NMR Structure Determination With The NMR Assignments and Molecular Modeling Tools in Hand: All we...

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?

4.1Å4.1Å

2.9Å2.9Å

NOENOE

CCHH

NHNH

NHNH

CCHHJJ

NOENOE- a through space correlation (<5Å)- a through space correlation (<5Å)- distance constraint- distance constraint

Coupling Constant (J)Coupling Constant (J)

- - through bond correlationthrough bond correlation- dihedral angle constraint- dihedral angle constraint

Chemical ShiftChemical Shift

- - very sensitive to local changes very sensitive to local changes in environmentin environment- dihedral angle constraint- dihedral angle constraint

Dipolar coupling constants (D)Dipolar coupling constants (D)

- - bond vector orientation relative bond vector orientation relative to magnetic fieldto magnetic field- alignment with bicelles or viruses- alignment with bicelles or viruses

DD


Nuclear Overhauser Effect (NOE)Nuclear Overhauser Effect (NOE)

Nuclear Overhauser Effect (NOE, ) – 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)

i = (I-Io)/Io

where Io is thermal equilibrium intensity

N N

N+

N-X

X A

A

irradiate

Populations and energy levels of a homonuclear AX system (large chemical shift difference)

Observed signals only occur from single-quantum transitions


N+½

I

I S

S

Populations and energy levels immediately following saturation of the S transitions

N+½N-½

N-½

Saturated(equal population)

Saturated(equal population)

saturate

W1

A

W1A

W1X

W1X

W2

W0

Observed signals only occur from single-quantum transitions

Relaxation back to equilibrium can occur through:Zero-quantum transitions (W0)Single quantum transitions (W1)Double quantum transitions (W2)

N-½

N+½

N+½

N-½


W1

A

W1A

W1X

W1X

W2

W0

N-½

N+½

N+½

N-½

021

02

2 WWW

WWA

A

Xi

Steady-state NOE enhancement at spin A is

a function of all the relaxation pathways

If only W1, no NOE effect at HA

If W0 is dominant, decrease in intensity at HA negative NOEIf W2 is dominate, increase in intensity at HA positive NOE

For homonuclear (X=A), maximum enhancement is ~ 50% For heteronuclear (X=A), maximum enhancement is ~50%(X/A)

Intensity of NOE “builds-up” as a function of the mixing time (m)

Solomon Equation:


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 (c) recall: rotational correlation is the time it takes a molecule to rotate one radian (360o/2)

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 c.

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

c ~ 10ns (macromolecule)

c ~ 10ps (small molecule)

1/c

Nuclear Overhauser Effect (NOE)Nuclear Overhauser Effect (NOE)Mechanism for Relaxation

• Spectral density is constant for w << 1/c c decreases, o also decreases and T1 increases at 1/c ≈ o there is a point inflection

– W2 falls off first since it it the sum of two transitions relaxation rates via dipolar coupling are:

Extreme narrowing limit: 1/c >>o then o2c

2 <<1)

62262

62260

62261

12

))(1(

12

2

))(

3

3

)1(

3

rrW

rrW

rrW

c

cXA

c

c

cXA

c

c

cA

cA

NOE is dependent on the distance (1/r6) separating the two dipole coupled nuclei

Important: the effect is time-averaged!

2D NOESY (Nuclear Overhauser Effect)2D NOESY (Nuclear Overhauser Effect)

Relative magnitude of the cross-peak is related to the distance (1/r6) between the protons (≥ 5Ǻ).

NOE is a relaxation factor that builds-up duringThe “mixing-time (tm)

2D NOESY Spectra at 900 MHz2D NOESY Spectra at 900 MHz Lysozyme Ribbon DiagramLysozyme Ribbon Diagram


How DO We Go From the NOESY Data to A Structure?


We Need to Assign Each NOE Cross-Peak to A Specific 1H-1H Pair from Assignment Table.

