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Accurate computational design of three-dimensional protein crystals

Nature Materials volume 22, pages 1556–1563 (2023)Cite this article

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Abstract

Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain–side-chain interactions across protein–protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein–protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.

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Fig. 1: Hierarchical crystal design strategy.
Fig. 2: Computational design and experimental characterization of F4132-1, F4132-2 and I432-1 crystals.
Fig. 3: Engineering crystal properties.
Fig. 4: Scaffolding of 3D AuNP superlattices using the designed protein crystals.

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Data availability

All data are available in the main text or as supplementary materials. Design scripts examples and design models (cages including T32-15, T32-15-6H, O43-2, T33-ECY54, T33-ECY55, T33-ECY59, T33-ECY66, T33-ECY67, O43-UWN453, O43-ZL1, O43-ZL7 and O32-ZL4, and crystal contact dihedrals of F4132-1-0, F4132-2, I432-1, F4132-2-ex1, F4132-2-ex2 and F4132-2-ex3) are available in the supplementary information files and through Zenodo79. SAXS and hyperspectral data are available as source data files. Crystallographic datasets have been deposited in the PDB (accession codes 8CUU, 8CUV, 8CUW, 8CUX, 8CWS, 8CUS, 8CUT, 8CWZ and 8FAR). CryoEM maps and corresponding atomic models have been deposited in the PDB (accession codes 8CWY and 8SZZ) and the Electron Microscopy Data Bank (accession codes EMD-27031 and EMD-40926). Source data are provided with this paper.

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Acknowledgements

We thank F. Busch and V. Wysocki at Ohio State University for support with native mass spectrometry experiments. We thank X. Zuo and T. Jun at Argonne National Laboratory for help with the SAXS measurements. We thank R. M. Haynes at the Pacific Northwest Cryo-EM Center for help with cryoEM data collection. We thank J. Du, S. Zhang and J. De Yoreo at the University of Washington for help with the crystallization measurements and for discussions. We thank I. Kopyeva and C. DeForest at the University of Washington for help with the mechanical measurements and for discussions. We thank D. Oberthür, V. Kremling, J. Sprenger, E. Scheer, B. Klopprogge and H. Chapman at the Center for Free-Electron Laser Science, Hamburg, Germany, for investigating the crystals on a free-electron laser. We thank S. Weaver, K. Patel and T. Gonen at the University of California, Los Angeles, for help with the microED. We thank S. Dickinson, N. Bethel and M. Wu at the University of Washington for help with cryoEM sample preparation and for screening. We thank S. Caldwell at the University of Washington for help with analysing the crystallographic data. We thank X. Wang at the University of Washington for suggestions on collecting X-ray crystallography data. We thank T. Huddy, R. Kibler, N. Bethel, A. Khmelinskaia, D. Zambrano and R. Haas at the University of Washington for providing potential protein building blocks. We thank H. Bai, R. Kibler, T. Huddy, H. Pyles, C. Xu and A. Ljubetic at the University of Washington for help with scripting and software. We thank R. Ravichandran for help with the bioreactor production of proteins. We thank J. Decarreau for help with the optical and fluorescence microscope imaging. We also thank members of the Baker lab and Institute for Protein Design, particularly J. Bale, N. King and F. Dimaio, for useful discussions. This work was supported with funds provided by the Howard Hughes Medical Institute (W.S. and D.B.), an Amgen gift (S.W.), Novo Nordisk (W.Y.), the Institute for Protein Design Directors Fund (M.J.B.), the Audacious Project at the Institute for Protein Design (Z.L., A.K.B., A.J.B., Q.D., R.D., A.F. and D.B.), the Open Philanthropy Project Improving Protein Design Fund (Y.H., H.N., N.I.E. and D.B.), the Synergistic Discovery and Design project HR001117S0003 of the Defense Advanced Research Projects Agency under contract FA8750-17-C-0219 (H.H. and D.B.), postdoctoral scholarships from the Washington Research Foundation (J.M.L. and H.H.), the Nordstrom Barrier Institute for Protein Design Directors Fund (A.E.), a Human Frontiers Science Program Long Term Fellowship (A.C.), a Public Health Service National Research Service Award (T32GM007270) from the National Institute of General Medical Sciences (NIGMS; U.N.), a Graduate Research Fellowship Program grant from the National Science Foundation (NSF DGE-1762114 to E.C.Y.) and a US Department of Energy (DOE), Office of Science, grant DE-SC0018940 (A.K. and D.B.). We thank the staff of the APS Northeastern Collaborative Access Team beamlines, APS beamline 24-ID-C, which are funded by the NIGMS from the National Institutes of Health (NIH; P30 GM124165), and the APS 12-ID-B SAXS beamline. This research used resources of APS, which is a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We also thank the ALS beamline 8.2.2/8.2.1 and SIBYLS Beamline 12.3.1 at Lawrence Berkeley National Laboratory. This research used ALS resources, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231 and the Integrated Diffraction Analysis Technologies grant. The ALS-ENABLE beamlines are supported by the NIH through NIGMS (Grant No. P30 GM124169-01). The Berkeley Center for Structural Biology is supported in part by the NIH, NIGMS and the Howard Hughes Medical Institute. A portion of this research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Center for Cryo-EM at Oregon Health & Science University and accessed through the Environmental Molecular Sciences Laboratory (grid.436923.9), which is a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work (M.Y.Y. and D.S.G.) was supported with funds provided by the DOE, Office of Science, Office of Basic Energy Sciences, as part of the Energy Frontier Research Centers program of the Center for the Science of Synthesis Across Scales under Award Number DE-SC0019288, located at University of Washington.

