NMR Structure Determination With The NMR Assignments and Molecular Modeling Tools in Hand: All we need are the experimental constraints Distance constraints between atoms is the primary structure determination factor. Dihedral angles are also an important structural constraint What Structural Information is available from an NMR spectra?

How is it Obtained?

How is it Interpreted?

NMR Structure Determination

Nuclear Overhauser Effect (NOE)Nuclear Overhauser Effect (NOE, h) the change in intensity of an NMR resonance when the transition of another are perturbed, usually by saturation.

Saturation elimination of a population difference between transitions (irradiating one transition with a weak RF field)hi = (I-Io)/Io where Io is thermal equilibrium intensityaaabbabbNNN+dN-dXXAAirradiatePopulations and energy levels of a homonuclear AX system (large chemical shift difference)Observed signals only occur from single-quantum transitions

Nuclear Overhauser Effect (NOE)aaabbabbN+dIISSPopulations and energy levels immediately following saturation of the S transitionsN+dN-dN-dSaturated(equal population)Saturated(equal population)saturateaaabbabbW1AW1AW1XW1XW2W0Observed signals only occur from single-quantum transitionsRelaxation back to equilibrium can occur through:Zero-quantum transitions (W0)Single quantum transitions (W1)Double quantum transitions (W2)N-dN+dN+dN-d

Nuclear Overhauser Effect (NOE)aaabbabbW1AW1AW1XW1XW2W0N-dN+dN+dN-dSteady-state NOE enhancement at spin A is a function of all the relaxation pathwaysIf only W1, no NOE effect at HAIf W0 is dominant, decrease in intensity at HA negative NOEIf W2 is dominate, increase in intensity at HA positive NOE

For homonuclear (gX=gA), maximum enhancement is ~ 50% For heteronuclear (gX=gA), maximum enhancement is ~50%(gX/gA)Intensity of NOE builds-up as a function of the mixing time (tm)Solomon Equation:

Nuclear Overhauser Effect (NOE)Mechanism for Relaxation Dipolar coupling between nuclei interaction between nuclear magnetic dipoles local field at one nucleus is due to the presence of the other depends on orientation of the whole molecule in solution, rapid motion averages the dipolar interaction in crystals, positions are fixed for single molecule, but vary between molecules leading range of frequencies and broad lines. Dipolar coupling, T1 and NOE are related through rotational correlation time (tc) recall: rotational correlation is the time it takes a molecule to rotate one radian (360o/2p) Relaxation or energy transfers only occurs if some frequencies of motion match the frequency of the energy transition.

The available frequencies for a molecule undergoing Brownian tumbling depends on tc.

The total power available for relaxation is the total area under the spectral density function.

Nuclear Overhauser Effect (NOE)Mechanism for Relaxation Spectral density is constant for w >wo then wo2tc2

2D NOESY (Nuclear Overhauser Effect)Relative magnitude of the cross-peak is related to the distance (1/r6) between the protons ( 5).NOE is a relaxation factor that builds-up duringThe mixing-time (tm)

2D NOESY Spectra at 900 MHzLysozyme Ribbon DiagramNMR Structure Determination How DO We Go From the NOESY Data to A Structure?

NMR Structure Determination We Need to Assign Each NOE Cross-Peak to A Specific 1H-1H Pair from Assignment Table.10.5 ppm to 7.65 ppm ...Assignment TableWhat H assignments for the protein are consistent with 10.5 ppm & 7.65 ppm?In some cases, the chemical shifts associated with the NOE cross-peak are unique and an assignment is straight-forward. More likely is the occurrence that the assignment is ambiguous multiple possible assignments to one or both of the chemical shifts.

COL assignment table frag

ResidueNCOCaCbOthers

D1120.1 (8.08)179.153.8 (4.37)39.9 (3.00,0.58)

E2128.9 (9.93)176.656.0 (4.55)30.9 (2.11,1.78)Cg 36.2(2.75,2.41)

D3116.1 (8.90)176.556.2 (4.73)38.9 (2.70,2.34)

E4113.3 (7.45)175.652.9 (4.71)27.2 (1.23,0.63)Cg 34.8(2.57,1.79)

R5123.8 (7.97)173.154.3 (4.43)28.9 (1.84,1.47)Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)

W6127.8 (9.27)177.155.4 (5.50)30.8 (3.11)

