Docking Molecular Docking Molecular Structures into EMStructures into EM

Lecture 2Lecture 2

Another Rigid Body Docking Another Rigid Body Docking ExampleExample

Rossman 2000, Fitting atomic models into Rossman 2000, Fitting atomic models into electron microscopy maps, Acta Cryst electron microscopy maps, Acta Cryst D56,1341-1349 D56,1341-1349 - nice description of 3 examples - nice description of 3 examples

Rossman 2000Rossman 2000

Human rhinovirus- about 90 of 100 known serotypes use the cell Human rhinovirus- about 90 of 100 known serotypes use the cell surface glycoprotein ICAM-1 or CD54 as a receptorsurface glycoprotein ICAM-1 or CD54 as a receptorstructure of ICAM-1 has been determined crystallographicallystructure of ICAM-1 has been determined crystallographicallyHRV-ICAM complex only determined via cryo-EM. May be HRV-ICAM complex only determined via cryo-EM. May be impossible to determine crystal structure because binding of ICAM impossible to determine crystal structure because binding of ICAM is a recognition event that initiates virus break-up and the release of is a recognition event that initiates virus break-up and the release of the virus RNA into host cell cytoplasmthe virus RNA into host cell cytoplasmICAM-1 usually functions to promote intercellular adhesion and ICAM-1 usually functions to promote intercellular adhesion and signalling in response to inflammationsignalling in response to inflammation

HRV14-Fab17-IA

Rossman 2000Rossman 2000glycosolated structures of ICAM-1 with glycosolated structures of ICAM-1 with various elbow angles were fitted into EM various elbow angles were fitted into EM map manuallymap manuallymodels were refined as rigid bodies in models were refined as rigid bodies in reciprocal space with respect to difference reciprocal space with respect to difference maps obtained by subtraction of HRV maps obtained by subtraction of HRV density from complex density using program density from complex density using program X-PLORX-PLORthe refinement process was reproducible to the refinement process was reproducible to better than 1.5 A between equivalent atomsbetter than 1.5 A between equivalent atomsdetermine atomic interaction between virus determine atomic interaction between virus and receptorand receptor

Automated Rigid-Body DockingAutomated Rigid-Body Docking

Originally, docking was done manuallyOriginally, docking was done manually

However, a variety of computational However, a variety of computational docking algorithms have been developed docking algorithms have been developed to perform reliable and reproducible fitting to perform reliable and reproducible fitting of rigid structures into low-resolution mapsof rigid structures into low-resolution maps

Correlation CoefficientsCorrelation Coefficients

The most widely used method for fitting atomic The most widely used method for fitting atomic structures into low-resolution EM maps is a systematic structures into low-resolution EM maps is a systematic maximization of the density cross-correlation of atomic maximization of the density cross-correlation of atomic models with electron density maps. models with electron density maps. packages used for this: COAN, DOCKEM and EMFITpackages used for this: COAN, DOCKEM and EMFITThe best results are obtained when the surface edges of The best results are obtained when the surface edges of individual components in a complex are well defined, and individual components in a complex are well defined, and where there are only small regions of densities that where there are only small regions of densities that cannot be assigned uniquely to a single component. cannot be assigned uniquely to a single component. However, at low resolution, standard correlation However, at low resolution, standard correlation coefficients lie within a small range – problem of false coefficients lie within a small range – problem of false positives in the fitting, particularly where the map positives in the fitting, particularly where the map includes densities not accounted for by docked includes densities not accounted for by docked compoundscompounds

Correlation Coefficients: Density Correlation Coefficients: Density FilteringFiltering

Several density filtering operations have Several density filtering operations have been proposed to overcome these been proposed to overcome these difficulties:difficulties: One approach involves the use of a mask to One approach involves the use of a mask to

block the overlapping region between the block the overlapping region between the densities arising from the individual docked densities arising from the individual docked components and the target map in components and the target map in calculations of the correlation coefficient ( calculations of the correlation coefficient ( RosemanRoseman, 2000, 2000). ).

Correlation Coefficients: Density Correlation Coefficients: Density FilteringFiltering

Another approach involves altering the Another approach involves altering the functional form of the compared densities by functional form of the compared densities by applying a filter that enhances detection of applying a filter that enhances detection of contourscontours (or surface) in the maps being (or surface) in the maps being comparedcompared

Filtering maximizes Filtering maximizes both density and contourboth density and contour overlap and therefore enhances numerical overlap and therefore enhances numerical scoring contrast between potential solutionsscoring contrast between potential solutions

Density FilteringDensity Filtering

Density FilteringDensity Filtering

Correlation Coefficients: Density Correlation Coefficients: Density FilteringFiltering

A drawback of density filtering is that the A drawback of density filtering is that the significant levels of noise that are present significant levels of noise that are present in low-resolution maps derived from in low-resolution maps derived from electron microscopy can be amplified by electron microscopy can be amplified by certain density filtering approaches, which certain density filtering approaches, which may increase the likelihood of "false-may increase the likelihood of "false-positive" fits of density. positive" fits of density.

