On 15 Apr 2013, at 17:19, Michel Fodje michel.fo...@lightsource.ca wrote: I imagine somebody accidentally deleted a space between P 21 21 2 and 18 and tried to fix it by adding it back, between 1 and 8. As this has now been mentioned twice in this discussion it should probably be noted for the archives that the last number in the CRYST1 record is not the space group number... See http://deposit.rcsb.org/adit/docs/pdb_atom_format.html#CRYST1 for details Huw
Re: [ccp4bb] salt or not?
I may be biased, but the only way to really be sure is to shoot them. If you see no spots at all, be sure to do a wide oscillation (rotation during the exposure) shot as well. It is not unlikely for a salt crystal to be oriented so that no relps are on the Ewald sphere, giving no spots. But, if you sweep through 180 degrees during the exposure you will at least have a nice, pretty symmetric diffraction pattern to look at for a moment before the disappointment sets in. -James Holton MAD Scientist On 4/15/2013 3:18 AM, Careina Edgooms wrote: Dear ccp4 I have been performing trials on a protein DNA complex for a while now and have not seen any crystals form. Today I checked an old plate (over a month old) and I see 4 large crystals. *excitement* Three of them look tetragonal in shape (like a pyramid) and one of them looks hexagonal. I do not know if they are salt or protein. There is calcium chloride in the buffer. They feel quite soft to touch. They do not cause much birefringence. One of them does not seem to absorb much izit. It did go a bit blue but not entirely. How can I tell if this crystal is protein or not? Do you think its worth trying to see how it diffracts? Also, does Izit affect diffraction/ protein structures at all? Could I use a crystal with Izit in a diffraction experiment and ultimately to get the structure? Best Careina
Re: [ccp4bb] salt or not?
Protein-DNA complex crystal with channels too small for the dye is *extremely* unlikely, imho. Original message From: Ulrike Demmer ulrike.dem...@biophys.mpg.de Date: 04/15/2013 8:48 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] salt or not? Dear Careina, altough your crystals does't take up the Izit dye it sounds promising. The uptake of Izit depends on the solvent channels of the protein molecule - sometimes the dye just can't enter the molecule. Concerning the Calciumchloride - if the concentration is not too high and without other ingredients which could cause less solube salt there is the possiblity that you have got protein crystals. I once had a condition whith 34 % MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting crystals To speak from my experience I think 1 month after setting up the trays the drops should not be dried out. If the wells are sealed properly new crytals can appear even after 1 year. You should definately check the diffraction then you will know for sure. Cheers, Ulrike
Re: [ccp4bb] Off-topic: PDB statistics
On Mon, 15 Apr 2013, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? There is no way to tell the PDB I already had phases for this structure because it's just a soak or co-crystallization of a new ligand in a well-studied crystal form. Sometimes those are labeled as MR sometimes isomorphous sometimes some other string no label at all. I have tried annotating such depositions as known structure but it gets changed to something else. Anyhow I could easily believe there are 19000 of these. Ethan Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Off-topic: PDB statistics
The only way to really do creative stats on the PDB is to just download the whole thing. It is a sobering thought to realize just how tiny it is! Less than 17 GB. Once you've got it all on your hard disk you can start writing little programs to look for different things. I have posted mine for doing the method count here: http://bl831.als.lbl.gov/~jamesh/pickup/pdb_method_count_notes.txt For those of you who don't speak awk, basically what I do is first look at the method used to determine the structure entry, but when that is NULL or otherwise uninformative you can do a number of things. If the entry lists another PDB entry in methods, it is probably a molecular replacement solution. Also, if the software used to solve it was PHASER or MOLREP, or ARP/wARP, or COMO etc., then I'm willing to bet it is an MR solution too. On the other hand, if the software was SOLVE or AUTOSHARP, then I imagine they probably were doing MAD/SAD. Even if they didn't know it. Currently, the script produces this list: 1 COCRYSTAL 1 FIBER-DIFFRACTION 1 MOLPREP 1 N? 1 P 1 SEE CITATION 1 UNCONVENTIANAL 1 UNNECESSARY 2 