T7109.9 (9.32)174.859.6 (4.85)71.3 (4.34)Cg 20.2(0.73)

N8115.2 (8.42)174.550.9 (5.06)38.7 (3.13,2.70)

N9116.7 (7.88)173.352.1 (4.94)38.0 (3.33,3.01)

F10110.1 (7.57)177.258.2 (4.46)38.5 (2.63,2.67)

R11121.2 (8.03)175.055.8 (4.06)29.7 (1.83,1.67)Cg 27.4(1.45,1.35);Cd 43.1(3.13)

E12119.0 (8.56)177.952.3 (3.79)27.4 (1.16,-0.05)Cg 36.6(2.19,1.97)

Y13122.1 (8.28)173.261.9 (3.80)41.2 (2.99,2.52)

N14121.5 (8.45)175.754.7 (4.89)39.2 (2.26)

L15127.7 (9.02)176.958.0 (4.48)41.2 (1.96,1.64)Cg 27.2(1.20);Cd 27.8(0.87);Cd 27.8(0.78)

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NMR Structure Determination We Need to Assign Each NOE Cross-Peak to A Specific 1H-1H Pair from Assignment Table....Assignment TableTo determine the structure, an assignment has to be made for each of the thousands of observed NOE cross peaks A lot of manual effort!Ambiguity in assignments also arises from errors in peak position and correlation to assignment table. Need to match assignments with an error range that is dependent on the resolution associated with each axis. Typically, 1H errors may range from 0.02 to 0.06 ppm and 13C, 15N from 0.25 to 1.0 ppm.

An NOE cross-peak of 4.25 0.02 ppm to 2.21 0.02 ppm may be consistent with a dozen or more possible combinations!

COL assignment table frag

ResidueNCOCaCbOthers

D1120.1 (8.08)179.153.8 (4.37)39.9 (3.00,0.58)

E2128.9 (9.93)176.656.0 (4.55)30.9 (2.11,1.78)Cg 36.2(2.75,2.41)

D3116.1 (8.90)176.556.2 (4.73)38.9 (2.70,2.34)

E4113.3 (7.45)175.652.9 (4.71)27.2 (1.23,0.63)Cg 34.8(2.57,1.79)

R5123.8 (7.97)173.154.3 (4.43)28.9 (1.84,1.47)Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)

W6127.8 (9.27)177.155.4 (5.50)30.8 (3.11)

T7109.9 (9.32)174.859.6 (4.85)71.3 (4.34)Cg 20.2(0.73)

N8115.2 (8.42)174.550.9 (5.06)38.7 (3.13,2.70)

N9116.7 (7.88)173.352.1 (4.94)38.0 (3.33,3.01)

F10110.1 (7.57)177.258.2 (4.46)38.5 (2.63,2.67)

R11121.2 (8.03)175.055.8 (4.06)29.7 (1.83,1.67)Cg 27.4(1.45,1.35);Cd 43.1(3.13)

E12119.0 (8.56)177.952.3 (3.79)27.4 (1.16,-0.05)Cg 36.6(2.19,1.97)

Y13122.1 (8.28)173.261.9 (3.80)41.2 (2.99,2.52)

N14121.5 (8.45)175.754.7 (4.89)39.2 (2.26)

L15127.7 (9.02)176.958.0 (4.48)41.2 (1.96,1.64)Cg 27.2(1.20);Cd 27.8(0.87);Cd 27.8(0.78)

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NMR Structure Determination Peak-Picking is Very Challenging in A Protein NOESY Spectra Spectra Can be very crowded with a number of overlapping peaks Automatic peak-picking fails in these crowded regions the quality of the structure is inherently dependent on the quality of the peak-picking assignments are made from the peak lists wrong peak position or picked noise peaks results in a mis-assignment that results in an incorrect structure distance constraints that results in a local distortion in the structureHow Many Peaks?What is each Peaks Position?Which are Noise Peaks?

