Blueprinting extendable nanomaterials with standardized protein blocks

Abstract

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures¹. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

Fig. 1. Overview of THR protein blocks and interaction modules.

a, Building a house frame from standardized wooden building blocks. b, THR internal geometry. Blocks are constructed from idealized straight α-helices with an angle of rotation between adjacent helices of Δθ; the remaining degrees of freedom that contribute to the repeat trajectory are also indicated. c, Changing Δθ (while holding the other parameters constant) specifically changes the curvature of the repeat trajectory. d, Single-chain THR modules. e, THR interaction modules. Image of house frame (a, top) by mmaxer, Can Stock Photo.

Fig. 2. 1D and 2D shapes from THRs.

a,b, The linear THR designs (rainbow) are nearly identical to the experimentally determined structures (grey). Side-chain sticks between α-carbon and β-carbon are shown to indicate helical phasing. a, Left: the 2.5-Å-resolution crystal structure of the short, linear THR1 has a 0.8 Å CA RMSD to the design. The inset below shows repeat packing in the THR interior. Right: the 2.7-Å-resolution crystal structure of the tall, linear THR5 has a 0.6 Å CA RMSD to the design. b, Bottom: Comparison of the stair-stepping linear THR4 design model to the cryo-EM structure (determined as part of a nanocage assembly; Supplementary Fig. 16). The CA RMSD between the cryo-EM structure and the design model is 1.0 Å. c, C₄ and C₃ polygons generated from four-helix turn module THRs as illustrated on the left. C₄ square 90_C4_B (middle) and C₃ triangle 120_C3_A (right) oligomers with representative ns-EM 2D class averages for comparison (raw EM micrographs are in Supplementary Fig. 1f). Chain breaks are at the ends of the rainbow sections. Scale bar, 4 nm (for the design models); class averages are not to scale. d, Uncapped curve THRs generate cyclic ring oligomers. The 12-repeat ring design (tested as C₄) R12B has a cryo-EM 3D reconstruction overlaid on the model; the two are nearly identical. A 2D class average with the individual straight helices resolved is shown left of the ring. e, The 20-repeat ring design (tested as C₄) R20A has an ns-EM reconstruction density overlaid on the model, and a raw micrograph is shown inside. Scale bar, 10 nm. f, The 30-repeat ring design (tested as C₆) R30A represented in a similar manner to e. Scale bar in e, 10 nm (for the design models with reconstruction maps overlaid in d–f); class averages are not to scale. The asymmetric unit is coloured in rainbow.

Fig. 3. Design of strutted double rings.

a,b, Two different size rings built from curve THRs for which integral multiples generate the same rotation can be concentrically nested and connected by struts. a, Three repeats of an outer ring (12° per repeat) are combined with two repeats of an inner ring (18° per repeat) that both generate a 36° rotation. Connection of the two pieces with a linear THR generates a C₁₀ single-component ring (strut_C10_8); an asymmetric unit is highlighted in the second ring image. An ns-EM 3D reconstruction in C₁₀ symmetry is shown overlaid with the design model next to 2D class averages and a representative micrograph. b, Five repeats of an outer ring (12° per repeat) are combined with three repeats of an inner ring (20° per repeat) that both generate a 60° rotation. Connection of the two pieces with a linear THR and an additional chain break in the outer ring generates a C₆ two-component ring (strut_C6_21); an asymmetric unit is highlighted in the second ring image with the two chains in different colors. A cryo-EM 3D reconstruction in C₆ symmetry is shown overlaid with the design model next to cryo-EM 2D class averages and a representative ns-EM micrograph (additional cryo-EM details are provided in Supplementary Fig. 8c). Scale bars, 20 nm (a,b). An asymmetric unit is outlined on top of the design model, and repeats are sectioned with dashed lines.