UNCONVENTIONAL 7 RIP 54 N/A 92 SIR 260 AI 355 MIRAS 434 SIRAS 689 OTHER 1058 MIR 5012 MAD 5335 SAD 8021 NULL 58293 MR Clearly, there's a few new ones I need to clean up, but some of them are funny. What is the method of COCRYSTAL anyway? Co-crystallized with a ligand? Or a heavy atom? There are plenty of entries that I can't figure out automatically (NULL + OTHER), but I imagine most of them are MR. But yes, Tim and Thierry are right, there is definitely confusion and disagreement about what exactly constitutes molecular replacement. I have been somewhat draconian here and lumped MR and direct refinement together because frankly it is just too hard to tell them apart from the PDB alone. In fact, there are 260 PDB entries that claim they were solved by AB INITIO methods, but did they really use direct methods with no prior phase information at all? Or did they do MAD/SAD and thought that meant ab initio? Probably examples of all. The resolution of the structure might be helpful in this case, but sometimes even reading the paper doesn't help. I do agree though that the use of molecular replacement should be true to the term as Rossmann coined it, where the 6D search of a model against the new dataset was actually required to solve the structure. Lots of people run molrep (or molprep!?) for each and every dataset, even when they are doing ligand soaks. Never really have understood why. However, I'm sure the day is not far off when phenix.refine or the like will check if the starting R factor is too high and just automatically invoke a run of MR to see if something clicks. Maybe even trying alternative space groups, just in case you screwed that up too. There may also soon be automaitc runs of BALBES based on the sequence information of the input file. Eventually, the difference between different methods gets muddied. Then your average depositor will be even more unsure about what to put into REMARK 200, and people like me will be endlessly asking for command-line options that turn these features off. -James Holton MAD Scientist On 4/15/2013 6:48 AM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Off-topic: PDB statistics
On Mon, Apr 15, 2013 at 11:47 AM, James Holton jmhol...@lbl.gov wrote: However, I'm sure the day is not far off when phenix.refine or the like will check if the starting R factor is too high and just automatically invoke a run of MR to see if something clicks. I think the latest Phaser code actually does the reverse: if the R-factor is already relatively low, it just outputs the search model. The more problematic (and very common) situation is where the structures are slightly isomorphous and rigid-body plus restrained refinement alone could work, but MR might work better - I don't think anyone has comprehensively evaluated this. We usually just run Phaser because compared to rebuilding and refinement, it's simply not that much of a bottleneck. -Nat
[ccp4bb] New Opening at Microlytic: Technical Key Account Manager USA
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[ccp4bb] Post-doc positions
Two post-docs positions are available in St Andrews, start date this summer. The project is the structural and functional characterisation of membrane proteins involved in sugar lipid polymer synthesis and transport in bacteria. It is funded by the Wellcome Trust. The ideal candidates have transformative powers, e.g. purifying proteins in a morning and crystallising proteins simply by looking; basically all round superstars. Failing that, I am looking for experience in protein chemistry especially membrane proteins. Industrial experience is as valid as academic. Strong preference will be given to successful experience with membrane protein biochemistry. You must have submitted a PhD before you can start work. A technician will assist these two positions. St Andrews is is located in Scotland and is an equal opportunity slave driver. Full details are available on the unbelievably expensive and cumbersome University job site. (Its main role seems to me is to act as a filter in discouraging applicants) https://www.vacancies.st-andrews.ac.uk/ The reference you need is Research Fellows - ME1326, it is listed under the school of chemistry. Sorry, but you do need to apply through this Byzantine system. Informal contacts are most welcome, email me naism...@st-andrews.ac.uk (subject post-doc). best Jim Jim Naismith BSRC, North Haugh j...@st-andrews.ac.uk The University of St Andrews is a charity registered in Scotland : No SC013532k Google scholar is free and tracks your outputs, mine are http://scholar.google.co.uk/citations?hl=enuser=fLmKKQMJ
