Re: [ccp4bb] nmr question
Structure calculation of biomolecules by NMR relies on unambiguous assignment of all protons in the biomolecule so as to get enough inter-proton distance contraints (from NOEs). Two protons that are in close proximity (less than 5 ang) through space will have a dipolar interaction that is manifested as the NOE (Nuclear Overhauser Effect). More distance constraints from NOEs - better the structure. This is the traditional approach. As you go to larger macromolecules, there are two main problems - first the number of protons in the molecule increases resulting in spectral overlap - so there is a problem of resolution - being able to resolve two proton frequencies that lie very close to each other or may overlap perfectly. The problem of resolution can be overcome by isotopic labeling (incorporating 15N, 13C, specific labeling of amino acids), going to higher field strengths of NMR magnets for data collection, and greater dimensionality of spectra (3D and 4D data are now routine). The main problem however is that of rotational correlation time which increases with molecular weight. Larger molecules tumble more slowly, thereby increasing the correlation time and decreasing the relaxation rates of nuclei, especially protons. The relaxation time constant T2, is inversely proportional to the apparant linewidth of NMR resonances - larger the linewidth, more the degeneracy in the spectra. In addition, as the T1 and T2 times get shorter, there is less magnetization left at the end of a 3D experiment for detection, since most of the magnetization has already decayed during the experiment. There are some NOE-independent methods (such as the use of residual dipolar couplings) or recent hybrid methods that rely on chemical shifts to overcome this problem. Perdeuteration also helps to increase the relaxation times. Currently 25-30 kDa is the upperlimit for structure calculation by NMR, although there are a few NMR structures solved for very large proteins or complexes where the spectral dispersion is excellent and they are stable enough that one can raise the temperature to increase the correlation time. These structures also relied heavily on NOE-independent approaches. The power of NMR is of course is in studying dynamics, and NMR has been used to study motions in some large systems (the 670 kDa proteasome is one example) where it was possible to get sequence specific assignments for a region in the protein. Hope that helps - Roopa ** Roopa Thapar Research Scientist Hauptman-Woodward Institute Assistant Professor, SUNY, Buffalo 700 Ellicott Street Buffalo, NY 14203 Tel: 716-898-8687 (Office); 716-898-8659 (Lab) * From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem Evdokimov [artem.evdoki...@gmail.com] Sent: Thursday, June 30, 2011 11:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] nmr question There are about 20 structures solved by NMR with chain lengths above 300 residues. Some of them are solved by combination of restraints and modeling (e.g. 2010 Nature paper describing implementation of Rosetta modeling coupled with backbone-only data, by Raman/Montelione/Baker et al.). Principal problems from the perspective of a non-specialist are: a) separation between resonances - complete (multidimensional!) overlaps are ruinous for assignment b) intensity of each resonance - for larger molecules there is less moles of resonating nuclei c) relaxation issues in large proteins (the need for expensive and hard to do deuterium labeling is the result of this) I am sure that true NMR experts would have much more to say on the subject :) Artem On Thu, Jun 30, 2011 at 5:20 PM, Lena Griese lena.gri...@googlemail.commailto:lena.gri...@googlemail.com wrote: Dear members, I know that it is not possible to solve a structure by nmr of more than approx. 30 kda. But I have to admit that I dont know why. What exactly is overlapping? With best regards, Lena
Re: [ccp4bb] generate large symmetry model
Thank you to all for the help. There’s obviously more than one way to skin a cat! In the end pdbcur did the job for me. Great! Dave David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS 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 martyn.w...@stfc.ac.uk Sent: 30 June 2011 15:08 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] generate large symmetry model As well as pdbset, you can use pdbcur. A combination of keywords genunit, symop and symcommit. I’d probably use genunit (applicable to whatever spacegroup you have), followed by a second call using symop/symcommit to apply unit cell translations. Cheers Martyn From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hargreaves, David Sent: 30 June 2011 13:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] generate large symmetry model Does anyone have a rigorous method (or script) for generating an extended lattice e.g 3x3x3 unit cells from any pdb file? Any help gratefully received, Dave David Hargreaves Associate Principal Scientist _ AstraZeneca DECS, CPSS 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 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. -- 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.
[ccp4bb] Measuring dimensions of channels
Dear, Does anybody know a conventional method/program for measuring the dimensions (e.g. in Å), not the volume, of solvent channels in metal organic frameworks (MOF's)..? Thank you very much Kristof --- Kristof Van Hecke, PhD Biomolecular Architecture Celestijnenlaan 200F 3001 Heverlee (Leuven) Belgium ---
Re: [ccp4bb] Measuring dimensions of channels
Hello Kristof, you could place dummy atoms with coot where you think the channel ends and measure the distance between the dummy atoms - that's probably as accurate as any automated tool because it takes you (human) experience into account. Tim On Fri, Jul 01, 2011 at 12:12:21PM +0200, Kristof Van Hecke wrote: Dear, Does anybody know a conventional method/program for measuring the dimensions (e.g. in Å), not the volume, of solvent channels in metal organic frameworks (MOF's)..? Thank you very much Kristof --- Kristof Van Hecke, PhD Biomolecular Architecture Celestijnenlaan 200F 3001 Heverlee (Leuven) Belgium --- -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A pgpzfl9qbtCey.pgp Description: PGP signature
Re: [ccp4bb] nmr question
...they are stable enough that one can raise the temperature to increase the correlation time. I wonder whether NMR people have tried using hyperthermophile proteins to be able to essentially boil the sample to decrease the rotational correlation time constant? There is even an organism I read about in a Science brevia paper whose peak doubling time is at autoclave temperatures... JPK
Re: [ccp4bb] nmr question
'NMR people' do use thermophilic proteins but I have never heard of anyone boiling samples :) By increasing temperature I meant going to around 37-40 deg C. On 7/1/11 12:26 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: ...they are stable enough that one can raise the temperature to increase the correlation time. I wonder whether NMR people have tried using hyperthermophile proteins to be able to essentially boil the sample to decrease the rotational correlation time constant? There is even an organism I read about in a Science brevia paper whose peak doubling time is at autoclave temperatures... JPK
Re: [ccp4bb] nmr question
NMR people do collect NMR data beyond 40 deg C. Many membrane protein structures are determined at 55-60 deg .C (the membrane proteins may not be very large but in micelles -combined weight is large). When the protein is stable and the 1H-15N-HSQC (fingerprint region) does not change with temp (meaning temp has no effect on the protein), one can go higher temp. Yes, it certainly helps reducing line width and if you combine TROSY based experiments, the resolution increases dramatically. Smita Mohanty Roopa Thapar rtha...@hwi.buffalo.edu 7/1/2011 11:47 AM ‘NMR people’ do use thermophilic proteins but I have never heard of anyone boiling samples :) By increasing temperature I meant going to around 37-40 deg C. On 7/1/11 12:26 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: ...they are stable enough that one can raise the temperature to increase the correlation time. I wonder whether NMR people have tried using hyperthermophile proteins to be able to essentially boil the sample to decrease the rotational correlation time constant? There is even an organism I read about in a Science brevia paper whose peak doubling time is at autoclave temperatures... JPK