10.5 ppm to 7.65 ppm

Residue N CO Ca Cb OthersD1 120.1 (8.08) 179.1 53.8 (4.37) 39.9 (3.00,0.58)E2 128.9 (9.93) 176.6 56.0 (4.55) 30.9 (2.11,1.78) Cg 36.2(2.75,2.41)D3 116.1 (8.90) 176.5 56.2 (4.73) 38.9 (2.70,2.34)E4 113.3 (7.45) 175.6 52.9 (4.71) 27.2 (1.23,0.63) Cg 34.8(2.57,1.79)R5 123.8 (7.97) 173.1 54.3 (4.43) 28.9 (1.84,1.47) Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)W6 127.8 (9.27) 177.1 55.4 (5.50) 30.8 (3.11)T7 109.9 (9.32) 174.8 59.6 (4.85) 71.3 (4.34) Cg 20.2(0.73)N8 115.2 (8.42) 174.5 50.9 (5.06) 38.7 (3.13,2.70)N9 116.7 (7.88) 173.3 52.1 (4.94) 38.0 (3.33,3.01)F10 110.1 (7.57) 177.2 58.2 (4.46) 38.5 (2.63,2.67)R11 121.2 (8.03) 175.0 55.8 (4.06) 29.7 (1.83,1.67) Cg 27.4(1.45,1.35);Cd 43.1(3.13)E12 119.0 (8.56) 177.9 52.3 (3.79) 27.4 (1.16,-0.05) Cg 36.6(2.19,1.97)Y13 122.1 (8.28) 173.2 61.9 (3.80) 41.2 (2.99,2.52)N14 121.5 (8.45) 175.7 54.7 (4.89) 39.2 (2.26)L15 127.7 (9.02) 176.9 58.0 (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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Assignment Table

What 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.


We Need to Assign Each NOE Cross-Peak to A Specific 1H-1H Pair from Assignment Table.

Residue N CO Ca Cb OthersD1 120.1 (8.08) 179.1 53.8 (4.37) 39.9 (3.00,0.58)E2 128.9 (9.93) 176.6 56.0 (4.55) 30.9 (2.11,1.78) Cg 36.2(2.75,2.41)D3 116.1 (8.90) 176.5 56.2 (4.73) 38.9 (2.70,2.34)E4 113.3 (7.45) 175.6 52.9 (4.71) 27.2 (1.23,0.63) Cg 34.8(2.57,1.79)R5 123.8 (7.97) 173.1 54.3 (4.43) 28.9 (1.84,1.47) Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)W6 127.8 (9.27) 177.1 55.4 (5.50) 30.8 (3.11)T7 109.9 (9.32) 174.8 59.6 (4.85) 71.3 (4.34) Cg 20.2(0.73)N8 115.2 (8.42) 174.5 50.9 (5.06) 38.7 (3.13,2.70)N9 116.7 (7.88) 173.3 52.1 (4.94) 38.0 (3.33,3.01)F10 110.1 (7.57) 177.2 58.2 (4.46) 38.5 (2.63,2.67)R11 121.2 (8.03) 175.0 55.8 (4.06) 29.7 (1.83,1.67) Cg 27.4(1.45,1.35);Cd 43.1(3.13)E12 119.0 (8.56) 177.9 52.3 (3.79) 27.4 (1.16,-0.05) Cg 36.6(2.19,1.97)Y13 122.1 (8.28) 173.2 61.9 (3.80) 41.2 (2.99,2.52)N14 121.5 (8.45) 175.7 54.7 (4.89) 39.2 (2.26)L15 127.7 (9.02) 176.9 58.0 (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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Assignment Table

To 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!

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 structure

How Many Peaks?What is each Peak’s Position?

Which are Noise Peaks?


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 Ǻ removes all possible assignments with distances ≥ 6Ǻ

• 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


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 Approach

“Two-Spin” Approximation – the observed volume or intensity of a NOE cross-peak is directly related to the distance between the 1H-1H pair

6

1)(

rIV

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)


Spin Diffusion – in the limit of c >> 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:

Strong 2.5 0.7 0.2 for NH-NH constraints use: 2.5 0.7 0.6

Medium 3.0 1.2 0.3 for NOEs with NH use: 3.0 1.2 0.5Weak 4.0 2.2 1.0Very Weak 5.0 2.0 1.0the lower limit is always set to slightly less than twice the hydrogen van der Waals radius (1.8Å)

• 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 community

NOEs 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


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 Å for Leu H-H 2.52 Å for Phe or Tyr H-H

Serious 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 distortions

Violated constraint

What would happen to the structure if both Leu had to be 3Å to the Ala ?

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 structure

Experimental Volumes from NOESY spectra

J. Am. Chem. Soc. 1990, 112, 6803-6809


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 process

Can relate cross-relaxation rates() 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 matrix

Very 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 (c)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


Two Very Important Facts to Remember • NOEs Reflect the Average Distance• Protein Structures Are Dynamic

We visualize protein structures as a static image

In reality, protein undergoes wide-ranges of motions (snapshots of 100 BPTI conformations)

J. Mol. Biol. (1999) 285, 727±740

NMR Structure Determination 22D, D, 1313C, C, 1515N Labeled ProteinN Labeled Protein

Resonance AssignmentsResonance Assignments Secondary StructureSecondary Structure