Vector QuantizationVector Quantization

Automated docking is sometimes combined with Automated docking is sometimes combined with vector distributions by vector quantization of the vector distributions by vector quantization of the atomic modelatomic modelvector quantizationvector quantization is a data compression is a data compression technique used in image and speech processing technique used in image and speech processing applicationsapplicationsVector quantization offers a flexible way to Vector quantization offers a flexible way to develop a reduced representation of 3D develop a reduced representation of 3D biological data from a variety of biophysical biological data from a variety of biophysical sources sources SitusSitus package can be used for this package can be used for this

Vector QuantizationVector Quantization

data is represented by a data is represented by a small number of small number of “codebook” vectors to “codebook” vectors to reduce the combinatorial reduce the combinatorial complexity of the complexity of the structural comparisonstructural comparison

an actin monomer an actin monomer (backbone trace + atoms) (backbone trace + atoms) encoded by four encoded by four codebook vectorscodebook vectors : :

Vector QuantizationVector Quantization

In rigid-body docking, vector quantization is In rigid-body docking, vector quantization is used to discretize both high- and low-resolution used to discretize both high- and low-resolution data sets. data sets.

Pairs of corresponding codebook vectors are Pairs of corresponding codebook vectors are then identified and superimposed by a least-then identified and superimposed by a least-squares fit. The codebook vector least-squares squares fit. The codebook vector least-squares fit results in a superposition of the corresponding fit results in a superposition of the corresponding high- and low-resolution data sets:high- and low-resolution data sets:

Core WeightingCore Weighting

"core-weighting" approach - the construction of a complex structure "core-weighting" approach - the construction of a complex structure from many components is simplified to a series of single component from many components is simplified to a series of single component fitting procedures. fitting procedures. The "core" region of a structure is defined as the part whose density The "core" region of a structure is defined as the part whose density distribution is unlikely to be altered by the presence of adjacent distribution is unlikely to be altered by the presence of adjacent components. components. The "surface" region is the part that is accessible or can interact The "surface" region is the part that is accessible or can interact with other components. The region enclosed by the accessible with other components. The region enclosed by the accessible surface thus belongs to the core region. surface thus belongs to the core region.

Core WeightingCore Weighting

The single component fitting is conducted The single component fitting is conducted using a grid-threading Monte Carlo using a grid-threading Monte Carlo (GTMC) method that identifies the global (GTMC) method that identifies the global maximum state (best fit) among a series of maximum state (best fit) among a series of local maximum states determined by short local maximum states determined by short Monte Carlo searches originating at a Monte Carlo searches originating at a variety of grid points. variety of grid points.

Flexible DockingFlexible Docking

Large macromolecular assemblies often Large macromolecular assemblies often undergo large functional rearrangementsundergo large functional rearrangements

in some cases, the X-ray structure does in some cases, the X-ray structure does not correspond to the EM structurenot correspond to the EM structure

This complicates the fitting, as not only This complicates the fitting, as not only orientation, but conformational orientation, but conformational rearrangements must be consideredrearrangements must be considered

Flexible DockingFlexible Docking

flexible fitting is often done by domain flexible fitting is often done by domain segmentation followed by fitting of each segmentation followed by fitting of each domain as a rigid body block (see earlier domain as a rigid body block (see earlier example with actin/myosin)example with actin/myosin)

However, large conformational changes However, large conformational changes often involve tightly coupled motions often involve tightly coupled motions between domains between domains

Flexible Docking: Vector Flexible Docking: Vector Quantization in SitusQuantization in Situs

Flexible docking combines vector quantization with Flexible docking combines vector quantization with molecular mechanics simulations where conformational molecular mechanics simulations where conformational changes require flexible fitting. Codebook vectors are changes require flexible fitting. Codebook vectors are used as constraints for the fitting.   used as constraints for the fitting.  

The colored regions in the following diagram are the so-called Voronoi (nearest neighbor) cells whose centroids should coincide with the generating vectors:  

Flexible Docking: Vector Flexible Docking: Vector QuantizationQuantization

Fitting of the Fitting of the centroids to the centroids to the low-resolution low-resolution vector positions vector positions induces the induces the desired desired conformational conformational change. change. it is possible to it is possible to constrain the constrain the distances distances between the between the vectors to vectors to reduce the effect reduce the effect of noise and of noise and experimental experimental limitations on limitations on the vector the vector positions:positions:

Flexible Fitting: Normal Mode Flexible Fitting: Normal Mode AnalysisAnalysis

novel method for quantitative flexible docking of novel method for quantitative flexible docking of structures into EM mapsstructures into EM maps

in many cases, functional rearrangements of in many cases, functional rearrangements of macromolecules can be described by a small macromolecules can be described by a small number of low-frequency normal modesnumber of low-frequency normal modes

