RDC-Assisted Dimer Structure Determination - Revision history

Jma at 21:02, 6 January 2010

2010-01-06T21:02:51Z

← Older revision		Revision as of 21:02, 6 January 2010
Line 50:		Line 50:
	== <br>'''References''' ==		== <br>'''References''' ==

	1. Levy, E.D., ~~et al~~., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>[http://www.ncbi.nlm.nih.gov/pubmed/10617446?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 2. Al-Hashimi, H.M., Bolon, P.J., and Prestegard, J. H.  Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15040978?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 3. Valafar, H. and Prestegard, J. H.  REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.]<br>4. Moont, G., H.A. ~~Gabb~~, and M.J.E. ~~Sternberg,~~ Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.		[http://www.ncbi.nlm.nih.gov/pubmed/17112313?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=10 1. Levy, E.D., Pereira-Leal, J.B., Chothia, C., and Teichmann, S.A. 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.]<br>[http://www.ncbi.nlm.nih.gov/pubmed/10617446?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 2. Al-Hashimi, H.M., Bolon, P.J., and Prestegard, J. H.  Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15040978?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 3. Valafar, H. and Prestegard, J. H.  REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.]<br>[http://www.ncbi.nlm.nih.gov/pubmed/10328272?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 4. Moont, G., Gabb, H.A., and Sternberg, M.J.E.  Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.]

Jma at 20:59, 6 January 2010

2010-01-06T20:59:17Z

← Older revision		Revision as of 20:59, 6 January 2010
Line 50:		Line 50:
	== <br>'''References''' ==		== <br>'''References''' ==

	1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>2. Al-Hashimi, H.M., P.J. ~~Bolon~~, and J.H. ~~Prestegard,~~ Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.<br>3. Valafar, H. and J.H. ~~Prestegard,~~ REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.		1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>[http://www.ncbi.nlm.nih.gov/pubmed/10617446?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 2. Al-Hashimi, H.M., Bolon, P.J., and Prestegard, J. H.  Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.]<br>[http://www.ncbi.nlm.nih.gov/pubmed/15040978?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=6 3. Valafar, H. and Prestegard, J. H.  REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.]<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.

Jma at 20:55, 6 January 2010

2010-01-06T20:55:41Z

← Older revision		Revision as of 20:55, 6 January 2010
Line 50:		Line 50:
	== <br>'''References''' ==		== <br>'''References''' ==

	1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>2. Al-Hashimi, H.M., P.J. Bolon, and J.H. Prestegard, Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.<br>3. Valafar, H. and J.H. Prestegard, REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.		1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>2. Al-Hashimi, H.M., P.J. Bolon, and J.H. Prestegard, Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.<br>3. Valafar, H. and J.H. Prestegard, REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.

	~~-- XuWang - 18 Jul 2009~~

	~~-- Edited by JimAramini - Nov 2009~~

Jma at 19:32, 10 December 2009

2009-12-10T19:32:51Z

← Older revision		Revision as of 19:32, 10 December 2009
Line 1:		Line 1:

	== '''Introduction''' ==		== '''Introduction''' ==

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	=== <br>Part 1: Obtaining the symmetry axis orientation ===		=== <br>Part 1: Obtaining the symmetry axis orientation ===

	Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis ~~(Figure 1)~~. For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis ~~(Figure 2)~~. The stationary axis is therefore the symmetry axis of the dimer.		Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis. For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis. The stationary axis is therefore the symmetry axis of the dimer.

	~~==== Figure 1: ====~~

	~~==== ====~~

	~~==== Figure 2: ====~~

	~~<br><br>~~Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.		Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.

	=== <br>Part 2: Grid search ===		=== <br>Part 2: Grid search ===

Jma: moved RDC-assisted dimer structure determination to RDC-Assisted Dimer Structure Determination

2009-11-05T22:15:49Z

moved RDC-assisted dimer structure determination to RDC-Assisted Dimer Structure Determination

← Older revision	Revision as of 22:15, 5 November 2009
(No difference)

Jma at 22:07, 5 November 2009

2009-11-05T22:07:28Z

← Older revision		Revision as of 22:07, 5 November 2009
Line 1:		Line 1:
	~~='''RDC-assisted dimer structure determination'''=~~
	== '''Introduction''' ==		== '''Introduction''' ==

	Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>		Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>

	== '''Theoretical Background''' ==		== '''Theoretical Background''' ==
Line 24:		Line 24:
	=== <br>Part 1: Obtaining the symmetry axis orientation ===		=== <br>Part 1: Obtaining the symmetry axis orientation ===

	Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure 1). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure 2). The stationary axis is therefore the symmetry axis of the dimer.		Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure 1). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure 2). The stationary axis is therefore the symmetry axis of the dimer.