NMR Structure Determination How Do We Resolve These Peak-Picking and Ambiguity Issues? Spread out the data into 3D and 4D symmetry can help remove ambiguities Use 1H-15N and 1H-13C connections Iterative analysis of the NOE data use structure and distance filter to remove ambiguities

NMR Structure Determination Iterative Analysis of NOESY Data, How Does It Work? Assign All unique or unambiguous NOEs Calculate Initial Structure with All the Data possible Use the Structure to Filter Ambiguous Assignments possible assignment has to be 6 removes all possible assignments with distances 6 Calculate New Structure With New constraints identify & correct violated constraints repeat NOE analysis repeat process until all NOEs correctly assigned and a quality structure is obtained

NMR Structure Determination Using PIPP to Analyze NOESY Data read in your initial structure(s) read in your NMR assignment list click on a peak and PIPP tells you: the possible assignments chemical shift errors relative to assignment table minimum, average and standard deviation distances

NMR Structure Determination After Assigning an NOE Peak to 1H-1H pair, Need to Assign a Distance Constraint There are Two Generally Accepted Approaches: Two-Spin Approximation Relaxation Matrix ApproachTwo-Spin Approximation the observed volume or intensity of a NOE cross-peak is directly related to the distance between the 1H-1H pair This approximation only holds true for the linear part of the NOE build-up curve (short-mixing times) when spin diffusion is not a significant component of the observed volume (intensity)

NMR Structure Determination Spin Diffusion in the limit of wtc >> 1 (biomolecules), the rate of transfer of the spin energy between nuclei becomes much larger than the rate of transfer of energy to the lattice. The observed NOE cross-peak volume between Hi & Hj is potentially increased by a HiHkHj & HiHlHj energy transfer.Effectively, the spin energy diffuses through all possible paths between all possible spin systems. Spin diffusion is always present, the magnitude depends on the mixing time and the efficiency of any particular pathway. The longer the path, the smaller the energy that is transferred

NMR Structure Determination As We Discussed Before, One Common Approach Uses a Qualitative Binning of NOE Intensities generally cluster NOE volumes into strong, medium, weak and very weak The following rules apply: Strong2.5 0.7 0.2 for NH-NH constraints use: 2.5 0.7 0.6Medium3.0 1.2 0.3 for NOEs with NH use: 3.0 1.2 0.5Weak4.0 2.2 1.0Very Weak5.0 2.0 1.0the lower limit is always set to slightly less than twice the hydrogen van der Waals radius (1.8) NOEs for methyls are scaled down by 1/3 Uses reasonably short mixing-time (100-150msec) and allows quantity of distance constraints to correct for spin-diffusion effects remove or move to lower bin violated constraint that may arise from spin-diffusion alternative is to leave all constraints as observed with corresponding contribution to violation energy and potential structure distortion point of debate in NMR communityNOEs are observed between the Ala methyl and both Leu methyls. Structure indicates a violation to one methyl-methyl pair NOE probably a result of spin diffusion Violated constraint

NMR Structure Determination Two-Spin Approximation Instead of binning into strong, medium, weak and very weak, can assign a relative distance

Use a reference volume with a known fixed distance to calibrate all volumes 2.52 for Leu Ha-Hb 2.52 for Phe or Tyr Hd-HeSerious Problems: obtain a highly precise distance that ignores all the inherent errors associated with the accuracy of measuring volumes, spin-diffusion and dynamics. Short, inaccurate distance constraints cause severe local structure distortionsViolated constraintWhat would happen to the structure if both Leu d had to be 3 to the Ala b?

NMR Structure Determination Relaxation Matrix Approach Takes Into Account Spin-Diffusion removes manual and potentially biased approach to identify spin-diffusion issues From the structure, calculates a spin relaxation matrix to correct for spin-diffusion contributions to observed volumes There are a number of programs that perform this analysis (CORMA, FIRM, MardiGras, MORASS, etc)Calculate from structureExperimental Volumes from NOESY spectraJ. Am. Chem. Soc. 1990, 112, 6803-6809

NMR Structure Determination Relaxation Matrix Approach Takes Into Account Spin-Diffusion Merge the cross-relaxation rates calculated from the structure with experimental volumes obtain distance matrix to calculate new structure iterate processCan relate cross-relaxation rates(G) with experimental NOE volumes (V)

NMR Structure Determination Problems with Relaxation Matrix Approach Errors and Failures with Matrix Calculations Do not obtain complete experimental NOESY volume matrixVery difficult to accurately measure diagonal peaks Significant errors in measuring experimental volumes Peak overlaps and degenerate assignments Missing peaks, limits of S/N Noise Assume Uniform Dynamics (tc)Poor Assumption Different regions of protein structure have very different local dynamics Contributions to relaxation rates by dynamics can be much more significant than spin-diffusion Output of calculation is a very specific distance constraint But, may have high errors (volume, dynamics) Large structure distortion that is propagated through iteration

