[ccp4bb] Off-topic: NMR and crystallography
Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of off-board, to allow for a good discussion. Thank you. Theresa
Re: [ccp4bb] Off-topic: NMR and crystallography
Well, if you do NMR you avoid the possible bottlenecks of having to obtain well-diffracting crystals, and having to phase the protein (i.e. obtain SeMet protein crystals or suitable heavy atom derivatives; or a suitable MR model). But instead, you'll need to prepare labelled protein (15N and/or 13C), which is expensive and for which your protein needs to be able to be expressed in minimal medium, and your protein will need to be very soluble, monodisperse (in general monomeric) and stable in a minimal NMR-compatible buffer for data collections lasting for hours. Assigning all the protons and calculating the final structure can also be months of work, while a high-resolution crystal structure can be finished in days, if the above-mentioned bottle-necks can be overcome. On 9 Jun 2013, at 17:36, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of off-board, to allow for a good discussion. Thank you. Theresa
Re: [ccp4bb] Off-topic: NMR and crystallography
Just to add on that, regarding membrane proteins: I noticed some recent publications on solid-state NMR for membrane proteins, not sure about solution NMR for this class. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mark van Raaij [mjvanra...@cnb.csic.es] Sent: Sunday, June 09, 2013 8:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off-topic: NMR and crystallography Well, if you do NMR you avoid the possible bottlenecks of having to obtain well-diffracting crystals, and having to phase the protein (i.e. obtain SeMet protein crystals or suitable heavy atom derivatives; or a suitable MR model). But instead, you'll need to prepare labelled protein (15N and/or 13C), which is expensive and for which your protein needs to be able to be expressed in minimal medium, and your protein will need to be very soluble, monodisperse (in general monomeric) and stable in a minimal NMR-compatible buffer for data collections lasting for hours. Assigning all the protons and calculating the final structure can also be months of work, while a high-resolution crystal structure can be finished in days, if the above-mentioned bottle-necks can be overcome. On 9 Jun 2013, at 17:36, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of off-board, to allow for a good discussion. Thank you. Theresa
Re: [ccp4bb] Off-topic: NMR and crystallography
I would agree with Mark. It would be also good to state that neither of us is cross-trained, but are one-trick dogs as far as NMR vs X-rays goes. Still, I think that if you get any protein in good amounts, try to crystallize it (there are even good facilities for that these days, and funding to set up crystallization through Biostruct-X), and if that fails, consider NMR. Many good NMR labs try to crystallize their targets first - if it crystallizes its cheaper, easier and the structure is of higher resolution. Disclaimer: I am not saying NMR is useless - quite the contrary. But, I cant see why you would do an NMR structure of something that crystallizes in a straightforward cheap crystallization screen. I see why you might do NMR to answer many different questions. I will leave the membrane proteins question to experts - but these days we all see many membrane proteins coming from X-rays and as far as NMR goes you will be on the solid state regime and not solution as far as I understand: great promise, but again I suspect the impact to be different than 'structure solution'. A. On 9 Jun 2013, at 19:33, Mark van Raaij wrote: Well, if you do NMR you avoid the possible bottlenecks of having to obtain well-diffracting crystals, and having to phase the protein (i.e. obtain SeMet protein crystals or suitable heavy atom derivatives; or a suitable MR model). But instead, you'll need to prepare labelled protein (15N and/or 13C), which is expensive and for which your protein needs to be able to be expressed in minimal medium, and your protein will need to be very soluble, monodisperse (in general monomeric) and stable in a minimal NMR-compatible buffer for data collections lasting for hours. Assigning all the protons and calculating the final structure can also be months of work, while a high-resolution crystal structure can be finished in days, if the above-mentioned bottle-necks can be overcome. On 9 Jun 2013, at 17:36, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? I would appreciate replies to the board, instead of off-board, to allow for a good discussion. Thank you. Theresa