	==== Figure 1: ====		==== Figure 1: ====

	==== ====		==== ====

	==== Figure 2: ====		==== Figure 2: ====

	<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.		<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.

	=== <br>Part 2: Grid search ===		=== <br>Part 2: Grid search ===

Jma at 17:38, 4 November 2009

2009-11-04T17:38:23Z

← Older revision		Revision as of 17:38, 4 November 2009
Line 59:		Line 59:
	1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>2. Al-Hashimi, H.M., P.J. Bolon, and J.H. Prestegard, Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.<br>3. Valafar, H. and J.H. Prestegard, REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.		1. Levy, E.D., et al., 3D complex: A structural classification of protein complexes. Plos Computational Biology, 2006. 2(11): p. 1395-1406.<br>2. Al-Hashimi, H.M., P.J. Bolon, and J.H. Prestegard, Molecular symmetry as an aid to geometry determination in ligand protein complexes. Journal of Magnetic Resonance, 2000. 142(1): p. 153-158.<br>3. Valafar, H. and J.H. Prestegard, REDCAT: a residual dipolar coupling analysis tool. Journal of Magnetic Resonance, 2004. 167(2): p. 228-241.<br>4. Moont, G., H.A. Gabb, and M.J.E. Sternberg, Use of pair potentials across protein interfaces in screening predicted docked complexes. Proteins-Structure Function and Genetics, 1999. 35(3): p. 364-373.

	-- ~~Main.~~XuWang - 18 Jul 2009		-- XuWang - 18 Jul 2009

			-- Edited by JimAramini - Nov 2009

Jma at 17:37, 4 November 2009

2009-11-04T17:37:53Z

← Older revision		Revision as of 17:37, 4 November 2009
Line 24:		Line 24:
	=== <br>Part 1: Obtaining the symmetry axis orientation ===		=== <br>Part 1: Obtaining the symmetry axis orientation ===

	Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure~~ ??~~). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure~~ ??~~). The stationary axis is therefore the symmetry axis of the dimer.<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.		Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure 1). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure 2). The stationary axis is therefore the symmetry axis of the dimer.

			==== Figure 1: ====

			==== ====

			==== Figure 2: ====

			<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.

	=== <br>Part 2: Grid search ===		=== <br>Part 2: Grid search ===

Jma at 18:40, 3 November 2009

2009-11-03T18:40:14Z

← Older revision		Revision as of 18:40, 3 November 2009
Line 1:		Line 1:
	='''RDC-assisted dimer structure determination'''=		='''RDC-assisted dimer structure determination'''=
	== ~~<br>~~ '''Introduction''' ==		== '''Introduction''' ==

	Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>		Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>

	== '''Theoretical Background''' ==		== '''Theoretical Background''' ==

Jma at 18:39, 3 November 2009

2009-11-03T18:39:44Z

← Older revision		Revision as of 18:39, 3 November 2009
Line 1:		Line 1:
			='''RDC-assisted dimer structure determination'''=
	== <br> '''Introduction''' ==		== <br> '''Introduction''' ==

	Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>		Protein homo-oligomers occur in nature frequently and some oligomerization processes can influence biological processes significantly. In fact, according to Levy et al. a high percentage of proteins are capable of forming homo-oligomers [1]. The prevalence of homo-oligomers has made elucidation of their complex structures a major imperative for the structural genomics community. However, solving homo-oligomer structures has always been difficult using traditional NOE-based techniques in solution NMR. This is primarily because obtaining unambiguous intermolecular distance constraints using NOE is limited by both sample preparation and NMR methodology. The traditional approach uses isotopic filtering methods to distinguish between intermolecular and intramolecular NOEs. This requires the preparation of samples that contains a mixture of isotopically labeled and unlabeled proteins. Owing to the probability of oligomer association, only half of the protein can form oligomers with both labeled and unlabeled components. This immediately results in a 50% loss in signal intensity. The isotope-filtering/editing experiment used to obtain the NOEs also suffers from low sensitivity due to the presence of many filtering elements. Further more, the nature of the homo-oligomer implies that the same set of residues will be involved in all intermolecular NOEs. This increases the likelihood of intermolecular NOEs between sequential residues or identical residues, thus lowering the number of unambiguous intermolecular NOEs in many situations.<br><br>The disadvantages of the NOE approach in solving homo-oligomer structures have become the motivation for the development of a complementary approach for solving homo-oligomer structures using NMR. The one approach taken by us in this study uses residual dipolar couplings (RDCs) to obtain orientation of the symmetry axis relative to the monomer structure. This information will then be used to model the dimer structure using a simple grid search algorithm to find the suitable binding interface.<br>

