Protein Layers And Their Use In Electron Microscopy

Abstract

Protein layers ( 1 ) repeating regularly in two dimensions comprise protein protomers ( 2 ) which each comprise at least two monomers ( 5 ), ( 6 ) genetically fused together. The monomers ( 5 ), ( 6 ) are monomers of respective oligomer assemblies ( 3 ), ( 4 ) into which the monomers are assembled to assembly of the protein layer. The first oligomer assembly ( 3 ) belongs to a dihedral point group of order O, where O equals ( 3 ), ( 4 ) or ( 6 ) and has a set of O rotational symmetry axes of order ( 2 ). The second oligomer assembly ( 4 ) has a rotational symmetry axis of order ( 2 ). Due to the symmetry of the oligomer assemblies ( 3 ), ( 4 ), the rotational symmetry axes of each second oligomer assembly ( 4 ) is aligned with one of said set of O rotational symmetry axes of a first oligomer assembly ( 3 ) with ( 2 ) protomers being arranged symmetrically therearound. Thus, an 2-fold fusion between the oligomer assemblies ( 3 ), ( 4 ) is produced and the arrangements of the rotational symmetry axes of the oligomer assemblies ( 3 ), ( 4 ) cause the protein layer to repeat regularly. The protein layer has many uses, for example to support molecular entities for biosensing, x-ray crystallography or electron microscopy.

1 . A method of performing electron microscopy of a molecular entity, comprising:
 providing a protein layer having a structure which repeats regularly in two dimensions and which supports molecular entities each attached at a predetermined position in the repeating structure of the protein layer; and   performing electron microscopy of the protein layer having the molecular entities supported thereon to derive an image.   
     
     
         2 . A method according to  claim 1 , wherein the protein layer comprises protein protomers which each comprise at least two monomers genetically fused together, the monomers each being monomers of a respective oligomer assembly, the protomers comprising:
 a first monomer which is a monomer of a first oligomer assembly belonging to a dihedral point group of order O, where O equals 3, 4 or 6, and having a set of O rotational symmetry axes of order  2  extending in two dimensions; and   a second monomer genetically fused to said first monomer which second monomer is a monomer of a second oligomer assembly having a rotational symmetry axis of order  2 ,   the first monomers of the protomers are assembled into said first oligomer assemblies and the second monomers of the protomers are assembled into said second oligomer assemblies, said rotational symmetry axis of said second oligomer assemblies of order  2  being aligned with one of said set of rotational symmetry axes of order  2  of one of said first oligomer assemblies with two protomers being arranged symmetrically therearound.   
     
     
         3 . A method according to  claim 1 , wherein said step of providing a protein layer which supports molecular entities comprises making the protein layer and subsequently attaching the molecular entities thereto. 
     
     
         4 . A method according to  claim 3 , wherein the step of attaching the molecular entities to the protein layer is performed in solution. 
     
     
         5 . A method according to  claim 1 , further comprising, prior to the step of performing electron microscopy, aligning the molecular entities with respect to the protein lattice. 
     
     
         6 . A method according to  claim 5 , wherein the step of aligning the molecular entities with respect to the protein lattice comprises applying an electric field to the protein lattice. 
     
     
         7 . A method according to  claim 6 , wherein the step of aligning the molecular entities with respect to the protein lattice comprises cooling the protein lattice to a minimum energy state. 
     
     
         8 . A method according to  claim 1 , further comprising performing data analysis of the image. 
     
     
         9 . A method according to  claim 8 , wherein the data analysis is a two-dimensional crystallographic data analysis. 
     
     
         10 . A method according to  claim 8 , wherein the data analysis comprises identifying the components of the protein lattice and subtracting them from the image derived in said step of performing electron microscopy to derive an image of the molecular entities, and performing a single particle reconstruction of the image of the molecular entities. 
     
     
         11 . A protein layer which repeats regularly in two dimensions,
 the protein layer comprising protein protomers which each comprise at least two monomers genetically fused together, the monomers each being monomers of a respective oligomer assembly, the protomers comprising:   a first monomer which is a monomer of a first oligomer assembly belonging to a dihedral point group of order O, where O equals 3, 4 or 6, and having a set of O rotational symmetry axes of order  2  extending in two dimensions; and   a second monomer genetically fused to said first monomer which second monomer is a monomer of a second oligomer assembly having a rotational symmetry axis of order  2 ,   the first monomers of the protomers are assembled into said first oligomer assemblies and the second monomers of the protomers are assembled into said second oligomer assemblies, said rotational symmetry axis of said second oligomer assemblies of order  2  being aligned with one of said set of rotational symmetry axes of order  2  of one of said first oligomer assemblies with two protomers being arranged symmetrically therearound.   
     
