recent advances established electron microscopy as a central technique for studying the structures of macromolecular assemblies in different functional states in vitro and in vivo . because the resolution of an electron microscopy density map is relatively low , fitting of atomic resolution component structures into the density map of the whole assembly is essential .
recent advances have established electron microscopy ( em ) as a central technique for studying the structures of macromolecular assemblies in different functional states in vitro and in vivo ( 1 ) .
the resolution of an em density map is typically better than 25  , and can be as high as 4  for highly symmetric structures ( 2,3 ) . in most cases , however , the resolution is insufficient to construct a full atomic model of a protein complex . to this end ,
fitting of atomic resolution structures into an em density map of the whole assembly is essential ( 48 ) . in the past decade ,
different algorithms have been developed for fitting a single protein subunit into its density map ( 920 ) .
most methods use a variant of the cross - correlation coefficient as the quality - of - fit measure ( 21 ) .
the position of a protein subunit inside the density map is sampled either exhaustively or by matching precalculated geometric features .
methods for fitting multiple components of large assemblies have also been recently described ( 2225 ) . in particular , we have developed the multifit module of the integrative modeling platform ( imp , http://www.salilab.org/imp/ ) software package ( 23,26 ) .
multifit simultaneously positions protein subunits into a density map of a protein assembly by combining geometric criteria commonly used in molecular docking and quality - of - fit criteria commonly used in em fitting .
the method was validated in the 2010 em modeling challenge ( http://ncmi.bcm.edu/challenge/ ) . here , we present a web interface to multifit .
the server takes as input a set of protein structures in the pdb format and an em density map in the mrc format .
the output is an ensemble of models ranked by their quality of fit to the density map .
multifit is a method for simultaneously fitting atomic - resolution protein structures into their assembly density map at resolutions as low as 25  .
the input is a set of atomic structures of proteins and an em density map of their assembly .
the component positions and orientations are optimized with respect to a scoring function that includes the quality - of - fit of components in the map , the protrusion of components from the map envelope and the shape complementarity between pairs of components .
the scoring function is optimized by an exact inference optimizer domino ( discrete optimization of multiple interacting objects ) that efficiently finds the global minimum within a discrete sampling space .
specifically , the optimization algorithm is composed of four stages , each sampling assembly models at increasingly higher resolution and accuracy . in 
anchor graph segmentation stage , an unlabeled segmentation of the density map into regions is calculated using a gaussian mixture model ; the segmented regions correspond approximately to the subunits in the complex . in  fitting - based assembly configuration stage , a set of coarse assembly models is found by an enumeration over possible assignments of subunits to regions , followed by simultaneous local fitting of the subunits in the corresponding regions . in  docking - based pose refinement stage ,
configuration stage is refined by simultaneous local optimization of the interfaces between pairs of interacting subunits as sampled by local pairwise docking . in 
refinement stage is further refined using a local monte carlo / conjugate gradients minimization procedure .
users can explore the ensemble of solutions generated by the first three stages and then refine a subset of the ensemble using a downloaded version of multifit . for cyclic symmetric complexes ,
the symmetry is imposed within the optimization procedure for improved efficiency , such that only symmetric models are sampled . in particular , in  fitting - based assembly configuration and  docking - based pose refinement , only cyclic symmetric models consistent with the symmetry of the density map are sampled ( 26 ) .
the multifit web server requires as input a set of protein structures in the pdb format , an em density map of their assembly in the mrc format , and a few parameters ( figure 1 ) .
the parameters for the density map include : ( i ) resolution (  ) ( 27 ) ; ( ii ) voxel spacing on the grid representing the map (  ) ; and ( iii ) the contour level that results in the volume accommodating the molecular mass of the complex .
these parameters are included for maps deposited in the em data bank ( emdb ) ( 28 ) . 