Re: [ccp4bb] Off-topic: NMR and crystallography
On Sunday, 09 June 2013, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? A relevant study is the comparison by Yee et al (2005) JACS 127:16512. http://pubs.acs.org/doi/abs/10.1021/ja053565+ They tried to solve 263 small proteins using both NMR and crystallography. 43 only worked for NMR 43 only worked for X-ray 21 could be solved either way So you could say it was a toss-up, but consider that - As the size gets larger, NMR becomes increasingly impractical - 156 (60%) weren't solved by either NMR or crystallography. What is the relative cost of the failed attempt? Ethan
Re: [ccp4bb] Off-topic: NMR and crystallography
Just to add some more information on the issue ... Take also a look at the following article: Does NMR mean not for molecular replacement? Using NMR-based search models to solve protein crystal structures Chen YW, Dodson EJ, Kleywegt GJ. Structure (2000) 8, 213-220 Martin _ Dr. Martin Martinez-Ripoll Research Professor xmar...@iqfr.csic.es Department of Crystallography Structural Biology www.xtal.iqfr.csic.es Telf.: +34 917459550 Consejo Superior de Investigaciones Científicas Spanish National Research Council -Mensaje original- De: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] En nombre de Ethan Merritt Enviado el: domingo, 09 de junio de 2013 20:13 Para: CCP4BB@JISCMAIL.AC.UK Asunto: Re: [ccp4bb] Off-topic: NMR and crystallography On Sunday, 09 June 2013, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? A relevant study is the comparison by Yee et al (2005) JACS 127:16512. http://pubs.acs.org/doi/abs/10.1021/ja053565+ They tried to solve 263 small proteins using both NMR and crystallography. 43 only worked for NMR 43 only worked for X-ray 21 could be solved either way So you could say it was a toss-up, but consider that - As the size gets larger, NMR becomes increasingly impractical - 156 (60%) weren't solved by either NMR or crystallography. What is the relative cost of the failed attempt? Ethan
Re: [ccp4bb] Off-topic: NMR and crystallography
Hi Theresa, Per your question about determination of membrane proteins - solution NMR is quite capable of delivering structures of proteins in the presence of detergents, such as the KcsA channel (see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242490/). You should note, though, that many membrane proteins are helical, and these proteins are notoriously difficult to determine via NMR (see this article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2862971/). Even so, if it won't crystallize, and you want it really bad, then go for it! Chen
Re: [ccp4bb] Off-topic: NMR and crystallography
The title of my PhD thesis was NMR of inorganic hydrides but I soon realized that I was out of my depth with the theory so switched to something easier to understand (gas phase electron diffraction). However this involved taking the (somewhat dangerous) samples by train to Durward Cruickshank's machine in Glasgow (later Manchester). Then I managed to find a Weissenberg camera so I was able to do some crystallography. Two points that don't seem to have been mentioned yet: State of the art In-house NMR equipment is an order of magnitude more expensive than in-house X-ray equipment (but of course if you need a synchrotron the real costs might be different). Many NMR structures are more modelled than experimentally determined, the number of independent experimental data can be quite small. But the good news is that force fields and modelling software are improving. George On 06/09/2013 08:12 PM, Ethan Merritt wrote: On Sunday, 09 June 2013, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? A relevant study is the comparison by Yee et al (2005) JACS 127:16512. http://pubs.acs.org/doi/abs/10.1021/ja053565+ They tried to solve 263 small proteins using both NMR and crystallography. 43 only worked for NMR 43 only worked for X-ray 21 could be solved either way So you could say it was a toss-up, but consider that - As the size gets larger, NMR becomes increasingly impractical - 156 (60%) weren't solved by either NMR or crystallography. What is the relative cost of the failed attempt? Ethan -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
[ccp4bb] Beamtime @ SLS
=== SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY AT SLS === Proposal application deadline: Saturday, June 15, 2013 Periods: September 1, 2013 - December 31, 2013 (Normal / Test proposals) September 1, 2013 - August 30, 2015 (Long-term proposals) Proposal submission: http://www.psi.ch/sls/px-beamlines-call-for-proposals Travel support: http://www.psi.ch/useroffice/sls-elisa-biostruct PSI DUO application for iphone: http://itunes.apple.com/ch/app/psi-duo/id375328818?mt=8 What's New? - SLS PX software (DA+) in all three beamlines - X06SA New diffractometer with on-axis microscope and fast camera - X06DA Multi-axis goniometer (PRIGO) - X06DA In-situ X-ray diffraction screening for any SBS format plate - X10SA Micro-beam with apertures (10 x 10, 30 x 30 micron^2) - Faster Sample changer (CATS) operation X06SA Beamline features (http://www.psi.ch/sls/pxi/pxi) - Undulator beamline with flux of 2x10^12 photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 17.5 keV (2.07 - 0.71 Å) - Focused beam size and divergency: HRD - 85x10 microns and 0.35x0.06 mrad; MD2 - 25x5 microns and 0.5x0.4 mrad - PILATUS 6M pixel detector at the High Resolution Diffractometer, allowing continuous, fine phi-sliced data acquisition (25 frames per second) with 20 bit dynamic range (see http://pilatus.web.psi.ch/ or www.dectris.com for further information) - MAR225 CCD at Micro-Diffractometer MD2, allowing data collection with a focussed beam size of 25 x 5 micrometers, and smaller beam size with triple-aperture assembly ( 5 x 5, 10 x 10, 20 x 20 micrometer). X06DA Beamline features (http://www.psi.ch/sls/pxiii/pxiii) - Super-bending magnet beamline with flux of 5x10¹¹ photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 17.5 keV (2.07 - 0.71 Å) - Focused beam size and divergency: 80x45 microns and 2x0.5 mrad (with possibility to reduce horizontal divergency to 0.4 mrad) - Mini-hutch design for fast manual mounting - PILATUS 2M (60 Hz, 450 um Si sensor) - Multi-axis goniometer (PRIGO) for crystal re-orientation - New phasing protocols with energy interleaving and multi-orientation strategy - In-situ X-ray diffraction screening (with any SBS format plate) available during users shifts (R. Bingel-Erlenmeyer, et al., Crystal Growth Design 2011, 11, 916) X10SA Beamline features (http://www.psi.ch/sls/pxii/pxii) - Undulator beamline with flux of 2x10^12 photons/sec at 12.4 keV (1Å) and fully tunable from 6.0 to 20 keV (2.07 - 0.62 Å) - Focused beam size and divergency: 50x10 microns and 0.6x0.1 mrad - Micro-beam with apertures (10 x 10, 30 x 30 micrometer) - PILATUS 6M pixel detector Best regards, The MX group at SLS __ Meitian Wang Swiss Light Source at Paul Scherrer Institut CH-5232 Villigen PSI - http://www.psi.ch/sls/ Phone: +41 56 310 4175
Re: [ccp4bb] Off-topic: NMR and crystallography
Many NMR structures are more modelled than experimentally determined, the number of independent experimental data can be quite small. But the good news is that force fields and modelling software are improving. George The quality of NMR structures/models depend heavily on the number of experimental NOE distance restraints per residue. Naturally, for inner core, stable regions of the protein this number is quite high (10 NOE or more per residue) but for flexible regions it's (very) low with NOEs possibly missing altogether in such regions. The model derived from NMR data suffer from similar problems that the crystal structures do. Crystal structures yield good looking density, low B's in ordered regions, bad looking density, sometime even missing density altogether and very high B in flexible regions. The NMR equivalent would be good superposition of the models in ordered regions and bad superposition (shown as a pile of noodle in NMR models) in flexible regions. This is also expressed in the rmsd column of PDBs from NMR models. As for the 'resolution' of NMR models - this is a debatable question. Some NMR people claim that a good NMR structure with 10 NOE/residue is equivalent to ~2A resolution structure. Maybe. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 On 06/09/2013 08:12 PM, Ethan Merritt wrote: On Sunday, 09 June 2013, Theresa Hsu wrote: Dear all A question for the cross-trained members of this forum - for small sized proteins, is NMR better than crystallography in terms of data collection (having crystals in the first place) and data processing? How about membrane proteins? A relevant study is the comparison by Yee et al (2005) JACS 127:16512. http://pubs.acs.org/doi/abs/10.1021/ja053565+ They tried to solve 263 small proteins using both NMR and crystallography. 43 only worked for NMR 43 only worked for X-ray 21 could be solved either way So you could say it was a toss-up, but consider that - As the size gets larger, NMR becomes increasingly impractical - 156 (60%) weren't solved by either NMR or crystallography. What is the relative cost of the failed attempt? Ethan -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
