Top Banner
Solution structures by NMR
114

Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Dec 22, 2015

Download

Documents

spin slide

nmr slide

d slide

1h1h slide

dimension slide

glyval slide

hh hh slide

molecular weight slide

Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Solution structures by NMR

Page 2: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

• • Structure Structure •• MobilityMobility interactionsinteractions}}

NMR is a powerful method to address these NMR is a powerful method to address these problemsproblems

• • 19841984 1 1 (first structure!) (first structure!) •• 19901990 25 25 •• 19941994 8080 (first paramagnetic structure!) (first paramagnetic structure!) •• 19981998 125 125 •• 20002000 200200

NMR structures per yearNMR structures per yearyearyear

•• Protein-protein Protein-protein •• Protein-ligandProtein-ligand

Structural Biology

Page 3: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

NMRResonance assignment

Structure calculations

3D structure

NOE intensities and J couplings (plus other structural constraints)

Conversion of NMR data in distances and

angles

Structure refinement:

REM, RMD

Structural constraints

Structure determination through NMRStructure determination through NMR

Page 4: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 5: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 6: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 7: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Assignment of homonuclear spectra

Page 8: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

TOCSY Total Correlation Spectroscopy

Esperimento 2D analogo al COSY, utile per misurare gli accoppiamenti scalari “consecutivi”

Page 9: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Spin System•Group of spins that are connected by scalar (through bond) spin-spin couplings (J)

•In proteins each amino acid is composed of one or more spin systems with the

peptide bond separating them

•Spin systems of the non-labile H atom in H-C-R can be classified as

AX (H-CH: Gly), AMX (H-CH2: Ser, Cys, Asp, Asn), etc.

Page 10: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Gly Val

Page 11: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Identification of spin systems through TOCSY

HHNN

H H ,,

Ala Long chainLong chain(Ile)(Ile)

7.08.0

2.0

4.0

Page 12: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Identification of aromatic residues through TOCSY

2 Trp, 1 Phe, 1 Tyr are identified

TrpTrp

PhePhe

TyrTyr

Page 13: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sistemi di spin - GLY

GLY

D(1H)

D(

1H)

HN H1,H2

Page 14: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sistemi di spin - ALA

GLY

D(1H)

D(

1H)

HN H, CH3

Page 15: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sistemi di spin - VAL

GLY

D(1H)

D(

1H)

HN CH3 ,CH3HH

Page 16: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sistemi di spin

Alcuni esempi di aminoacidi e dei relativi sistemi di spin

Page 17: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sistemi di spinAlcuni esempi di aminoacidi e dei relativi sistemi di spin

Page 18: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 19: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 20: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 21: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 22: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 23: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 24: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 25: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 26: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

NMR Tecniques and molecular weight

Page 27: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Multifrequency NMR Multifrequency NMR experimentsexperiments

To make full use of multidimensional NMR, To make full use of multidimensional NMR, isotope labeled samples are neededisotope labeled samples are needed

Multifrequency NMR experimentsMultifrequency NMR experiments

For each frequency dimension a different type For each frequency dimension a different type of coupling can be detectedof coupling can be detected

Page 28: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Extending to the 3rd dimension

Page 29: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Multi-Dimensional NMR: why should we go to higher dimensions?

By introducing a third dimension we are improving the resolution and reducing the overlap problems

F1

F3

F2

15N

1H

1H

if now take an homonuclear

experiment and you label 1HN signals

with the 15N frequency of their

own amide nitrogen, you create

a 3D

Page 30: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

By introducing a third dimension we are increasing spectral resolution and reducing the overlap problems

F1

F3

F2

15N

1H

Multi-Dimensional NMR: why should we go Multi-Dimensional NMR: why should we go to higher dimensions?to higher dimensions?

