received by the International Bureau on 30 October 2018 (30.10.2018)

CLAIMS

1 , A process of solving a three-dimensional structure of a target protein comprising: non-genetically associating a target protein with a preformed multimeric self-assembling protein structure to form a target complex, and

subjecting said target complex to cryo-electror) microscopy whereby the multimeric self- assembling protein structure serves as a scaffold for solution of a three-dimensional, structure of said target protein, optionally wherein said target protein has a molecular weight of 200 kilodaltons or less, and optionally a value of resolution of said three-dimensional structure is less than 20 angstroms.

2_r The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures, wherein one or more of said protein substructures comprises a capture sequence, the capture sequence expressed at the N-terminus of the protein substructure or within 10 amino acids from the N-terminus of the protein substructure,

3. The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures_} wherein one or more of said protein substructures comprises a linker and a capture sequence, the linker covalently bonding the capture sequence to the protein substructure.

4. The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures wherein each of said protein substructures is identical in primary amino acid sequence.

5. The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures wherein each of said protein substructures comprises an amino acid sequence that is 70% or greater identical to any one of SEQ ID NOs: 1- 6.

6. The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures, wherein one or more of said protein substructures comprises a capture sequence, the capture sequence expressed at or near the N-terminus of the protein substructure, the capture sequence comprising the sequence of SEQ ID NO: 8, SEQ ID NO: 9, btotin, or avidin.

7. The process of claim. 1. wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures, wherein one or more of said protein substructures comprises a linker and a capture sequence, the linker covalently bonding the capture sequence to the protein substructure, the linker a flexible linker.

8. The process of claim 7 wherein the flexible linker is comprises a niultimer of the amino acid sequence GGS₃ GSS, or combinations thereof.

9. The process of claim 1 wherein said multimeric self-assembling protein structure comprises a plurality of protein substructures, wherein one or more of said protein substructures comprises a linker and a capture sequence, the linker covalently bonding the capture sequence to the protein substructure, the linker a rigid linker.

10. The process of claim 9 wherein the rigid linker comprises one or more stabilizing disulfide bonds, one or more repeats of SEQ ID NO: 10, an amino acid sequence comprising 3 or more proline residues, an amino acid sequence comprising 1 or more sequences of PPA, or a combination thereof.

11. The process of any one of claims 1 -10 wherein the multimeric self-assembling protein structure comprises a multimer of any one of SEQ ID NOs: 1 -6.

12. The process of claim 11 wherein, the multimer is a 60-mer.

13. The process of claim 11 wherein the multimeric self-assembling protein structure forms a dodecahedron.

14. The process of any one of claims 1-10 wherein the target protein has a molecular weight of less than 200 kDa.

15. The process of any one of claims 1-10 wherein the target protein has a molecular weight of less than 150 kDa.

16. The process of any one of claims H O wherein the target protein has a molecular weight of less than 120 kDa.

17. The process of any one of claims 1 -10 wherein the target protein comprises a tag, the tag suitable for high affinity binding to a capture sequence on the multimeric self-assembling protein structure.

18. The process of claim 17 wherein the tag comprises SEQ ID NO: 20, SEQ ID NO: 21, biotia, or avidin.

19. The process of any one of claims 1-10 wherein said target protein sequence comprises the RNA-bindmg protein, cytosolic Poly-A Binding Protein (PABP) (optionally SEQ ID NO: 16), a DNA-binding protein, of the ApiAP2 specific transcription factor family (optionally SEQ ID NO: 1 7), a binding domain of tristetraprolin (TTP) of a NOT family protein (optionally SEQ ID NO: 18), or a RNA-recognition motif of the Upregulated in Infectious Sporozoites 12 (UIS12) protein (optionally SEQ ID NO: 19).

20. The process of any one of claims 1-10 wherein said target protein is saturated onto said a multimeric self-assembling protein structure to form a target complex at a level of 50% or greater.

21. The process of any one of claims 1 -1 0 wherein said target protein is saturated onto said a multimeric self-assembling protein structure to form a target complex at a level of 90% or greater,

22. The process of any one of claims 1. -1 0 wherein said multimeric self-assembling protein structure to form a target complex is in an aqueous buffer comprising at or greater than 100 mM of a salt.