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Author notes

  1. These authors contributed equally: Zhe Li, Shunzhi Wang, Una Nattermann.

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Zhe Li, Shunzhi Wang, Una Nattermann, Asim K. Bera, Andrew J. Borst, Matthew J. Bick, Erin C. Yang, William Sheffler, Hannah Nguyen, Alex Kang, Radhika Dalal, Joshua M. Lubner, Yang Hsia, Hugh Haddox, Alexis Courbet, Quinton Dowling, Marcos Miranda, Natasha I. Edman, Wei Yang, Connor Weidle & David Baker

  2. Institute for Protein Design, University of Washington, Seattle, WA, USA

    Zhe Li, Shunzhi Wang, Una Nattermann, Asim K. Bera, Andrew J. Borst, Matthew J. Bick, Erin C. Yang, William Sheffler, Hannah Nguyen, Alex Kang, Radhika Dalal, Joshua M. Lubner, Yang Hsia, Hugh Haddox, Alexis Courbet, Quinton Dowling, Marcos Miranda, Andrew Favor, Ali Etemadi, Natasha I. Edman, Wei Yang, Connor Weidle & David Baker

  3. Graduate Program in Biological Physics, Structure & Design, University of Washington, Seattle, WA, USA

    Una Nattermann & Erin C. Yang

  4. Department of Chemistry, University of Washington, Seattle, WA, USA

    Muammer Y. Yaman & David S. Ginger

  5. X-Ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA

    Byeongdu Lee & Soenke Seifert

  6. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Greg L. Hura & Banumathi Sankaran

  7. HHMI, University of Washington, Seattle, WA, USA

    Alexis Courbet & David Baker

  8. Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA, USA

    Andrew Favor

  9. Medical Biotechnology Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran

    Ali Etemadi & Babak Negahdari

  10. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA

    Natasha I. Edman

  11. Medical Scientist Training Program, University of Washington, Seattle, WA, USA

    Natasha I. Edman

  12. Department of Chemistry, University of Massachusetts Lowell, Lowell, MA, USA

    Michael B. Ross

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  1. Zhe Li
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U.N., Z.L., S.W., E.C.Y. and D.B. were responsible for the conceptualization of the study. Z.L., S.W., U.N., W.S. and D.B. presented the methodology. Z.L., S.W., U.N., E.C.Y. and R.D. undertook the investigations. Z.L., S.W., A.J.B. and C.W. performed the CryoEM. Z.L., S.W., U.N., A.K.B., M.J.B., H.N., A.K. and B.S. performed the X-ray crystallography. S.W., B.L., S.S. and G.H. performed the SAXS. M.Y.Y. and D.S.G. performed the hyperspectra and SEM. M.B.R. carried out the simulations. Z.L., U.N., E.C.Y., J.M.L., Y.H., Q.D., M.M., A.F., B.N., N.I.E. and W.Y. prepared the building blocks. Z.L., S.W., U.N., W.S., Y.H., A.C., Q.D. and A.E. provided the design protocols. H.H. carried out the Rosetta score function calculations. Z.L., S.W., U.N., A.J.B., W.Y. and D.B. provided the visualization. D.B. was responsible for funding acquisition. D.B. supervised the study. Z.L., S.W., U.N. and D.B. wrote the original draft of the manuscript. Z.L., S.W., U.N., A.K.B., A.J.B., M.Y.Y., E.C.Y., H.H., M.B.R. and D.B. reviewed and edited the manuscript.

Corresponding author

Correspondence to David Baker.

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Competing interests

Z.L., S.W., U.N., E.C.Y., W.S., J.M.L., Y.H., B.S. and D.B. are inventors on a provisional patent application submitted by the University of Washington for the design, composition and function of the proteins created in this study.