1H

Page 31: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

3D HSQC-NOESY

Sequential assignment

Different slices are shown

Page 32: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Assignment of heteronuclear spectra:

Similarly, the above method of sequential assignment can be used to assign heteronuclear 3D spectra such as 15N NOESY-HSQC and 15N TOCSY-HSQC, because in each 15N plane of such a spectrum only the NOESY or TOCSY signals from that proton are visible which is directly attached to the regarded nitrogen atom. Therefore, a 15N plane is a subspectrum of the respective 2D spectrum. The biggest difference from the 2D method results from the different frequency range of 15N spectra: Only the signals of those protons are detected that interact with a 15N-bound proton. Therefore, only frequencies between 12 to 5 ppm are detected in the acquisition dimension. The sidechain region to the right of the water signal is missing in a 15N-NOESY-HSQC or 15N-TOCSY-HSQC.

Page 33: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Identification of spin systems through TOCSY

HHNN

H H ,,

Ala Long chainLong chain(Ile)(Ile)

7.08.0

2.0

4.0

Page 34: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

NMR Tecniques and molecular weight

Page 35: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 36: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

J couplings for backbone resonances

Page 37: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

The Concept of Triple Resonance

Page 38: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Triple Resonance

J couplings

Relaxation rates

Page 39: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

HNCO

H – N - CO

Trasferisco da Hn ad NOsservo N prima dimensioneTrasferisco da N a COOsservo CO Seconda dimensioneTrasferisco da CO a N, da N ad HnOsservo Hn Terza dimensione

Page 40: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sequential asignment using the HNCA and HNCOCA experiments

Page 41: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Assignment with triple resonance spectra:

offer a third approach to sequential assignment, which does not need any knowledge about spin systems. Instead the sequential correlations via 1J and 2J couplings are used to establish connectivities between amino acids. The general strategy can be explained using the HNCA spectrum as an example:

A HNCA spectrum has three frequency axes: 1H, 15N and 13C. It correlates an amide proton with the Calpha atom of the 'own' and in most cases also with the Calpha of the preceeding amino acid. A 1H/15N projection of a HNCA looks like an HSQC: Each signal represents a single amino acid. At the frequency of each amide proton there are two cross signals in the Calpha dimension: One from the intraresidual and one from the interresidual Calpha atom. Using these cross signals a chain of correlations through the whole amino acid sequence can be established, just like building a chain of dominoes. However, proline residues interrupt that chain due to their missing amide proton.

Page 42: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Assignment procedure

Page 43: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sequential assignment in triple resonance experiments. HNCA

13C

1HBackbone assignment of 6 residues using 13C

Page 44: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Transfer without acquisition

Page 45: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Side chain Assignment

Page 46: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 47: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

CO

HN

HNN

HNN

HNN

Sequential assignment using triple resonance experiments

HNCO & HNCACO experiments

Page 48: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

13C Chemical shift as a tool for Assignment

Page 49: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

NMR structural characterization of the target protein

Approaches to the Structure Determination of Proteins

•For proteins of up to 30 kDa, use 13C/15N-labelling

•For proteins of higher molecular weight, use fractional deuteration and 13C/15N-labelling

•For proteins of 100 kDa and above, use selective protonation and 13C/15N-labelling

Page 50: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 51: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

CO

HN

HNN

HNN

HNN

Sequential assignment using triple resonance experiments

HNCO & HNCACO experiments

Page 52: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Further NMR experiments

•Side-chain assignments

•Measurement of NOE contacts

Page 53: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 54: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Almeno qui

Page 55: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Classical constraints for Classical constraints for structure determination structure determination

DistancesDistances

,,,,

Vector Vector orientationorientation

33J couplings J couplings Chemical shiftsChemical shifts

Residual dipolar couplings Residual dipolar couplings Cross Correlation effectsCross Correlation effects

NOENOEH-bondsH-bonds

{{

{{

{{

Page 56: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

N

Side chain Torsion angles.

