docking molecular structures into em. introduction detailed models of proteins are required in order...

Docking Molecular Docking Molecular Structures into EMStructures into EM

IntroductionIntroduction

Detailed models of proteins are required in Detailed models of proteins are required in order to elucidate the structure-function order to elucidate the structure-function relationshiprelationship

methods for directly imaging large methods for directly imaging large complexes at atomic level are not currently complexes at atomic level are not currently available (and are not likely to be soon).available (and are not likely to be soon).


atomic models provide detailed 3D data, but are atomic models provide detailed 3D data, but are taken out of their physiological context and are taken out of their physiological context and are often available only as smaller fragments of often available only as smaller fragments of larger unitslarger units

Datasets obtained by 3D-EM techniques reveal Datasets obtained by 3D-EM techniques reveal a good approximate of the macromolecular a good approximate of the macromolecular shape (in the form of surface) as well as the shape (in the form of surface) as well as the internal arrangement of mass within the surface. internal arrangement of mass within the surface.


pseudo-atomic-resolution models of pseudo-atomic-resolution models of macromolecular complexes can be macromolecular complexes can be generated by combining high-resolution generated by combining high-resolution data (XRC and NMR) for assembly data (XRC and NMR) for assembly components with lower-resolution data components with lower-resolution data (EM) for the whole complex(EM) for the whole complex

Qualitative Docking: Manual FittingQualitative Docking: Manual Fitting

the fit of the atomic model into the the fit of the atomic model into the isosurface envelopes is judged by eye and isosurface envelopes is judged by eye and correlated manually until the fit “looks correlated manually until the fit “looks best”best”

“ “ a jigsaw-puzzle”a jigsaw-puzzle”

Manual FittingManual Fitting

If components are large molecules with If components are large molecules with distinctive shapes at the resolution of the distinctive shapes at the resolution of the construction, manual fitting can often be construction, manual fitting can often be performed with relatively little ambiguityperformed with relatively little ambiguity

manual fitting is widely used:manual fitting is widely used: Baker TS and Johnson JE, 1996, Baker TS and Johnson JE, 1996, Low Low

resolution meets high – towards a resolution resolution meets high – towards a resolution continuum from cells to atomscontinuum from cells to atoms, Curr Opin , Curr Opin Struct Biol 6:585-95Struct Biol 6:585-95

Manual Fitting Example 1Manual Fitting Example 1

Rayment et al, 1993, Science, 261:58-65Rayment et al, 1993, Science, 261:58-65


muscle contraction occurs when thin actin muscle contraction occurs when thin actin filaments and thick myosin filaments slide filaments and thick myosin filaments slide past one anotherpast one another

Impossible to create a structural model for Impossible to create a structural model for how muscle contracts (contractile cycle) how muscle contracts (contractile cycle) without knowledge of the 3D structures of without knowledge of the 3D structures of the components and how they interactthe components and how they interact


Rayment et al. obtained a model for rigor Rayment et al. obtained a model for rigor complex of F actin and myosin head by complex of F actin and myosin head by combining the molecular structures (XRC) of the combining the molecular structures (XRC) of the individual proteins with electron density maps individual proteins with electron density maps obtained via cryo-EMobtained via cryo-EM XRC myosin S1 structure to 2.8 XRC myosin S1 structure to 2.8 Å resolutionÅ resolution coordinates for F actin obtained from x-ray structure coordinates for F actin obtained from x-ray structure

of G actin by fitting and refining model to x-ray fiber of G actin by fitting and refining model to x-ray fiber data from F actin gelsdata from F actin gels

data combined using low-resolution (~30 Å) electron data combined using low-resolution (~30 Å) electron density maps of Factin alone and myosin-actin density maps of Factin alone and myosin-actin complex calculated from images recorded from cryo-complex calculated from images recorded from cryo-EM. Manual building using FRODOEM. Manual building using FRODO


1.1. actin model positioned in F actin actin model positioned in F actin envelopeenvelope

2.2. actin envelope replaced with actin-actin envelope replaced with actin-myosin envelope and myosin structure myosin envelope and myosin structure rotated and translated into placerotated and translated into place

myosin head highly asymmetric, therefore myosin head highly asymmetric, therefore easy to position molecule unambiguously in easy to position molecule unambiguously in envelopeenvelope


clear that myosin head must clear that myosin head must be close to actin – leads to be close to actin – leads to model for molecular basis model for molecular basis for muscle contraction:for muscle contraction:


even though resolution of EM is only even though resolution of EM is only ~30 ~30 Å, the accuracy of the fitted results is Å, the accuracy of the fitted results is higher – ambiguity of ~5 Åhigher – ambiguity of ~5 Å


collision at the site of actin-S1 interface collision at the site of actin-S1 interface viewed not as shortcoming of model, but viewed not as shortcoming of model, but an indication that there may be a an indication that there may be a conformational change, which may conformational change, which may contribute to understanding structural contribute to understanding structural basis of contractile cyclebasis of contractile cycle


J. Virol, 1993, 67:1148-58J. Virol, 1993, 67:1148-58

HRV major cause of the common coldHRV major cause of the common coldover 100 serotypesover 100 serotypesstructure of HRV14 known to atomic structure of HRV14 known to atomic resolution – icosahedral, 300 resolution – icosahedral, 300 ÅÅcapsid is built from 60 copies of four capsid is built from 60 copies of four viral proteins – VP1, VP2, VP3 and VP4viral proteins – VP1, VP2, VP3 and VP4

Manual Fitting Example 2Manual Fitting Example 2EM graph of virus with EM graph of virus with complexed neutralizing complexed neutralizing antibodiesantibodiesimmunogenic sites all occur at immunogenic sites all occur at the rim of a 12 the rim of a 12 ÅÅ deep canyon deep canyon that encircles each of the that encircles each of the icosahedral fivefold axes. The icosahedral fivefold axes. The canyon is the site at which the canyon is the site at which the cell surface receptor proteins cell surface receptor proteins bind. Arrangement allows bind. Arrangement allows naturally-occurring mutants to naturally-occurring mutants to circumvent immune recognition circumvent immune recognition without affecting receptor without affecting receptor recognition.recognition.