Fig. 4. Modular construction of protein nanocages from THRs.

a, In box: regular nanocages are constructed from curve THR rings with linear arms projecting outwards that can be linearly extended (left) and designed handshake C₂ interfaces (right) that hold the rings at the angle required for the desired polyhedral symmetry. THR chains that are fused into one chain are shown in blue, with identical backbone and sequence areas used for concatenation indicated in dotted outlines. b–e, C₂ handshake modules generating the required angles (top left in each panel, b–e) are combined with either C₃ (b–d) or C₄ (e) versions of the ring (shown below the box in a) to construct nanocages in b–e. Design models are shown as helix cylinders, with the asymmetric unit in blue, and the remaining copies in purple. Each cage is overlaid with the 3D cryo-EM reconstruction or ns-EM (marked with an asterisk here and in subsequent figures) reconstruction, with representative paired 2D classes on the right (left is experimental; right is computed from design model). The blue dots indicate the location of the handshake angle in the cage. b, The T₃ tetrahedral design cage_T3_101 uses a C₃ ring and a 70.5° C₂ handshake. c, The O₃ octahedral design cage_O3_20 uses a C₃ ring and a 109.5° C₂ handshake. d, The I₃ icosahedral design cage_I3_8 uses a C₃ ring and a 138.2° C₂ handshake. e, Th O₄ octahedral design cage_O4_34 uses a C₄ ring and a 90.0° C₂ handshake. Scale bars, 20 nm (b), 27 nm (c), 52 nm (d) and 22 nm (e).

Fig. 5. Extendable THR-based nanomaterials.

a, An extendable C₄ square. Left: the base design (sC4); right, an expanded version with six additional helices per chain (sC4_+6). Far left and right: representative cryo-EM micrographs (scale bars, 100 nm) with adjacent 2D class averages. A 3D reconstruction is superimposed on the base size design model. Chain breaks are in the red heterodimer region. The length of the side of the square (bar labeled x) is indicated beneath the 2D class averages. b, Expandable O₄ octahedral handshake nanocage, cage_O4_34. Top left to right, design models and cryo-EM or ns-EM reconstructions (the asterisk denotes ns-EM on +8 and +12) of designs extended by 0, 4, 8 and 12 helices. Bottom (left): ns-EM 2D class averages along three symmetry axes (rows) for increasing size designs (columns). The inserted helices are shown in yellow for each case. Scale bars, 30 nm (for 2D class averages). The length of a side of each cube (x, measured between the outside corners of the handshake) determined from the EM map volumes is indicated beneath the class averages. c, An expandable two-component O₄₃ nanocage cage_O43_129. Bottom row: the second size (+4; lowest deviation cryo-EM reconstruction) is shown with a symmetrized design model. For the other sizes, individual oligomers were fitted into the EM density (cryo-EM for +0 and +8 sizes; ns-EM for +12 size) so that rotational deviations from the ideal designs can be analysed (Supplementary Fig. 22). The extendable trimer component is in blue and the constant tetramer component is in red. Right, above 3D cage models: ns-EM 2D class averages along the three symmetry axes for each of the four sizes. The dimension x is the experimentally determined distance between the centre of mass of neighbouring C₄ and C₃ components. Scale bar, 25 nm (for 2D class averages).

Fig. 6. Designed train track fibres.

a, Components of the train track designs: a branch module, a split module and a C₂ interface module. b, Train track designs. The asymmetric unit is outlined in the top left design; red and blue are unique protein chains. Taking advantage of the extensibility of the building blocks, four different train track designs were created, starting with the design at the top left. Top right: increasing the spacing between rungs by expanding the split module. Bottom left, increasing the length of the rungs by expanding the C₂ module. Bottom right: increasing both rung spacing and length by expanding both the split and C₂ modules. Inserted segments are in yellow. c, An ns-EM micrograph of the design at the top left of b. Scale bar, 25 nm. d, ns-EM 2D class averages of the four designs in b, shown in the same overall layout. The design models are overlaid on the class averages at 1:1 scale. Scale bar, 25 nm.