NMR Structure Determination Two Very Important Facts to Remember NOEs Reflect the Average Distance Protein Structures Are Dynamic

We visualize protein structures as a static imageIn reality, protein undergoes wide-ranges of motions (snapshots of 100 BPTI conformations)J. Mol. Biol. (1999) 285, 727740

NMR Structure Determination We have already discussed labeling the protein, data collection and the resonance assignmentNext Step, is to identify which residues adopt which secondary structure present

NMR Structure Determination Protein Secondary Structure and NOE Patterns a-HelixSequential NOEs observed in 3D 15N-edited NOESY are indicative of a-helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns a-HelixSequential NOEs observed in 3D 15N-edited NOESY are indicative of a-helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns a-HelixSequential NOEs observed in 3D 15N-edited NOESY are indicative of a-helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns a-HelixSequential NOEs observed in 3D 15N-edited NOESY are indicative of a-helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns a-HelixSequential NOEs observed in 3D 15N-edited NOESY are indicative of a-helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns b-SheetAcross strand NOEs observed in 3D 15N-edited NOESY and 3D 13C-edited NOESY are indicative of b-sheet

NMR Structure Determination Protein Secondary Structure and NOE Patterns b-SheetAcross strand NOEs observed in 3D 15N-edited NOESY and 3D 13C-edited NOESY are indicative of b-sheet

NMR Structure Determination Protein Secondary Structure and NOE Patterns b-SheetAcross strand NOEs observed in 3D 15N-edited NOESY and 3D 13C-edited NOESY are indicative of b-sheet

Protein Secondary Structure and NOE Patterns TurnsSequential NOEs observed in 3D 15N-edited NOESY are indicative of turns Similar to a-helix, shorter amino acid stretches connect b-strands

NMR Structure Determination

NMR Structure Determination Protein Secondary Structure and NOE Patterns TurnsSequential NOEs observed in 3D 15N-edited NOESY are indicative of turns Similar to a-helix, shorter amino acid stretches connect b-strands

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts Chemical shift differences between Ca,Cb random-coil values and experimentally observed values yields secondary structure chemical shift.Helix: DCa ~ 3 ppm DCb ~ -1 ppm

b-strand: DCa ~ -2 ppm DCb ~ 3 ppm

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts TALOS +Shen et al. (2009) J. Biomol NMR 44:213

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts TALOS+ Given the Ca, Cb Chemical shift assignments and primary sequence Compares the secondary chemical shifts against database of chemical shifts and associated high-resolution structure comparison based on triplet of amino acid sequences present in database structures with similar chemical shifts and secondary structure Provides potential f , y backbone torsion constraints

Issues: May not provide a unique solution, two or more sets of f , y are possible. Can not initially use TALOS results if ambiguous. Can add constraint latter if consistent with structure.

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts TALOS+TALOS may provide relatively tight error bounds associated with the predicted f,y.

It is better being more conservative by using minimal errors of:

f 30y 50c 20

NMR Structure Determination Protein Secondary Structure and 3JHNa Karplus relationship between f and 3JHNa f =180o 3JHNa = ~8-10 Hz b-strand f = -60o 3JHNa = ~3-4 Hz a-helixVuister & Bax (1993) J. Am.Chem. Soc. 115:7772

NMR Structure Determination Protein Secondary Structure and 3JHNa Karplus relationship between f and 3JHNa Measure 3JHNa for a protein using HNHA Ratio of cross-peak to diagonal intensity yields coupling constant Common approach to measure coupling constants in complex protein NMR spectraJ. Am. Chem. Soc. 1993,115, 7772-7777

NMR Structure Determination Protein Secondary Structure and NH Exchange Rates Relatively Slower Exchanging NHs are involved in Hydrogen Bonds or Buried Hydrogen Bonds are an important component of secondary structuresNH Exchange Rate

NMR Structure Determination Protein Secondary Structure and NH Exchange Rates Measure Relatively Slow Exchanging NHsExchange NMR sample into 100% D2O and measure series of 1H-15N HSQC as a function of time. Observed slowly exchanging NHs are correlated with secondary structure.Secondary structures identified from NOEs, chemical shifts and 3JHNa Assign hydrogen-bond distance constraintJ. Mol. Biol. (1997) 271, 472-487