Protein structure and dihedral anglesProtein structure and dihedral angles

Page 57: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

NOE class

distance [Å]

upper bound

[Å]

very strong 2.3 2.5

strong 2.8 3.1

medium 3.1 3.4

weak 3.5 3.9 very

weak 4.2 5.0

In this procedure, all non-sequential signals which are visible in the NOESY spectra have to be assigned, the number of which easily exceeds 1000 in a medium-sized protein (ca. 120 amino acids). It is distinguished between cross peaks of protons no more than five amino acids apart in the protein sequence (medium range NOE's) and those which are more than five amino acids apart (long range NOE's). The former are mainly indicative of the protein backbone conformation and are used for secondary structure determination, whereas the latter are an expression of the global structure of the protein and therefore contain the main information used for tertiary structure calculation. In addition to interproton distances the phi-dihedral angles of the protein backbone can be determined from a COSY spectrum or a HNCA-J spectrum (a variant of the HNCA spectrum, from which the coupling constants of the N-Calpha bonds can be determined). Dihedral angles are connected with the coupling constants via the Karplus equation .

Contraints for Structure Calculation

So far, the emphasis has been on identification of the observed signals in the spectra and their correlation with the amino acid protons giving rise to the signals. Afterwards, one has to extract the data which are relevant for the structure. Of special importance in this respect are proton-proton distances, which can be estimated from the signal intensities in the 2D NOESY, 3D 15N-NOESY-HSQC and 3D 13C-NOESY-HSQC spectra .

Signal intensity depends on the distance r between two nuclei i and j, according to:

NOEij ~ 1/rij6

Distances are derived from the spectra after calibration against NOE signals for known distances (such as distances in elements of secondary structure) and grouped into a few classes. An upper and a lower bound of distance is assigned to each class. The lower bound is often set to the sum of the van der Waals radii of the two protons.

Page 58: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

. .. .. .

.. .. .

.. .. .. . .

.

.. .

....

C constant is initially determined from NOE’s between protons at fixed distance

log V

log r

log V = log C - n·log r

 

nr

CV where C is a constant and n can vary from 4 to 6.

Classes of constraints:1.             intra-residue (except NH, H e H)2.             sequential and intra-residue NH, H e H3.             medium range4.             long range backbone

CALIBA CALIBA (CALIBrAtion of NOE intensity versus distance constraints)(CALIBrAtion of NOE intensity versus distance constraints)

 The NOESY cross-peak intensities (V) are converted into upper distance limits (r) through the equation:

Güntert, Braun, and Wüthrich, et al

Page 59: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

N

Side chain Torsion angles.

Protein structure and dihedral anglesProtein structure and dihedral angles

Page 60: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of 3D protein and nucleic acid Calculation of 3D protein and nucleic acid structuresstructures

Güntert P., Mumenthaler C., Wüthrich K., J.Mol. Biol., 1997

Simulated annealing combined with molecular dynamics in torsion angle space

Numerical solution of the classical mechanical Lagrange equations of motion with torsion angles as generalized internal coordinates

The target function represents the potential energy of the system

A temperature bath to cross barriers between local minima is cooled down slowly from its initial high temperature

The NMR constraints are used as pseudopotential to calculate the velocity

The program DYANAThe program DYANA

VdWWddUE iiiiiipot 2020 cos1 + other constraint contributions

Page 61: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

• Covalent geometry • Torsion angles • Chirality • Planarity • Precision

•Restraint violations

Results are presented as plots suitable for publication

Structure quality through PROCHECKStructure quality through PROCHECK

Laskowski R A, MacArthur M W, Moss D S & Thornton J M (1993). J. Appl. Cryst., 26, 283-291.

Page 62: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Classical constraints for Classical constraints for structure determination structure determination

DistancesDistances

,,,,

Vector Vector orientationorientation

33J couplings J couplings Chemical shiftsChemical shifts

Residual dipolar couplings Residual dipolar couplings Cross Correlation effectsCross Correlation effects

NOENOEH-bondsH-bonds

{{

{{

{{

Page 63: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Strategies for Sequential Assignment

Using this cyclic procedure of alternatively connecting intraresidual TOCSY with interresidual NOESY cross peaks one can walk - ideally - along the entire length of the protein.

Problem: there are a few proline residues in most proteins.

Problem: there are a number of additional short proton proton distances which can occur as a result of certain elements of secondary structure.