	== '''Theoretical Background''' ==		== '''Theoretical Background''' ==
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	==== Implementation of the grid search ====		==== Implementation of the grid search ====

	Once the symmetry axis of the oligomer has been identified, the construction of the dimer models can then be carried out via a simple grid search. Computational modeling of dimer structures can be implemented with several approaches. In the absence of the symmetry axis information, the search is a three-dimensional site search that contains a large number of possibilities. But with the symmetry information, the search is restricted to a simple two dimensional search on the plane orthogonal to the symmetry axis. This is because movements parallel to the axis will break the symmetry thus can not be allowed in order to preserve the orientation of the symmetry axis.<br>The grid search can be implemented with a variety of programs. The implementation used in this protocol uses the program VMD, which contains an extensive set of functions that allows the convenient manipulation of PDB coordinates and is capable of calling outside programs to perform molecular dynamics and simulate RDCs. During the grid search, the PDB coordinates of the monomer in the alignment tensor frame is first imported and placed at the origin. An identical copy of the molecule is then made and rotated by 180 degrees around the symmetry axis. The copy is then positioned at regularly spaced points in the plane perpendicular to the symmetry axis. At each grid point, a preliminary analysis of the relations between the two molecules is carried out to judge the suitability of the model being a realistic dimer. The analysis involves mostly calculating the distance between the two molecules to ensure that there are no egregious clashes between the molecules yet they are close enough to have an intermolecular interaction surface. Models judged to be plausible will undergo a round of molecular dynamics involving only residues at the interface. This will improve the interfacial contact if the initial side chain conformation of the monomer is not optimal. All suitable models produced by the model will then be evaluated based on the suitability of the intermolecular interface and, if PEG RDCs are available, the agreement between predicted RDCs based on the dimer structure and the experimental RDCs.		Once the symmetry axis of the oligomer has been identified, the construction of the dimer models can then be carried out via a simple grid search. Computational modeling of dimer structures can be implemented with several approaches. In the absence of the symmetry axis information, the search is a three-dimensional site search that contains a large number of possibilities. But with the symmetry information, the search is restricted to a simple two dimensional search on the plane orthogonal to the symmetry axis. This is because movements parallel to the axis will break the symmetry thus can not be allowed in order to preserve the orientation of the symmetry axis.<br>The grid search can be implemented with a variety of programs. The implementation used in this protocol uses the program VMD, which contains an extensive set of functions that allows the convenient manipulation of PDB coordinates and is capable of calling outside programs to perform molecular dynamics and simulate RDCs. During the grid search, the PDB coordinates of the monomer in the alignment tensor frame is first imported and placed at the origin. An identical copy of the molecule is then made and rotated by 180 degrees around the symmetry axis. The copy is then positioned at regularly spaced points in the plane perpendicular to the symmetry axis. At each grid point, a preliminary analysis of the relations between the two molecules is carried out to judge the suitability of the model being a realistic dimer. The analysis involves mostly calculating the distance between the two molecules to ensure that there are no egregious clashes between the molecules yet they are close enough to have an intermolecular interaction surface. Models judged to be plausible will undergo a round of molecular dynamics involving only residues at the interface. This will improve the interfacial contact if the initial side chain conformation of the monomer is not optimal. All suitable models produced by the model will then be evaluated based on the suitability of the intermolecular interface and, if PEG RDCs are available, the agreement between predicted RDCs based on the dimer structure and the experimental RDCs.