NMR Structure Determination Protein Secondary Structure and NH Exchange Rates Measure Fast Exchanging NHs CLEANEX-PM experiment amount of exchange depends on mixing time Missing Peaks are slowly exchanging NHs Observed slowly exchanging NHs are correlated with secondary structure. Secondary structures identified from NOEs, chemical shifts and 3JHNa Assign hydrogen-bond distance constraint2D 1H-15N HSQCCLEANEX-PMNH exchange with H2O during spin-lockselects H2OJ. Am. Chem. Soc. (1997) 119, 6203J. Biomol. NMR (1998) 11:221

NMR Structure Determination Protein Secondary Structure and NH Exchange Rates Measure Fast Exchanging NHs CLEANEX-PM experiment amount of exchange depends on mixing time Can measure exchange rates by varying mixing time Missing Peaks are slowly exchanging NHs

NMR Structure Determination Protein Secondary Structure and NH Exchange Rates Assign Hydrogen-Bond Distance Constraints for Slowly Exchanging NHsFor a helix hydrogen bond constraints:constraint between Oi & Ni+42.8 0.4 0.5constraint between Oi & HNi+41.8 0.3 0.5

For b sheet hydrogen bond constraints areAcross strands:constraint between Oi & Nj2.8 0.4 0.5constraint between Oi & HNj1.8 0.3 0.5

NMR Structure Determination Protein Secondary Structure Summary Secondary Structure NOEs, slowly exchanging NHs, 3JNHa and secondary structure chemical shifts provide the foundation for detemining a protein NMR structure

NMR Structure Determination Protein Tertiary Structure Determination Include all the various structural information, disulphide bonds, hydrogen bonds, dihedral angles, chemical shifts, coupling constants, and NOES (distance constraints) to calculate (FOLD) the structure using simulated annealingDepiction of short-range and long range NOEsAnal. Chem. 1990, 62, 2-15

Simply Need to Complete the Assignment of All the Remaining NOEs in an Iterative Process to Obtain the Structure

NMR Structure Determination Automated Protein Structure Determination A number of efforts are on-going to automate the process (ARIA, AutoStructure, CYANA, Rosetta, etc) From assignment tables, NOE peak lists, coupling constants and slowly exchanging NHs.

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific AssignmentsAssign Hb1 and Hb2 chemical shifts and determine c1 dihedral angle

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific AssignmentsAssign Hb1 and Hb2 chemical shifts and determine c1 dihedral angle. Assign Val Hg1 and Hg2 chemical shifts and determine c1 dihedral angle. HNHB and HN(CO)HB experiments Relative intensities of Hb cross-peaks determines c1 and stereospecific assignmentHNHBHN(CO)HBCan include c1 dihedral constraints and replace HB# pseudoatoms with specific distance constraints.

Pseudoatoms require weaker upper bound constraints that can be tighten with specific assignment

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific AssignmentsAssign Leu Hd1 and Hd2 chemical shifts and determine c2 dihedral angle. based on c1 assignments Assign Ile c2 dihedral angle.

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific AssignmentsAssign Leu Hd1 and Hd2 chemical shifts and determine c2 dihedral angle. Assign Ile c2 dihedral angle. Long Range 13C-13C Correlation Relative intensities of Hd cross-peaks compared to diagonal determines 3JCaCd

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific Assignments Assign Hd1, Hd2 and He1,He2 chemical shifts and determine c2 dihedral angle for Phe and Tyr. Inferred assignment from structure. Phe and Tyr undergo rapid ring flip so Hd1, Hd2 and He1,He2 are equivalent degenerate chemical shifts c2 should be 90o or -90o, which are symmetrically equivalent

Based on which (90,-90) c2 is closer to the observed structure, add that c2 constraint and replace Hd#, He# pseudoatoms with stereoassignments that are consistent with the structure. Hd1He1

NMR Structure Determination Improving the Quality of NMR Structures Stereospecific Assignments Making stereospecific assignments increase the relative number of distance constraints while also tightening the upper bounds of the constraints There is a direct correlation between the quality of the NMR structure and the number of distance constraints more constraints higher the precision of the structure