The general work of Wuthrich and co-workers identified a whole range of secondary specific short proton proton distances that are summarized here:

Page 64: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Strategies for Sequential Assignment

Here are a number of characteristic distances that connect the two strands of a -sheet; short enough to appear as cross peaks in a NOESY spectrum.

These are - , amide- and amide-amide distances

-sheet specific NOEs in red and simple sequential NOEs in green.

Other regular elements of secondary structure, e.g. different types of -turns, 3-10 helices and parallel -strands, are characterized by similar patterns of short distances involving backbone protons.

Page 65: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

Results - The Structure FamilyAfter the structural calculations a family of structures is obtained instead of an exactly defined structure. This family spans out a relatively narrow conformational space. Therefore, the quality of a NMR structure can be defined by the mean deviation of each structure from this family (RMSD) from an energy minimized mean structure which has to be calculated previously. The smaller the deviation from this mean structure the narrower the conformational space. Another definition of RMSD is to compare pairwise the structures of a family and calculate the mean of these deviations. The RMSD is different for different parts of the protein structure: Regions with flexible structure or without secondary structure (loops) show a larger deviation than those with rigid and well defined secondary structure. This higher RMSD in loops results in first instance from the smaller number of distance constraints for these parts of the protein structure. Additionally it can originate from real flexibility, but this diagnosis can only be confirmed by measuring the relaxation times for the protein. A result of a structure calculation is shown here:

Page 66: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

Page 67: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

The idea of computer-aided structure calculation is to convert distance- and torsion-angle-data (constraints) into a visible structure. However, the experimentally determined distances and torsion angles by themselves are not sufficient to fully characterize a protein structure, as they are based on a limited number of proton-proton distances. Only the knowledge of empirical input data, such as bond lengths of all covalently attached atoms and bond angles, enables a reasonably exact structure determination.

Page 68: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

For this purpose, a randomly folded starting structure is calculated from the empirical data and the known amino acid sequence. The computer program then tries to fold the starting structure in such a way, that the experimentally determined interproton distances are satisfied by the calculated structures. In order to achieve this, each known parameter is assigned an energy potential, which will give minimal energy if the calculated distance or angle coincides with its input value. The computer program tries to calculate a structure with a possibly small overall energy.

Page 69: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

Without the experimentally determined distance- and torsion angle-constraints from the NMR spectra, the protein molecule can adopt a huge number of conformations due to the free rotation around its chemical bonds (except for the peptide bond, of course). the N-Calpha bond and the Calpha-CO bond. All these possible conformations are summed up in the so-called conformational space. Therefore, it is important to identify as many constraints as possible from the NMR spectra to restrict the conformational space as much as possible, thus getting close to the true structure of the protein. In fact, the number of constraints employed is more important than the accuracy of proton-proton distances, so that the classification above is sufficiently precise.

Page 70: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Calculation of Tertiary Structure

Energy PotentialsA starting structure is needed for a molecular dynamics calculation, which is generated from all constraints for the molecular structure, such as bond-lengths and bond-angles. This starting structure may be any conformation such as an extended strand or an already folded protein. During the simulation, it develops in a potential field under the influence of various forces, in which all information about the protein is summarized. Two classes of energy terms are distinguished: Eempirical and Eeffective:

V = Eempirical + Eeffective

with: Eeffective = ENOE + Etorsion,

and Eempirical = Ebond + Eangle + Edihedral + Evdw + Eelectr

Eempirical contains all information about the primary structure of the protein and also data about

topology and bonds in proteins in general.                                   The contributions of covalent bonds, bond-angles and dihedral angles towards Eempirical are approximated by a harmonic

function. In contrast, non-covalent van-der-Waals forces and electrostatic interactions are simulated by an inharmonic Lennard-Jones potential or Coulomb potential, respectively. E effective

takes the experimentally determined constraints into account. Angle constraints are introduced by a harmonic function analogous to that for the dihedral angles. For distance constraints, the energy potential will be set to zero, if the corresponding distance is within the given limits. If it is outside these limits, a harmonic energy potential is used, which tries to push the value of the distance into the limits.