the inputs are divided into three parts : ( i ) general information , ( ii ) density map information and ( iii ) protein complex information .
seven copies of the groel chaperon monomer [ pdb entry 1oel ( 33 ) ] are simultaneously fitted to its ring density map at 11.5- resolution [ emdb entry 1080 ( 34 ) ] using cyclic symmetry mode .
the input subunit pdb file and the input assembly density map used for this example can be obtained from the multifit web server help page .
the input parameters for the resolution , spacing , contour level and symmetry order obtained from emdb site are 11.5 , 2.7 , 0.852 and 7 , respectively . the optional parameters for x , y and z origins are set to 50 , 50 and 50 , respectively .
the top 20 assembly models of the groel chaperon complex are ranked according to the quality - of - fit score from top left to bottom right .
the user can click on the model thumbnail to open it using chimera for further analysis .
( c ) top scored structural model of the groel ring fitted into the density map .
the inputs are divided into three parts : ( i ) general information , ( ii ) density map information and ( iii ) protein complex information .
seven copies of the groel chaperon monomer [ pdb entry 1oel ( 33 ) ] are simultaneously fitted to its ring density map at 11.5- resolution [ emdb entry 1080 ( 34 ) ] using cyclic symmetry mode .
the input subunit pdb file and the input assembly density map used for this example can be obtained from the multifit web server help page .
the input parameters for the resolution , spacing , contour level and symmetry order obtained from emdb site are 11.5 , 2.7 , 0.852 and 7 , respectively . the optional parameters for x , y and z origins are set to 50 , 50 and 50 , respectively .
the top 20 assembly models of the groel chaperon complex are ranked according to the quality - of - fit score from top left to bottom right .
the user can click on the model thumbnail to open it using chimera for further analysis .
( c ) top scored structural model of the groel ring fitted into the density map .
the multifit web server operates in two modes : cyclic - symmetric and non - symmetric . in the cyclic - symmetric mode ,
the symmetry order should be provided ( 2 for dimer , 3 for trimer , etc . ) .
if the arrangement of the input monomers in its native complex follows a different type of symmetry , the user should use the downloaded version of multifit . in the non - symmetric mode ,
a list of subunit pdb files and the number of copies of each subunit are required .
the input density should be pre - segmented to contain only the input set of proteins .
the server also has an optional input parameter specifying an e - mail address to which a link to the results page will be sent once the job is completed . alternatively , the user can bookmark a web link to the results page at the time of data submission .
the status of the job ( queued , running or finished ) can be accessed on the queue page .
the computation is performed in real time and the server page is updated once the calculation has finished .
the typical running time is about 20 min for assemblies with tens of thousands of atoms .
the web server output page displays a table of the top 20 assembly models that best fit the assembly density map , along with their quality - of - fit scores ranked from top left to bottom right ( figure 1 ) .
multifit lists the optimal as well as suboptimal solutions ; when the latter have good scores and are different from the optimal solution , the user should be skeptical about all solutions and further analyze the ensemble .
each model can be saved as a pdb file and can also be directly opened with ucsf chimera ( 19 ) .
row i lists the transformation applied to each of the subunits , the model quality - of - fit score , and the geometric complementarity score for model i. this output file can be used as input to imp for further refinement and analysis .
it can also be used as input for refining symmetric complexes using the symmref method ( 29 ) .
the multifit web server requires as input a set of protein structures in the pdb format , an em density map of their assembly in the mrc format , and a few parameters ( figure 1 ) .
the parameters for the density map include : ( i ) resolution (  ) ( 27 ) ; ( ii ) voxel spacing on the grid representing the map (  ) ; and ( iii ) the contour level that results in the volume accommodating the molecular mass of the complex .
these parameters are included for maps deposited in the em data bank ( emdb ) ( 28 ) . 

the inputs are divided into three parts : ( i ) general information , ( ii ) density map information and ( iii ) protein complex information .
seven copies of the groel chaperon monomer [ pdb entry 1oel ( 33 ) ] are simultaneously fitted to its ring density map at 11.5- resolution [ emdb entry 1080 ( 34 ) ] using cyclic symmetry mode .
the input subunit pdb file and the input assembly density map used for this example can be obtained from the multifit web server help page .
the input parameters for the resolution , spacing , contour level and symmetry order obtained from emdb site are 11.5 , 2.7 , 0.852 and 7 , respectively . the optional parameters for x , y and z origins are set to 50 , 50 and 50 , respectively .
the top 20 assembly models of the groel chaperon complex are ranked according to the quality - of - fit score from top left to bottom right .
the user can click on the model thumbnail to open it using chimera for further analysis .