23. The process of claim 22 wherein the aqueous buffer comprises greater than 200 mM salt.

24. The process of claim 22 wherein the aqueous buffer comprises from 200 mM salt to 500 mM salt.

25. The process of any one of claims 1-10 wherein said value of resolution is less than. 10 A.

26. The process of any one of claims 1.-1 0 wherein said value of resolution is less than 5 A.

27. The process of any one of claims 1 -10 wherein said resolution is less than 3 A.

28. A three dimensional protein structure comprising:

a multimeric self-assembling nanocage, the nanocage comprising a plurality of protein substructures, one or more of the protein substructures comprising a linker and a capture sequence on an N-terminus; and

a target protein non-genetically fused, to said protein substructure, the target protein comprising a tag, the tag complementary to the capture sequence so that an association between the tag and the capture sequence covalently bonds said target protein to said protein substructure, or non-covalently bonds said target protein to said protein substructure with a KD or 10^"u M or lower.

29. The protein structure of claim 28 wherein said linker is intermediate the protein substructure and the capture sequence, the linker eovalently bonding the capture sequence to the protein substructure.

30. The protein structure of claim 29 wherein the linker eovalently bonding the capture sequence to the protein substructure, the linker a flexible linker.

31. The protein structure of claim 30 wherein the flexible linker is selected from the group consisting of a multimer of the amino acid sequence GGS, GSS, or combinations thereof.

32. The protein structure of claim 29 wherein the linker is a rigid linker.

33^. The protein structure of claim 32 wherein the rigid linker comprises one or more stabilizing disulfide bonds, one or more repeats of SEQ ID NO: 10, an amino acid sequence comprising 3 or more proline residues, an amino acid sequence comprising 1 or more sequences of PPA, or a combination thereof.

34. The protein structure of claim 28 wherein each of said protein substructures comprises an amino acid sequence that is 70% or greater identical to any one of SEQ ID NOs; 1- 6.

35. The protein structure of claim 28 wherein the capture sequence comprises the sequence of SEQ ID NO; 8. SEQ ID NO: % biotin, or avidin^,

36. The protein structure of any one of claims 28-35 wherein the multimeric self- assembling protein structure comprises a multimer of any one of SEQ ID NOs: 1-6.

37. The protein structure of any one of claims 28-35 wherein the multimer is a 60- mer.

38. The protein structure of any one of claims 28-35 wherein the multimeric self- assembling protein structure forms a dodecahedron.

39. The protein structure of any one of claims 28-35 wherein the target protein has a molecular weight of less than 200 kDa.

40. The protein structure of any one of claims 28-35 wherein the target protein has a molecular weight of less than 150 kDa.

41. The protein structure of any one of claims 28-35 wherein the target protein has a molecular weight of less than 120 kDa.

42. The protein structure of any one of claims 28-35 wherein the tag comprises SEQ ID NO: 20_s SEQ ID NO: 21, biotin, or avidin.

43. The protein structure of any one of claims 28-35 wherein said target protein is the RNA-binding protein, cytosolic Poly-A Binding Protein (PABP), a DNA-binding protein of the ApiAP2 specific transcription factor family, a binding domain of tristetraprolin (TIP) of a NOT family protein, or a RNA-recognition motif of the Upregulated in Infectious Sporozoites 12 (UIS12) protein.

44. The protein structure of any one of claims 28-35 wherein said target protein is saturated onto said a multimeric self-assembling protein structure to form a target complex at a level of 50% or greater.

45. The protein structure of any one of claims 28-35 wherein said target protein is saturated onto said a multimeric self-assembling protein structure to form a target complex at a level of 90% or greater.

46. The protein structure of any one of claims 28-35 protein structure is in an aqueous buffer comprising at or greater than 100 mM of a salt.

47. The protein structure of claim 46 wherein the salt is 200 mM to 500 niM.

48. The protein structure of any one of claims 28-35 wherein the target protein is an antigen.

49. The use of the protein structure of any one of claims 28-35 for immunizing a subject with said target protein.

50. The use of claim 49 wherein the multimerjc self-assembling protein structure comprises a multimer of any one of SEQ ID NOs: 1 -6.

51. The use of claim 49 wherein the multimer is a 60-mer.

52. The use of claim 49 wherein the multimeric self-assembling protein structure forms a dodecahedron.

53. The use of claim 49 wherein the target protein has a molecular weight of less than 200 kDa.

54. The use of claim. 49 wherein the target protein has a molecular weight of less than 150 kDa.

55. The use of claim 49 wherein the target protein has a molecular weight of Jess than 120 kDa.

56. The use of claim 49 wherein the tag comprises SEQ ID NO: 20, SEQ ID NO: 21, biotin, or avidin.

57. The use of claim. 49 wherein said target protein is saturated onto said a multimeric Self-asserabling protein structure to form a target complex at a level of 50% or greater.

58. The use of claim 49 wherein said target protein is saturated onto said, a multimeric self-assembling protein structure to form, a target complex at a level of 90% or greater.