Page 71: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

AGGFHRLIFTHWQDCSAAVHYLGGP………………..

Ogni aminoacido ha valori precisi di Distanze tra atomi.

Libreria

Page 72: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 73: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 74: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 75: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Sequenza primariaLibreria di aminoacidiLegame peptidico 180°

Distanze tra protoni intraresiduoDistanze tra protoni di residui consecutiviDistanze tra protoni di residui a breve distanza (i,i+4)Angoli diedri

Distanze tra prootni a madio e lungo raggio

Page 76: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 77: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Cosa Ottengo?

Page 78: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 79: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Target (penalty) Function

Ripeto il calcolo n volte

Per Ogni struttura calcolo il valore della funzione penalità

Seleziono le strutture che hanno il piu’ basso valore della funzione penalità

Page 80: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Target (penalty) Function

La somma delle violazioni dei vincoli sperimentali

E’ di fatto, impossibile ottenere una struttura che sia in grado di rispettare perfettamente l’insieme di tutti i vincoli sperimentali che noi imponiamo

Non ci sono solo I vincoli sperimentali, ma quelli derivanti dalla struttura di un polipeptide, (es: le violazioni di Van der Walls, gli angoli non permessi, etc..)

Page 81: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Target (penalty) Function

La somma delle violazioni dei vincoli sperimentali

Considero “buone” tutte quelle strutture che hanno il più basso valore della funzione penalità

Page 82: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Famiglia di strutturePerché ne considero 20 e non una sola?

In principio, esistono infiniti modi (conformazioni) che permettono di ottenere una bassa funzione penalità.

Non vi é nessun motivo per sceglierne una piuttosto che un altra

In linea di principio, la conformazione a piu’ bassa funzione penalità é considerabile “la migliore”, ma tutte le altre che hanno valore molto simile sono ugualmente valideQuindi, preferisco prendere in considerazione un numero fisso di conformazioni (20, o 30) che hanno la piu’ bassa penalità e vedere quanto esse sono simili tra loro

Page 83: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Famiglia di struttureRMSD

Se le strutture sono molto simili tra loro significa che tutte le conformazioni che ho considerato sono molto simili. Avro’ una struttura accurata

Page 84: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Famiglia di struttureRMSD

Se le strutture sono molto diverse tra loro significa che devo considerare come ugualmente “buone” conformazioni molto diverse. Avro’ una struttura molto poco accurata

Page 85: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 86: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 87: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Rappresentazione alternativa di una struttura in Rappresentazione alternativa di una struttura in soluzione, ovvero di una famiglia di strutturesoluzione, ovvero di una famiglia di strutture

Page 88: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Cyt c oxidized Cyt c reduced

Banci, Bertini, Bren, Gray, Sompornpisut, Turano, Biochemistry, 1997

Baistrocchi, Banci, Bertini, Turano, Bren, Gray, Biochemistry, 1996

Solution structure of oxidized and Solution structure of oxidized and reduced Cytochrome creduced Cytochrome c

Page 89: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Active site channel of Reduced Monomeric Active site channel of Reduced Monomeric Q133 Copper Zinc Superoxide dismutaseQ133 Copper Zinc Superoxide dismutase

Reduced Reduced Q133M2SODQ133M2SOD

Oxidized human Oxidized human SODSOD

Page 90: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

0 20 40 60 800.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

Paramagnetic structureDiamagnetic structure

RM

SD

back

bone

Residue Number

Bertini, Donaire, Jiménez, Luchinat, Parigi, Piccioli, Poggi, J.Biomol. NMR, 2001,21,85-98

Structure of CeStructure of Ce3+3+ substituted Calbindin D substituted Calbindin D9k9k

Page 91: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

The solution structure of a human The solution structure of a human SOD mutant SOD mutant

Page 92: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Stucture Calculation Structure Validation Structure Visualisation

Page 93: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Structure validation

Page 94: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

RMSD

How to overlay structures-entire-fragments- bb & all heavy atoms

Page 95: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

RMSD

Page 96: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

RMSD

Page 97: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Stereoviews

Page 98: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Average pairwise rmsd values calculated for backbone heavy atoms N, Ca, and C' ("Backbone"), all heavy atoms ("All heavy"), and backbone heavy atoms N, Ca, and C' together with heavy atoms of the best defined side-chains. The values for the DYANA structures are represented by red bars, and values for molecular dynamics refined (MDR) and energy-minimization refined (EMR) structures are displayed in green and gray, respectively. Standard deviations are indicated by vertical error bars.