Flexible Fitting: Normal Mode Flexible Fitting: Normal Mode AnalysisAnalysis

Normal mode refinement procedure relies Normal mode refinement procedure relies on real space correlation between the on real space correlation between the calculated electron density map from calculated electron density map from atomic model and EM mapatomic model and EM map

The fitting is performed by deforming the The fitting is performed by deforming the structure along a set of low-frequency structure along a set of low-frequency normal modes in order to maximize the normal modes in order to maximize the correlation coefficientcorrelation coefficient

SitusSitus

Situs is Situs is set of routinesset of routines for quantitative docking of 3D for quantitative docking of 3D data at variable resolutiondata at variable resolutionThe software supports both rigid-body and flexible The software supports both rigid-body and flexible docking using a variety of fitting strategies. docking using a variety of fitting strategies. topology-representing neural networks are used to topology-representing neural networks are used to correlate the high- and low-resolution data sets – a form correlate the high- and low-resolution data sets – a form of vector quantizationof vector quantizationSitus is developed by the Laboratory for Structural Situs is developed by the Laboratory for Structural Bioinformatics, Bioinformatics, biomachina.orgbiomachina.org. . (other public software for map fitting - COAN ( (other public software for map fitting - COAN ( Volkmann and Volkmann and HaneinHanein, 1999, 1999), EMfit ( ), EMfit ( RossmannRossmann et al., 2001 et al., 2001), EMAN ( ), EMAN ( JiangJiang et al., 2001 et al., 2001; ; LudtkeLudtke et al., 1999 et al., 1999))

Situs PapersSitus Papers

J Struc. Biol, 1999, J Struc. Biol, 1999, 125:185-195125:185-195

J Struc. Biol, 2001, J Struc. Biol, 2001, 133:193-202133:193-202

SitusSitus

microtubules decorated with kinesin-related ncd proteins are used to demonstrate utility of packagesSitusSitus step-by-step step-by-step tutorial on this tutorial on this availableavailable

Situs: Vector QuantizationSitus: Vector Quantization

Given a certain number of vectors, need to make sure Given a certain number of vectors, need to make sure that the vector positions are that the vector positions are statistically reliablestatistically reliable and and reproduciblereproducible. . Situs employs two quantization algorithms with different Situs employs two quantization algorithms with different characteristics:characteristics:

the topology-representing neural network (TRN) performs a the topology-representing neural network (TRN) performs a global stochastic search using random start vectors. Often a global stochastic search using random start vectors. Often a number of statistically independent TRN runs is repeated and number of statistically independent TRN runs is repeated and the results are clustered and averaged. Useful if no prior the results are clustered and averaged. Useful if no prior information exists about the vector positions information exists about the vector positions

Linde-Buzo-Gray (LBG, also called Linde-Buzo-Gray (LBG, also called kk -means) algorithms -means) algorithms performs only a single run of local gradient descent search. It is performs only a single run of local gradient descent search. It is useful if one wants to update existing vector positions, or if one useful if one wants to update existing vector positions, or if one wants to add distance constraints.wants to add distance constraints.

Situs: VIRTUAL REALITY Situs: VIRTUAL REALITY

developing a 3-dimensional graphics extension developing a 3-dimensional graphics extension for Situs, termed SenSitus, that can support for Situs, termed SenSitus, that can support virtual-reality devices such as stereo glasses, 3-virtual-reality devices such as stereo glasses, 3-dimensional trackers, and force-feedback dimensional trackers, and force-feedback (haptic) devices. (haptic) devices. A force-feedback device measures a user's A force-feedback device measures a user's hand position and exerts a precisely controlled hand position and exerts a precisely controlled force on the hand. force on the hand. forces are calculated according to the correlation forces are calculated according to the correlation coefficient of density maps and crystallographic coefficient of density maps and crystallographic data. data.

Docking Issues/ Open ProblemsDocking Issues/ Open Problems

estimation of fitting qualityestimation of fitting quality

validation of resultsvalidation of results

estimation of fitting errorsestimation of fitting errors

detection of ambiguitiesdetection of ambiguities

attempt at automated solutionsattempt at automated solutions

Possible Future DevelopmentsPossible Future Developments

parallel computing can speedup processparallel computing can speedup process

further development of methods to further development of methods to combine data from NMR, XRC and EMcombine data from NMR, XRC and EM

models derived by homology modelling models derived by homology modelling are increasingly being used for docking are increasingly being used for docking studies - integration of docking methods studies - integration of docking methods with structural database searcheswith structural database searches

The “Other” docking: The “Other” docking: ligands and proteinsligands and proteins

Not this year …!Not this year …!