HRV14-Fab17-IAHRV14


HRV14-Fab17-IA

crystal structures for crystal structures for both whole virus and both whole virus and Fab-17IA were knownFab-17IA were known

EM envelope served as EM envelope served as constraint for docking constraint for docking the Fab atomic model the Fab atomic model onto surface of HRVonto surface of HRV


translations of the Fab model translations of the Fab model by as little as 2-4 by as little as 2-4 Å moved Å moved substantial portions of the substantial portions of the model outside the EM model outside the EM envelope or caused the two envelope or caused the two models to overlapmodels to overlapdocking procedure lead to a docking procedure lead to a pseudo-atomic model of the pseudo-atomic model of the complex, from which complex, from which interacting surfaces could be interacting surfaces could be defineddefinedfrom this, a number of from this, a number of electrostatic interactions were electrostatic interactions were identified as contributing to the identified as contributing to the binding affinity of Fab for the binding affinity of Fab for the virus- confirmed by virus- confirmed by mutagenesismutagenesis

HRV14-Fab17-IA

Problems with Manual FittingProblems with Manual Fitting

Manual fitting does not always proceed to Manual fitting does not always proceed to happily- happily- Docking results may be invalid if the molecule Docking results may be invalid if the molecule

adopts a significantly different structure in adopts a significantly different structure in crystal and large complex (but may be proof crystal and large complex (but may be proof that a molecule HAS changed conformation)that a molecule HAS changed conformation)

Problems with Manual FittingProblems with Manual Fitting

divergent models of the same complex divergent models of the same complex docked by eye have also been reported:docked by eye have also been reported: e.g. Hoenger et al, 1998, J. Cell. Biol.e.g. Hoenger et al, 1998, J. Cell. Biol.

intracellular transport, flagella beating and other motile intracellular transport, flagella beating and other motile phenomena in cells are based on the interaction phenomena in cells are based on the interaction between microtubules and motor proteins such as between microtubules and motor proteins such as kinesins or dyneinkinesins or dynein

Hoenger et al, 1998, J. Cell. Biol.Hoenger et al, 1998, J. Cell. Biol.

motor proteins consist motor proteins consist of several domains – of several domains – a head, a stalk and a a head, a stalk and a tailtailcontroversial reports controversial reports on stochiometry of on stochiometry of binding of tubulin and binding of tubulin and kinesin – one head kinesin – one head per tubulin subunit or per tubulin subunit or twotwo

Hoenger et al, 1998, J. Cell. Biol.Hoenger et al, 1998, J. Cell. Biol.

Quantitative FittingQuantitative Fitting

objectiveobjective scoring functions can be used to assess the scoring functions can be used to assess the quality and refine the initial manual fit, or perform and quality and refine the initial manual fit, or perform and automated procedure:automated procedure:e.g. J. Structural Biology, 1999, 125,176-184e.g. J. Structural Biology, 1999, 125,176-184

global search with correlation between the calculated global search with correlation between the calculated electron density from atomic model and that observed electron density from atomic model and that observed by EMby EM

objective evaluation of the quality of the dockingobjective evaluation of the quality of the docking

Quantitative FittingQuantitative Fitting

The global search is followed by a statistical The global search is followed by a statistical analysis of the distribution of the fitting criterionanalysis of the distribution of the fitting criterion

results in definition of the confidence intervals results in definition of the confidence intervals that eventually lead to that eventually lead to solution setssolution sets

solution sets are small regions in parameter solution sets are small regions in parameter space that satisfy the data within the error space that satisfy the data within the error margin defined by the chosen confidence margin defined by the chosen confidence intervalinterval

Solution SetsSolution Sets

The size of the solution set can serve as a The size of the solution set can serve as a normalised goodness-of-fit criterion – the smaller normalised goodness-of-fit criterion – the smaller the set the better the data that determines the the set the better the data that determines the position of the fitted atomic structureposition of the fitted atomic structure

Solution sets allow for the use of standard Solution sets allow for the use of standard statistical tests, such as Student’s t-test, to statistical tests, such as Student’s t-test, to evaluate the differences between models in evaluate the differences between models in different functional states and to help model different functional states and to help model conformational changesconformational changes

Difference MappingDifference Mapping

Difference mapping Difference mapping between the density between the density calculated from the fitted calculated from the fitted model and the model and the reconstruction from reconstruction from electron microscopy can electron microscopy can locate portions of the locate portions of the structure not present in the structure not present in the crystal structure or identify crystal structure or identify conformational changesconformational changese.g. actin-myosin S1 e.g. actin-myosin S1 (Rayment 1993) – light (Rayment 1993) – light chain positional change on chain positional change on binding to actinbinding to actin

search volume for

S1 fragments

solution-set centre fit of

complete S1 structure –note bad fit

of white domain

map of density

difference between S1 and b cyan


motor domain of

S1 structure

solution-set centre fit of light chain

into remaining density

difference density

between light chain

and c


complete S1 structure –note bad fit

of white domain


motor domain of

S1 structure

search volume for S1 fragments

map of density difference between

S1 and b cyan

solution-set centre fit of light chain into remaining density difference

density between

light chain and c

docking molecular structures into em. introduction detailed models of proteins are required in order...

Documents