	== '''Experimental Procedure''' ==		== '''Experimental Procedure''' ==
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	=== <br>Part 1: Obtaining the symmetry axis orientation ===		=== <br>Part 1: Obtaining the symmetry axis orientation ===

	Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure ??). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure ??). The stationary axis is therefore the symmetry axis of the dimer.<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.		Information regarding the orientation of the symmetry axis relative to the monomers is embedded in the RDCs. To obtain this information, the structure of the monomer is required. Using the structure, alignment tensor orientation of the RDCs can be calculated. The most convenient way of calculating the alignment tensor is to use the program REDCAT [3]. Please refer to the “RDC Screening and Structure Refinement” section of the TWiki for details on how to prepare input files for and running REDCAT. For dimers, RDCs from two non-degenerate media are required.<br><br>Once the alignment tensors have been calculated, use the plot feature of REDCAT to plot the Sauson-Flamsteed projection of the tensor orientation (Tools->Plot->2D SF Plot). Trimers and higher order oligomers should have an axially symmetric alignment tensor with the non degenerate axis being the symmetry axis (Figure ??). For dimers, plot the two alignment tensors on the same graph. The two tensor orientations should be related to each other by a rotation around a stationary axis (Figure ??). The stationary axis is therefore the symmetry axis of the dimer.<br><br>Once the symmetry axis has been identified, the PDB coordinates of the structure in the alignment tensor frame can be obtained by using the rotate-PDB function in REDCAT, keeping in mind that the angles supplied by REDCAT only applies to the structure from which the alignment tensor is calculated.

	=== <br>Part 2: Grid search ===		=== <br>Part 2: Grid search ===
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	<br>The grid search uses only a single processor. However, as the search can be started in any place on the grid (albeit this is available for only one dimension), several runs covering non-overlapping areas of the grid can be started simultaneously.		<br>The grid search uses only a single processor. However, as the search can be started in any place on the grid (albeit this is available for only one dimension), several runs covering non-overlapping areas of the grid can be started simultaneously.

	After the search has been completed, a file containing statistics on each model generated will be produced. Some of the statistics will be used to evaluate the model later on.		After the search has been completed, a file containing statistics on each model generated will be produced. Some of the statistics will be used to evaluate the model later on.

	=== <br>Part 3: Evaluating the models ===		=== <br>Part 3: Evaluating the models ===
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	Residue pairing score is a simple but reliable way of evaluating the validity of the proposed interaction surface between two proteins. The concept was propose by Moont et al. [4] and utilizes empirical statistics of the probability of a pair of amino acids being close to each other in the interface. It is extremely helpful in removing models that have non-probable interaction surface, which can not be recognized by RDC prediction or geometric filters. However, before running the program, the PDB models generated by the grid search needs to be processed. First, the two monomers should be placed in separate PDB files. Then each monomer PDB will then be preprocessed with the script “preprocess-pdb.per”. The parsed PDB file is now suitable for use as input to the program rpdock. Higher residue pairing score usually indicates the interface has high agreement with experimental norm.		Residue pairing score is a simple but reliable way of evaluating the validity of the proposed interaction surface between two proteins. The concept was propose by Moont et al. [4] and utilizes empirical statistics of the probability of a pair of amino acids being close to each other in the interface. It is extremely helpful in removing models that have non-probable interaction surface, which can not be recognized by RDC prediction or geometric filters. However, before running the program, the PDB models generated by the grid search needs to be processed. First, the two monomers should be placed in separate PDB files. Then each monomer PDB will then be preprocessed with the script “preprocess-pdb.per”. The parsed PDB file is now suitable for use as input to the program rpdock. Higher residue pairing score usually indicates the interface has high agreement with experimental norm.

	Besides predicted RDC and residue pairing scores, other criteria such as van der waal energy of the model and the size of the binding surface can also be used as selection criteria. However, in our experience, predicted RDC and residue pairing score are the most selective. If NOE data is available, they can also serve as highly selective evaluators. Shape data from small angle X-ray scattering (SAXS) experiments can also be used as a replacement for back predicted RDCs. In fact, SAXS data may be more reliable than PALES-predicted RDCs since the shapes are directly measured by SAXS.		Besides predicted RDC and residue pairing scores, other criteria such as van der waal energy of the model and the size of the binding surface can also be used as selection criteria. However, in our experience, predicted RDC and residue pairing score are the most selective. If NOE data is available, they can also serve as highly selective evaluators. Shape data from small angle X-ray scattering (SAXS) experiments can also be used as a replacement for back predicted RDCs. In fact, SAXS data may be more reliable than PALES-predicted RDCs since the shapes are directly measured by SAXS.

	== <br>'''References''' ==		== <br>'''References''' ==