( c ) top scored structural model of the groel ring fitted into the density map .
the inputs are divided into three parts : ( i ) general information , ( ii ) density map information and ( iii ) protein complex information .
seven copies of the groel chaperon monomer [ pdb entry 1oel ( 33 ) ] are simultaneously fitted to its ring density map at 11.5- resolution [ emdb entry 1080 ( 34 ) ] using cyclic symmetry mode .
the input subunit pdb file and the input assembly density map used for this example can be obtained from the multifit web server help page .
the input parameters for the resolution , spacing , contour level and symmetry order obtained from emdb site are 11.5 , 2.7 , 0.852 and 7 , respectively . the optional parameters for x , y and z origins are set to 50 , 50 and 50 , respectively .
the top 20 assembly models of the groel chaperon complex are ranked according to the quality - of - fit score from top left to bottom right .
the user can click on the model thumbnail to open it using chimera for further analysis .
( c ) top scored structural model of the groel ring fitted into the density map .
the multifit web server operates in two modes : cyclic - symmetric and non - symmetric . in the cyclic - symmetric mode ,
the symmetry order should be provided ( 2 for dimer , 3 for trimer , etc . ) .
if the arrangement of the input monomers in its native complex follows a different type of symmetry , the user should use the downloaded version of multifit . in the non - symmetric mode ,
a list of subunit pdb files and the number of copies of each subunit are required .
the input density should be pre - segmented to contain only the input set of proteins .
the server also has an optional input parameter specifying an e - mail address to which a link to the results page will be sent once the job is completed . alternatively , the user can bookmark a web link to the results page at the time of data submission .
the status of the job ( queued , running or finished ) can be accessed on the queue page .
the computation is performed in real time and the server page is updated once the calculation has finished .
the typical running time is about 20 min for assemblies with tens of thousands of atoms .
the web server output page displays a table of the top 20 assembly models that best fit the assembly density map , along with their quality - of - fit scores ranked from top left to bottom right ( figure 1 ) .
multifit lists the optimal as well as suboptimal solutions ; when the latter have good scores and are different from the optimal solution , the user should be skeptical about all solutions and further analyze the ensemble .
each model can be saved as a pdb file and can also be directly opened with ucsf chimera ( 19 ) .
row i lists the transformation applied to each of the subunits , the model quality - of - fit score , and the geometric complementarity score for model i. this output file can be used as input to imp for further refinement and analysis .
it can also be used as input for refining symmetric complexes using the symmref method ( 29 ) .
with the growing number of macromolecular assemblies characterized by em , integrative modeling techniques are becoming increasingly useful for a mechanistic understanding of these assemblies ( 6,3032 ) .
the multifit web server was designed to provide a user - friendly web interface to the multifit module in the imp package , for fitting multiple protein structures into their assembly density map .
the clore foundation ph.d scholars program ( to k.l . ) ; the israel science foundation ( 1403/09 ) and the hermann minkowski - minerva center for geometry at tel aviv university ( h.j.w . ) ; and the sandler family supporting foundation , national institutes of health ( r01 gm54762 , u54 rr022220 , pn2 ey016525 and r01 gm083960 ) , hewlett - packard , netapp , ibm and intel ( to a.s . ) .
 advances in electron microscopy ( em ) allow for structure determination of large biological assemblies at increasingly higher resolutions . 
 a key step in this process is fitting multiple component structures into an em - derived density map of their assembly . here 
 , we describe a web server for this task . 
 the server takes as input a set of protein structures in the pdb format and an em density map in the mrc format . 
 the output is an ensemble of models ranked by their quality of fit to the density map . 
 the models can be viewed online or downloaded from the website . 
 the service is available at ; http://salilab.org/multifit/ and http://bioinfo3d.cs.tau.ac.il/. 
SIGNIFICANCE
INTRODUCTION
THE MULTIFIT METHOD
WEB SERVER
Input
Output
CONCLUSIONS
FUNDING