Low Resolution StructureLow Resolution Structure

High Resolution StructureHigh Resolution Structure

Dock LigandsDock Ligands

X-ray or Homology ModelX-ray or Homology Model

C.SC.SNOENOE

NMR Data CollectionNMR Data Collection

We have already discussed labeling the protein, data collection and the resonance assignment

Next Step, is to identify which residues adopt which secondary structure present


Protein Secondary Structure and NOE Patterns • -Helix

Sequential NOEs observed in 3D 15N-edited NOESY are indicative of -helix

NMR Structure Determination Protein Secondary Structure and NOE Patterns

• -SheetAcross strand NOEs observed in 3D 15N-edited NOESY and 3D 13C-edited NOESY are indicative of -sheet

Protein Secondary Structure and NOE Patterns • Turns

Sequential NOEs observed in 3D 15N-edited NOESY are indicative of turns Similar to -helix, shorter amino acid stretches connect -strands


NMR Structure Determination Protein Secondary Structure and NOE Patterns

• TurnsSequential NOEs observed in 3D 15N-edited NOESY are indicative of turns Similar to -helix, shorter amino acid stretches connect -strands

NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts

Chemical shift differences between C,C random-coil values and experimentally observed values yields secondary structure chemical shift.

Helix: C ~ 3 ppm C ~ -1 ppm

-strand: C ~ -2 ppm C ~ 3 ppm


1

2 3

4

I

II

III

IV


• TALOS +

Shen et al. (2009) J. Biomol NMR 44:213


• TALOS+ Given the C, C 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 , backbone torsion constraints

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


Protein Secondary Structure and Carbon Chemical Shifts• TALOS+

TALOS may provide relatively tight error bounds associated with the predicted ,

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

± 30 ± 50 ± 20

NMR Structure Determination Protein Secondary Structure and 3JHN

• Karplus relationship between and 3JHN

=180o 3JHN~8-10 Hz -strand = -60o 3JHN = ~3-4 Hz -helix

Vuister & Bax (1993) J. Am.Chem. Soc. 115:7772

NMR Structure Determination Protein Secondary Structure and 3JHN

• Karplus relationship between and 3JHN

Measure 3JHN 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 spectra

J. Am. Chem. Soc. 1993,115, 7772-7777


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 structures

NH 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 3JHN

Assign hydrogen-bond distance constraint

J. Mol. Biol. (1997) 271, 472-487


• 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 3JHN

Assign hydrogen-bond distance constraint

2D 1H-15N HSQC CLEANEX-PM

NH exchange with H2O during spin-lock

selects H2O

J. Am. Chem. Soc. (1997) 119, 6203J. Biomol. NMR (1998) 11:221


• 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


Protein Secondary Structure and NH Exchange Rates• Assign Hydrogen-Bond Distance Constraints for Slowly Exchanging NHs

For –helix hydrogen bond constraints:constraint between Oi & Ni+4 2.8 0.4 0.5constraint between Oi & HNi+4 1.8 0.3 0.5

For –sheet hydrogen bond constraints areAcross strands:constraint between Oi & Nj 2.8 0.4 0.5constraint between Oi & HNj 1.8 0.3 0.5

NMR Structure Determination Protein Secondary Structure Summary

• Secondary Structure NOEs, slowly exchanging NHs, 3JNH and secondary structure chemical shifts provide the foundation for detemining a protein NMR structure


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 annealing

Depiction of short-range and long range NOEs Anal. 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.


Improving the Quality of NMR Structures• Stereospecific Assignments

Assign H1 and H2 chemical shifts and determine 1 dihedral angle

NMR Structure Determination Improving the Quality of NMR Structures

• Stereospecific AssignmentsAssign H1 and H2 chemical shifts and determine 1 dihedral angle. Assign Val H1 and H2 chemical shifts and determine 1 dihedral angle. HNHB and HN(CO)HB experiments Relative intensities of H cross-peaks determines 1 and stereospecific assignment

HNHB

HN(CO)HB

Can include 1 dihedral constraints and replace HB# pseudoatoms with specific distance constraints.

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


Improving the Quality of NMR Structures

• Stereospecific AssignmentsAssign Leu H1 and H2 chemical shifts and determine 2 dihedral angle.

based on 1 assignments Assign Ile 2 dihedral angle.


• Stereospecific AssignmentsAssign Leu H1 and H2 chemical shifts and determine 2 dihedral angle. Assign Ile 2 dihedral angle. Long Range 13C-13C Correlation Relative intensities of H cross-peaks compared to diagonal determines 3JCC


• Stereospecific Assignments Assign H1, H2 and H1,H2 chemical shifts and determine 2 dihedral angle for Phe and Tyr. Inferred assignment from structure.