Page 99: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Target function analysis

Violations < threshold Energy terms

Page 100: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 101: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

PROCHECK The PROCHECK suite of programs provides a

detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of both the overall quality of the structure, as compared with well-refined structures of the same resolution, and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved, but also of existing structures and those being modelled on known structures.

Page 102: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

PROCHECK & PROCHECK-NMR The only input required for

PROCHECK is the PDB file holding the coordinates of the structure of interest. For NMR structures, each model in the ensemble should be separated by the correct MODEL and ENDMDL records

Page 103: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 104: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 105: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 106: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 107: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 108: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 109: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 110: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 111: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Model-by-model secondary structures

Page 112: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)
Page 113: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Plots of PROCHECK G-factor (all dihedrals) vs. MOLPROBITY Z-scores (1) calculated for x-ray crystal structures (circles) deposited in the PDB during 2000-2004 colored according to resolution [green: high-resolution (£ 1.8 Å); gray: medium-resolution (1.8 – 2.5 Å); red: low-resolution (2.5– 3.5 Å)] and NMR structures (yellow triangles) deposited in the PDB during 2000-2004 by other leading NMR groups.

Page 114: Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Related Documents
structure elucidation by modern nmr 92 Duddeck

structure elucidation by modern nmr 92 Duddeck

Category: Documents
Protein NMR Part II. 1. Protein Structures by NMR NMR, UNLIKE Xray crystallography and EM, DOES NOT experimentally produce a protein structure. NMR yields.

Protein NMR Part II. 1. Protein Structures by NMR NMR,...

Category: Documents
NMR study of lithium mobility in polymer electrolytes

NMR study of lithium mobility in polymer electrolytes

Category: Documents
NMR in biology: Structure, dynamics and energetics

NMR in biology: Structure, dynamics and energetics

Category: Documents
Artificial Neural Networks for NMR Structure Elucidation ...edoc.unibas.ch/511/1/DissB_7681.pdf · Artificial Neural Networks for NMR Structure Elucidation of Oligosaccharides Inauguraldissertation

Artificial Neural Networks for NMR Structure Elucidation...

Category: Documents
NMR studies of protein dynamics and structure

NMR studies of protein dynamics and structure

Category: Documents
Full wwPDB NMR Structure Validation Report ifiles.rcsb.org/pub/pdb/validation_reports/rs/2rsc/2rsc_full_validation.… · Full wwPDB NMR Structure Validation Report ... 6.4 Non-standard

Full wwPDB NMR Structure Validation Report...

Category: Documents
NMR Experiments for Structure Determination

NMR Experiments for Structure Determination

Category: Documents
Full wwPDB NMR Structure Validation Report i - …files.rcsb.org/pub/pdb/validation_reports/my/2myl/2myl_full... · Full wwPDB NMR Structure Validation Report ... 1.7.3 (157068),

Full wwPDB NMR Structure Validation Report i -...

Category: Documents
Protein Structure Determination by NMR - AUbionmr.chem.au.dk/download/tv/tmp/Protein_NMR.pdf · 2 Thomas Vosegaard Protein Structure Determination by NMR •Proteins •Primary, secondary,

Protein Structure Determination by NMR -...

Category: Documents
Solution structures by NMR. Structure Structure Mobility Mobility interactions } NMR is a powerful method to address these problems 19841 (first structure!)

Solution structures by NMR. Structure Structure Mobility...

Category: Documents
NMR INVESTIGATIONS OF PROTEIN STRUCTURE

NMR INVESTIGATIONS OF PROTEIN STRUCTURE

Category: Documents