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Nature Materials thanks Mauri Kostiainen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Design rules of 3D protein crystals.

a, Constrained degree of freedom (DOF): The angle of rotation at the designed dihedral crystal interface (Fig. 1a, right panel) must be precisely specified by the design process, where the C2 axis of the dihedral needs to coincide with the C2 axis of the space group. In this example, the disruptive effect (highlighted in red) of a 15-degree error in alignment on crystal assembly is illustrated; similar crystal lattice breakdowns occur with all deviations from the target alignment angle. b, Accessible secondary structure (SS): Dihedral interfaces with helices perpendicular to the symmetry axis (docked from T33-15 cage) are more designable than those with helices parallel to the symmetry axis (docked from T33-21 cage26). Interacting secondary structures are highlighted in red. c, Affinity and Specificity: Working interfaces have sufficient hydrophobic packing with specific polar interactions at the boundary. Highly hydrophobic interfaces destruct the designed self-assembly, including insoluble components and off-target assemblies.

Extended Data Fig. 2 Characterizations of the constituent cages of designed crystals.

a-d, T33-15-D3-4, e–h, T32-15, i-l, O43-2. a,e,i, SEC chromatograms of two oligomeric components (green and orange) and cages assembled via in-vitro mixing of components (blue). b,f,j, nsEM images (scale bars, 50 nm). c,g,k, overlay of the design model with 3D reconstructed nsEM density map/ cryoEM model (scale bars, 5 nm). d,h,l, SAXS profile and simulation results of cages.

Extended Data Fig. 3 Characterizations of new tetrahedral cages for crystal design.

a–e, from left to right, computational model, SEC chromatogram, SAXS profile, and nsEM images.

Extended Data Fig. 4 Characterizations of new octahedral cages for crystal design.

a–d, from left to right, computational model, SEC chromatogram, SAXS profile, and nsEM images.

Extended Data Fig. 5 Symmetric dockings of tetrahedral and octahedral cages into crystal lattices.

a, Two tetrahedral cages are docked along their C3 axis for crystal contacts of D3 dihedrals, which allow them to crystallize in the F4132 space group. b, Two octahedral cages are docked along their C3 axis for crystal contacts of D3 dihedrals, which allow them to crystallize in the I432 space group. See methods for detailed docking protocol.

Extended Data Fig. 6 Optical microscopy and cryoEM characterization of designed protein crystals.

a, Optical micrograph of F4132-1-0 crystals. b, Optical micrograph of F4132-1 crystals. c, CryoEM image of F4132-1 crystals. d, Optical micrograph of F4132-2-6H crystals. e, Optical micrograph of F4132-2 crystals. f, CryoEM image of F4132-2 crystals. g, Optical micrograph of I432-1 crystals. h, Optical micrograph of I432-1-CC crystals. i, CryoEM image of I432-1 crystals.

Extended Data Fig. 7 CryoEM data of the T32-15 cage.

a, Representative 2D class averages of the T32-15 cage. b, CryoEM local resolution map of the T32-15 cage (top) and built atomic model (bottom). Local resolution estimates range from ~2.5 Å at the core to ~4 Å along the crystal-contact forming helices. c, Map-to-model comparison within a low-resolution region (top) and a high-resolution region (bottom). d, Global FSC. e, Orientational distribution plot demonstrating full angular sampling.

Extended Data Fig. 8 Tuning the crystallization behavior of designed crystals by mutagenesis.

a, Mutations to the F4132-1 crystals. b, Mutations of F4132-2 crystals. c, Mutations and redesigns (orange) of I432-1 crystals. Top panels, crystal interface models based on X-ray structure. Interface side chains are hypothetically placed to demonstrate mutation sites. Bottom panels: optical micrographs of representative crystallization results. Scale bars, 100 µm.

Extended Data Fig. 9 Design pipeline for engineering crystal unit cell dimension.

The crystal contact of the F4132-2 crystal was redesigned with different DHR arm fusion. See Methods for the details of step a-g.

Extended Data Table 1 Comparison of properties between designed protein crystals and crystals from screening
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–33 and Tables 1–7.

Supplementary Data 1

PDB models of cages and dihedrals.

Supplementary Data 2

Example scripts for computational dockings and designs.

Source data

Source Data for Fig. 1

SAXS data and simulation of three designed crystals.

Source Data for Fig. 2

Analysis of PDB entry resolution, space groups and solvent content distribution.

Source Data for Fig. 3

SAXS data of crystals of engineered lattice size.

Source Data for Fig. 4

SAXS data and simulation of crystals with AuNPs.

Source Data for Fig. 5

Hyperspectral data of different AuNP superlattices.

Source Data for Fig. 6

Optical microscope measurement results of crystal sizes.

Source Data for Fig. 7

SAXS data of all designed protein cages reported in the extended data figures.

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Li, Z., Wang, S., Nattermann, U. et al. Accurate computational design of three-dimensional protein crystals. Nat. Mater. 22, 1556–1563 (2023). https://doi.org/10.1038/s41563-023-01683-1

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