Phe and Tyr undergo rapid ring flip so H1, H2 and H1,H2 are equivalent degenerate chemical shifts 2 should be 90o or -90o, which are symmetrically equivalent

Based on which (90,-90) 2 is closer to the observed structure, add that 2 constraint and replace H#, H# pseudoatoms with stereoassignments that are consistent with the structure.

H1

H1


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


• 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

½(3cos2-1)Magic Angle (54.7o) averages to zero


• 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


• 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

)/()( 33 ArrDNHD ABBANHHNABAB

unaligned aligned

DAB(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


Improving the Quality of NMR Structures• Residual Dipolar Coupling Constants (RDC)

DAB() = Da(3cos2- 1) + 3/2 Dr(sin2 cos2)]

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 Dxx

Da and Dr measure the magnitude (intensity) of the alignment. 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-axis

To use DAB in a structure calculation, need to know Da and Dr

Alignment frame of the protein

Orientation of bond-vector in protein alignment



Extremes of the histogram correspond to the alignment tensor components Dxx, Dyy, and Dzz

Dxx+Dyy+Dzz =0Dzz = 2Da

Dyy = - 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 average

Dxx most populated value in histogram

Dzz average of the high extreme values of RDC

Dyy average of the low extreme values of RDC

R is the rhombicity given by Dr/Da

Recreate “Powder Pattern” by histogram plot of all normalized RDCs


Observed RDC only limits bond vector to taco shaped curve on sphere

Refine RDCs against coordinate vector position that is free to move during


RDC provide long-range (> 5 Å) 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), 13:549-554

NMR Structure Determination Without RDCs With RDCs

Without RDCs With RDCs

Consistency of Structures with Experimental RDCs


Addition of RDCs do not induce dramatic changes in structure improves relative orientation/packing very valuable for proper alignment of complexes

http://protsci.highwire.org/content/vol13/issue2/images/large/73210-04f2_F4TT.jpeg

http://protsci.highwire.org/content/vol13/issue2/images/large/73210-04f3_L4TT.jpeg


Improving the Quality of NMR Structures• Water Refinement

protein structures generally calculated in vacuum. water has a significant effect on protein structures

explicit solvent model–MD simulation in box of water– box > 10 Å, 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 () 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, dielectric constant for a vacuum, of solvent

http://cmm.cit.nih.gov/intro_simulation/node8.html


• Water Refinement generalized Born model (GB) has been implemented in Xplor

compare structures in vacuum to water– no visible difference

Xia et al. (1996) J. Biomol. NMR 22:317


• 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 and angle distributions


• Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor script

topology

@TOPPAR:topamber.inp

@TOPPAR:amberpatches.pro

end

parameter @TOPPAR:paramber.gb.inp end

segment

name="ASN1"

molecule name=ASN number=1 end

end

patch NASN refe=nil=(resid 1) end

patch CASN refe=nil=(resid 1) end

vector do (charge = charge + .2306) (name ht%)

vector show sum (charge) (all)

segment

name="ASN1"

molecule name=ASN number=1 end

end

patch NTER refe="+"=(resid 1) end

patch CTER refe="-"=(resid 1) end

vector show sum (charge) (all)

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Read in the GB parameter and topology files (partial charges)

Set N- and C-terminal charges for protein


• Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor script (continued)

parameter

nbonds

atom cdie trunc

e14fac=0.8333333 ! use this to reproduce amber elec

cutnb 500. ctonnb 480. ctofnb 490. ! essentially no cutoff

tolerance=100. ! only build the nonbonded list once

nbxmod 5 vswitch

wmin=1.0

end

end

parameters nbonds

EPS=1. WEPS=80. offset=0.09 GBHCT ! GB parameters

end

End

@TOPPAR:volumes.amber

vector do (rmsd = rmsd * 0.9) (all) ! reduce volumes by 10%

flags include gbse gbin end

parameter reduce selection=(all) overwrite=true mode=average end end

energy end

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Set standard electrostatic parameters

Set GB parameters

Read in volumens and tell Xplor to use GB in calculation


• Water Refinement generalized Born model (GB) has been implemented in Xplor sample Xplor script (continued)

! weakly restrain Calpha position

vector do (harmonic = 0.5) (name ca)

coor disp=reference @asn.-80.10.pdb

constraints harmonic end

flags include harmonic end

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Weakly restrain Ca position during dynamics

Don’t allow large structural changes. Just make local changes to fix problems

NMR Structure Determination With The NMR Assignments and Molecular Modeling Tools in Hand: All we...

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Transcript of NMR Structure Determination With The NMR Assignments and Molecular Modeling Tools in Hand: All we...