View source for NMR Ensembles of Models

==Caveat==

I wrote the initial content for this page based on discussions I've had with NMR spectroscopists and crystallographers. Be warned that I am far from an expert, and have never done either NMR spectroscopy nor crystallography. Until this page is vetted by an expert, its content should be considered provisional, not authoritative! [[User:Eric Martz|Eric Martz]] 01:25, 26 June 2008 (IDT)

==Structure Determination by NMR==

About 14% of the entries in the [[Protein Data Bank]] were determined by nuclear magnetic resonance in solution (NMR) as of mid-2008. 85% were determined by X-ray crystallography, and <1% by other methods. NMR can only be used for relatively small macromolecules (see [[#Median_Size_of_Published_NMR_Structures|below]]).

NMR measurements yield a set of interatomic distances, called "restraints". Model building for NMR experiments typically starts with the complete protein or nucleic acid chain, including [[Hydrogen atoms]]. The distance restraints are then applied. The resulting model usually includes the entire protein and nucleic acid chains, unlike [[X-ray crystallography|X-ray crystallographic]] models that often lack the ends, and even loops in the middle of chains, due to  [[Disorder in protein crystals]].

Macromolecular structure determination by NMR is done in aqueous solution, and thus requires that the molecule be soluble. For more information, see ''Nature of 3D Structural Data''<ref>[http://www.pdb.org/pdb/static.do?p=general_information/about_pdb/nature_of_3d_structural_data.html Nature of 3D Structural Data]</ref> and ''NMR in Wikipedia''<ref>[http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance NMR in Wikipedia]</ref>.

==Displaying NMR Models==

===Display of NMR Models by Proteopedia===
{{STRUCTURE_1lcd|  PDB=1lcd  |  SCENE=  }}
Proteopedia shows '''all the models''' in ensembles of models from NMR experiments. This enables you to see where the models agree with each other, and where they differ. Each model is shown as a thin backbone trace (a line connecting alpha carbon atoms of amino acids, or phosphorus atoms in DNA or RNA chains). The backbone traces are colored by <font color="blue">'''Amino'''</font> to  <font color="red">'''Carboxy'''</font> "rainbow", a spectral sequence of colors starting at the amino terminus (or 5' terminus of nucleic acid chains) and ending at the carboxy terminus (or 3' terminus).

{{ColorKey N2CRainbow}}

'''Ligands''' ([[Hetero atoms]]) are also shown for all models, except that they are opaque only for model 1, and translucent for all other models. Ligand atoms are colored by element, using the [[CPK color scheme]]. Examples with hetero groups covalently linked to chain termini, with extremely variable positions, are [[1jsa]] and [[1dqc]]. [[1bah]] also has hetero groups in variable positions. [[1hpn]] has only hetero atoms.

The example at right shows the 3 models for [[1lcd]], a lac repressor domain bound to DNA, with one sodium ion. '''Water''' is present in this model, but for clarity, Proteopedia does not show water in its initial scene. <scene name='NMR_Ensembles_of_Models/Water/1'>Show water</scene>. (To hide water, click the ''initial scene'' green link just below the molecule.)

'''Disulfide bonds''' are shown as yellow rods connecting backbones, with the first model opaque, and all other models translucent. An example is [[1iw4]].

===Individual Models===

In order to view individual models, click on ''Jmol'' (lower right corner below the molecule) to '''open Jmol's menu'''. There, use the '''All N models''' item (where N is the total number of models in the ensemble). For example, clicking on 1.1: 1 will display only model 1, and the menu will now say ''model 1/N''. You can also use Jmol's menu to change the rendering and coloring.

===Animating NMR Ensembles===

When the models in an NMR ensemble are played like a movie, the resulting animation simulates thermal motion (although not all the motions is necessarily real -- see [[#Meaning_of_the_Variation_Between_Models|below]]). In order to animate the models, click on ''Jmol'' (lower right corner below the molecule) to '''open Jmol's menu'''. Choose '''Animation''', then '''Animation mode''', and click on '''Loop'''. Then choose ''Animation'' again, and click '''Play'''. You can change the speed of the animation with '''FPS''' (frames per second) on the ''Animation'' menu.  By default, there is a delay at the first and last models.