     
         12 . A protein layer according to  claim 11 , wherein the second oligomer assembly belongs to a dihedral point group of order  2  or to a cyclic point group of order  2 . 
     
     
         13 . A protein layer according to  claim 11 , wherein the protomers are homologous with respect to the monomers. 
     
     
         14 . A protein layer according to  claim 13 , wherein said second oligomer assembly belongs to a dihedral point group of order  2 . 
     
     
         15 . A protein layer according to  claim 13 , wherein the second oligomer assembly is a heterologous oligomer assembly of said second monomers and of third monomers, said protein layer further comprising said third monomers assembled with said second monomers into said second oligomer assembly. 
     
     
         16 . A protein layer according to  claim 15 , wherein the third monomers are monomers which have a binding site capable of binding to biotin or a peptide, and said second monomers are aptamers which are capable of binding to said binding site. 
     
     
         17 . A protein layer according to  claim 16 , wherein said third monomers are streptavidin. 
     
     
         18 . A protein layer according to  claim 16 , wherein said second monomers are Steptag I (SEQ ID NO. 3). 
     
     
         19 . A protein layer according to  claim 11 , wherein the protomers are heterologous with respect to the monomers. 
     
     
         20 . A protein layer according to  claim 19 , wherein the protein layer comprises protein protomers of two types,
 the first type of protomer comprising a first monomer which is a monomer of said first oligomer assembly belonging to a dihedral point group of order O, where O equals 3, 4, or 6, genetically fused to a second monomer which is a monomer of said second oligomer assembly, said second oligomer assembly being a heterologous oligomer assembly belonging to a cyclic point group of order  2 , and   the second type of protomer comprising a third monomer which is a monomer of said second oligomer assembly, genetically fused to a fourth monomer which is a monomer of a third oligomer assembly, said third oligomer assembly belonging to a dihedral point group of order  2  or O.   
     
     
         21 . A protein layer according to  claim 20 , wherein said oligomer assembly belongs to a dihedral point group of order O, said third oligomer assembly being the same as said first oligomer assembly. 
     
     
         22 . A protein layer according to  claim 11 , wherein each of said monomers of said respective oligomer assemblies either is a naturally occurring protein or is based on a naturally occurring protein with peptide elements being absent from, substituted in, or added to the naturally occurring protein without substantially affecting assembly of monomers of said respective oligomer assembly. 
     
     
         23 . A protein layer according to  claim 11 , wherein, in said protomers, said monomers are genetically fused via a linking group. 
     
     
         24 . A protein layer according to  claim 23 , wherein the linking group is oriented relative to the first and second monomers in the protomer in its normal form prior to assembly to reduce any difference in the assembled layer in either or both of the position and orientation of (a) the termini of said first monomers in their arrangement in said first oligomer assembly in its natural form symmetrically around said one of said set of rotational symmetry axes of order N of said first oligomer assembly, and (b) the termini of said second monomers in their arrangement in said second oligomer assembly in its natural form symmetrically around said rotational symmetry axis of order N of said second oligomer assembly. 
     
     
         25 . A protein layer according to  claim 11 , and having an array of molecular entities attached thereto. 
     
     
         26 . A protein layer according to  claim 25 , wherein a component of the protein layer has an affinity tag, the molecular entities being attached to respective affinity tags. 
     
     
         27 . A protein layer according to  claim 25 , wherein the molecular entity comprises a protein having a peptide affinity tag attached to a component of the protein layer. 
     
     
         28 . A protein layer according to  claim 25 , wherein the molecular entity comprises a protein, and both of a component of the protein layer and the molecular entity have respective affinity tags attached to each other. 
     
     
         29 . A protein layer according to  claim 25 , wherein the molecular entities are genetically fused within a component of the protein layer. 
     
     
         30 . A protein protomer comprising at least two monomers genetically fused together, the monomers each being monomers of a respective oligomer assembly into which the monomers are capable of self-assembly to assemble a protein layer which repeats regularly in two dimensions, wherein said protomer comprises:
 a first monomer which is a monomer of a first oligomer assembly belonging to a dihedral point group of order O, where O equals 3, 4 or 6, and having a set of O rotational symmetry axes of order  2  extending in two dimensions; and   a second monomer genetically fused to said first monomer which second monomer is a monomer of a second oligomer assembly having a rotational symmetry axis of order  2 .   
     
     
         31 . A polynucleotide encoding a protein protomer according to  claim 30 . 
     
     
         32 . A vector capable of expressing a protein protomer according to  claim 30 . 
     
     
         33 . A host cell comprising a vector according to  claim 32 . 
     
     
         34 . A method of performing x-ray crystallography, comprising:
 supporting an array of molecular entities on a protein layer according to  claim 11 , and   performing x-ray crystallography on the protein layer having the molecular entities supported thereon to derive an image.