Increasing Number of NOE Based Constraints

NMR Structure Determination Improving the Quality of NMR Structures Dipolar Coupling Constants Solid state NMR yields a powder pattern or a ensemble of chemical shifts due to different orientations relative to the magnetic field. CSA chemical shift anisotropy In liquids, isotropic tumbling averages the various orientations to zero Simulated in a solid by spinning the sample

w (3cos2q-1)Magic Angle (54.7o) averages to zero

NMR Structure Determination Improving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC) In liquids, dipolar coupling can become non-zero if isotropic tumbling is removed. proteins can be aligned by using bicelles, virus particles or polyacrylamide gels. mechanically impede the random tumbling of protein do not want and interaction between the protein and alignment media

NMR Structure Determination Improving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC) RDC can be measured for each bond vector in the protein RDCs are measured from a difference in the coupling constants between aligned and unaligned media. Different RDCs can be normalized based on ratios of bond lengths unalignedalignedDAB(NH) = 93.3 Hz 100.2 Hz = - 6.9 HzThe magnitude of RDC depends on the extent of the alignment of the protein and the orientation of the bond vector to BO

NMR Structure Determination Improving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC)DAB(q,f) = Da(3cos2q- 1) + 3/2 Dr(sin2q cos2f)]

where Da and Dr are the axial (**) and rhombic (2) components of the traceless diagonal tensor D given by 1/3[DZZ - (Dxx+ Dyy)/2] and 1/3(Dxx- Dyy), with Dzz > Dyy DxxDa and Dr measure the magnitude (intensity) of the alignment.q is the angle between the bond and the z-axis of the principal alignment frame (x, y, z) is the angle between the bond's projection in the x-y plane and the x-axisTo use DAB in a structure calculation, need to know Da and DrAlignment frame of the proteinOrientation of bond-vector in protein alignment

Improving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC)NMR Structure Determination Extremes of the histogram correspond to the alignment tensor components Dxx, Dyy, and DzzDxx+Dyy+Dzz =0Dzz = 2DaDyy = - Da(1 + 3/2 R)Dxx = - Da(1 - 3/2R)- use different normalized sets of RDCs (CH, NH, CaC, NC , NC, etc) to create one histogram , etc) - exclude residues with substantially lower order parameter than averageDxx most populated value in histogramDzz average of the high extreme values of RDCDyy average of the low extreme values of RDCR is the rhombicity given by Dr/DaRecreate Powder Pattern by histogram plot of all normalized RDCs

NMR Structure Determination Observed RDC only limits bond vector to taco shaped curve on sphereRefine RDCs against coordinate vector position that is free to move during Improving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC) RDC provide long-range (> 5 ) interaction RDCs identify angular orientation of bond vector to alignment axis No translational orientation of bond vector. RDC alone can not define a protein structure NOEs are necessary to define translational orientation of bond vectors Absolute alignment axis is unknown RDC are all relative to an alignment axis Allow the structure and RDC to be refined against a coordinate position that floats or changes during the simulation

Protein Science (2004), 13:549-554NMR Structure Determination Without RDCsWith RDCsWithout RDCsWith RDCsConsistency of Structures with Experimental RDCsImproving the Quality of NMR Structures Residual Dipolar Coupling Constants (RDC) Addition of RDCs do not induce dramatic changes in structure improves relative orientation/packing very valuable for proper alignment of complexes

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement protein structures generally calculated in vacuum. water has a significant effect on protein structures explicit solvent modelMD simulation in box of water box > 10 , keep solvent from edge 1000 to 10,000s water molecule Computationally expensive not usually done implicit solvent model treat solvent as a high dielectric continuous medium (e) around protein significantly faster different solvation models generalized Born model (GB)

where :where q point charge , r is the distance between the charges and a is the radius of ion, eo dielectric constant for a vacuum, e of solventhttp://cmm.cit.nih.gov/intro_simulation/node8.html

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement generalized Born model (GB) has been implemented in Xplor compare structures in vacuum to water no visible differenceXia et al. (1996) J. Biomol. NMR 22:317

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement generalized Born model (GB) has been implemented in Xplor subtle, but significant improvements compare structures in vacuum to water improves NH to CO hydrogen bonds improves f and y angle distributions

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor scriptRead in the GB parameter and topology files (partial charges)Set N- and C-terminal charges for protein

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor script (continued)Set standard electrostatic parametersSet GB parametersRead in volumens and tell Xplor to use GB in calculation

NMR Structure Determination Improving the Quality of NMR Structures Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor script (continued)Weakly restrain Ca position during dynamics

Dont allow large structural changes. Just make local changes to fix problems