==Multiple Model Ensembles from NMR==
===NMR Experiments Yield Multiple Models===

When a macromolecular structure is determined by nuclear magnetic resonance (NMR) in solution, the result is an '''ensemble of multiple molecular models''', each of which is consistent with the experimental data. The results of an NMR experiment are a large number of inter-atomic distance restraints, which are consistent with multiple models. This is in contrast to the result of an X-ray crystallographic experiment, which is a single model that best fits the empirical electron density. (In some cases where the resolution is very high, the model may include alternative positions for some atoms.)

The number of NMR models published depends upon the experiment and is up to the authors, and varies between 2 and over 100. The first model in the ensemble has no special significance (see [[#The Most Representative Model|the most representative model]]).

===Meaning of the Variation Between Models===

The '''variation between models''' in the ensemble can mean either of two things. The variation can represent actual '''flexibility and thermal motion''' that occurred during the NMR measurements in solution, typically at room temperature. Alternatively, the variation can simply mean '''uncertainty in the atomic positions''', namely, that an inadequate number of restraints were available to determine the positions of some atoms. Unfortunately, there is nothing comparable to the [[B value]] or [[Temperature value]] that quantitates the uncertainty of the position of each atom in crystallographic results. Hence, the only way to find out what the meaning of the variation between models is to contact the experimenters who authored the published ensemble of models.

Using appropriate methodologies, it is possible to determine both the average structure and its dynamic movements<ref>Simultaneous determination of protein structure and dynamics. Kresten Lindorff-Larsen, Robert B. Best, Mark A. DePristo, Christopher M. Dobson, and Michele Vendruscolo (2005). Nature 433:128. PMID:[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15650731 15650731].</ref>.

===The Most-Representative Model===
The '''most representative model''' is the model closest to the average model. A server called [http://www.ebi.ac.uk/msd-srv/pqs/pqs-nmr.html Olderado] reports the most representative model, and enables you to download it separately.

===The Minimized Average Model===
It is common to average the models from an NMR experiment, but in order for the result to be realistic, it must undergo some energy minimization in order to adjust covalent bond lengths and angles. The result is called a '''minimized average model'''. Sometimes, authors publish both the ensemble and the minimized average. For example [[2bbm]] appears to be the minimized average for the ensemble of 21 models in [[2bbn]], but without reading the original publication or contacting the authors, it is difficult to be sure (since the header of the [[PDB file format|PDB file]] does not say).

==Reliability of NMR Models==

NMR models are more likely to contain major errors <ref>Traditional biomolecular structure determination by NMR spectroscopy allows for major errors. Sander B. Nabuurs, Chris. A. E. M. Spronk, Geerten W. Vuister, and Gert Vriend. (2006). PLoS Computational Biology 2: [http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020009 Open Access Full Text] [http://proteinexplorer.org/favlit/nmr.htm Precis]. DOI: 10.1371/journal.pcbi.0020009</ref> than are crystallographic models that have good [[Resolution]] and [[Free R]] values.

==Median Size of Published NMR Structures==
Solution NMR is unable to determine atomic resolution protein structures for molecules in excess of about 30,000 Daltons. In fact, the median mass of NMR structures published in the [[Protein Data Bank]] is about 9 kD, with 90% less than 19 kD <ref>At Protein Explorer: [http://proteinexplorer.org/gpsi/chardata.htm Size and Redundancy of 43,000 Entries 
in the Protein Data Bank] (as of April 2007)</ref>. In contrast, the median mass of crystallographically determined structures is 45 kD, with 90% <145 kD.

==Alignment of Models==
NMR models are typically structurally aligned by the authors before publication. However, there are some exceptions, such as [[1qp6]], [[1dl0]], and [[1i25]], in which the individual models are not aligned. In such cases, one needs to look at [[#Individual_Models|individual models]] in order to understand the molecular structure.

The alignment can affect your perception of the variation between models. For example, calmodulin contains two [[EF-hands]] connected by a flexible linker. When calmodulin is not bound to a cognate peptide, the two EF-hands can move relative to each other, flexing the linker. In [[1cfc]], the N-terminal EF-hands are aligned, but the C-terminal EF-hands are in different orientations. Alternatively, had the C-terminal EF-hands been aligned, then the N-terminal EF-hands would be in variable orientations. And less plausibly, had a short center segment of the flexible linker been aligned, both ends would be in variable orientations.

==References and Websites==

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