Gaucher Disease: Task 10 - Normal mode analysis

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lab journal

With normal mode analysis (NMA), you are able to simulate or analyse the natural resonant movements of proteins. There are two different webserver available to do a NMA, WEBnm@ and ElNemo. They provide the following information.

WEBnm@

• Deformation energy
• atomic fluctuations
• correlation matrix
• atomic displacement

ElNemo

• amplitudes that were applied in the normal mode perturbation as DQMIN(DQSTEP)DQMAX (perturbation)
• animations of the top five modes in GIF format (small/large)
• distance fluctuations between all C-alpha atoms (CA-vari)
• RMSD with respect to the second reference structure (if given)
• visualization of the mean square displacement of all C-alpha atoms associated with a given mode (<R2>)
• frequency of a mode
• collectivity of a mode

WEBnm@

WEBnm@ uses only alpha-carbon atoms in the models for the normal mode analysis.

Movements

<figtable id="energy">

Mode	Deformation Energy	Visualisation	Movements
7	174.43	Mode 7	hing-moving
8	251.61	Mode 8
9	344.99	Mode 9
10	759.13	Mode 10	equally breathing of the whole structure
11	1099.96	Mode 11

Five best values of the deformation energy for the lowest-frequency non-trivial modes.

</figtable>

Domains

Motions of corelated residues of Glucocerebrosidase.

The Glucocerebrosidase shows a domain definition according to their chains in the vsualization of the corelated residue motion map on the right. Within the chains, which are identical, a very big domain and very small domain (<100 residues) at the end of the chains can be seen. While the the first consists of a high number of helices and a few sheets, the latter one includes only a few sheets. All domain databases agree with each other that the bigger domain has a glycosidic characteristics. However, they differ in their information about the second domain. While PFAM neglects a second domain, CATH classifies it as an alpha-mannosidase II and SCOP identifies a beta-glycanases. A correlation of motions between residues at the beginning and the end of a chain can be observed. Between the chains there is only a slight movement correlation observable. Some residues of the big domain in one chain correlate with a few other residues at the beginning and outside the domain of the other chain.

As chain A and B are identical, the following domain definition concentrates on one domain: PFAM

• 1 domain
• PF02055: Glycoside hydrolase family 30

CATH

• 2 domains
• 3.20.20.10: Glycosidases
• 2.60.40.1180: alpha-mannosidase II

SCOP

• 2 domains
• b.71.1.2: Composite domain of glycosyl hydrolase families 5, 30, 39 and 51
• c.1.8.3: beta-glycanases

ElNemo

ElNemo uses also only the C-alpha atoms of the structure for normal mode analysis, therefore it takes only the ATOM record from the PDB file, ignoring the HETATM record of a ligand. We ran ElNemo with our reference structure 1OGS without changing anything, so that the ligand could be ignored for the normal mode calculation.

For the normal mode analysis we specified a cutoff of 10 Å between the C-alpha atoms. A hundred modes were calculated (<R2>, frequency and collectivity information of all found modes). For the best five modes, i.e. with lowest frequency and the highest collectivity of the motions, more information is provided, including PDB files and animations in GIF format. B-factor analysis of ElNemo server yielded correlation of 0.662 for 1006 C-alpha atoms (main result page). The best five modes are presented in <xr id="elnemo_modes"/>.

<figtable id="elnemo_modes">

Mode	Frequency	Collectivity	Animations			Description
			a	b	c
7	1.00	0.7546				Hinge motion of the two identical chains.
8	1.22	0.7472				Twist motion of the both chains in opposite directions to the axis which connects the chains combined with a slight bending movement between the two domains (described in the WEBnm@ section) in each chain.
9	1.74	0.7765				Twist motion of the both chains in opposite directions to the axis which connects the chains, similar to that in mode 8 (but no bending between the domains).
10	3.36	0.7970				Breathing of the chains and a slight bending movement between the two domains in each chain.
11	4.30	0.8301				Movement of the chains towards each other and back in the perpendicular direction to the axis connecting them, combined with a slight twist, similar to that in modes 8 and 9. A slight breathing and a hinge movement between the two domains in each chain, similar to that in mode 10, is detectable.

Five lowest-frequency modes for the structure 1OGS created by ElNemo (cutoff used to identify elastic interactions=10).

</figtable>

As we can see from the animations of the best five modes, the most flexible regions are where the two chains connect to each other (make a twist, a hinge and a movement towards each other and back on perpendicular from the chain connecting axis) and inside the chains the regions between the two domains (SCOP and CATH) are most flexible (produce a sort of a hinge movement). Other regions inside the domains seem to be stable.

Discussion

As mentioned, both servers calculate the normal modes from the C-alpha atoms of a structure only. In principle, very many more normal modes could be calculated for a protein, if no C-alpha cutoff would have been used, because of the bend, angle and torsion movements between the connected atoms. However, it is very calculation intensive to find all this small movement and is a task of MD. In normal mode analysis we are interested only in "big range" movements, movement on the chain and domain levels.

As we do not have a structure completely without a ligand, but only structures with different, not native ligands. The reason for is that the complex with the native ligand, glucosylceramide, is too unstable and the enzyme processes the glucosylceramide instantly into the substrates, ceramide and glucose. Therefore, unfortunately we do not have structures with different conformations for glucocerebrosidase. For this reason, we could not do the option of calculation of normal modes with two different structures. Simply deleting of a ligand from the PDB structure would not result in a change of the main chain coordinates and thus have no effect on the normal modes.

TODO: Can you observe notable differences between the normal modes calculated by the different servers?

TODO: What are the advantages and disadvantages of NMA compared to MD?

Sources

WEBnm@:
WEBnm@ server
ElNemo:

1. ElNemo server
   
2. K. Suhre & Y.H. Sanejouand, ElNemo: a normal mode web-server for protein movement analysis and the generation of templates for molecular replacement. Nucleic Acids Research, 32, W610-W614, 2004.
   
3. K. Suhre & Y.H. Sanejouand, On the potential of normal mode analysis for solving difficult molecular replacement problems. Acta Cryst. D vol.60, p796-799, 2004 © International Union of Crystallography.