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Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

<div class='caption-title'>Scaffolding strategy and biophysical screening of MBP-GS fusion constructs.</div>
<div class='caption-title'>Negative-stain EM analysis of MBP-GS fusion proteins.</div>
<div class='caption-title'>Initial cryo-EM analysis of MBP-GS constructs Δ2 and Δ5.</div>
Coscia F, Estrozi LF, Hans F, Malet H, Noirclerc-Savoye M, Schoehn G, Petosa C
OpenAccess logo Sci Rep 6 30909 (2016)
Cited: 23 times
EuropePMC logo PMID: 27485862

Abstract

Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.

Articles - 5ldf mentioned but not cited (2)

  1. New tools for the analysis and validation of cryo-EM maps and atomic models. Afonine PV, Klaholz BP, Moriarty NW, Poon BK, Sobolev OV, Terwilliger TC, Adams PD, Urzhumtsev A. Acta Crystallogr D Struct Biol 74 814-840 (2018)
  2. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Coscia F, Estrozi LF, Hans F, Malet H, Noirclerc-Savoye M, Schoehn G, Petosa C. Sci Rep 6 30909 (2016)


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  6. Cryo-electron microscopy-based drug design. Cebi E, Lee J, Subramani VK, Bak N, Oh C, Kim KK. Front Mol Biosci 11 1342179 (2024)

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  2. Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. Liu Y, Gonen S, Gonen T, Yeates TO. Proc Natl Acad Sci U S A 115 3362-3367 (2018)
  3. A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. Liu Y, Huynh DT, Yeates TO. Nat Commun 10 1864 (2019)
  4. Generation of ordered protein assemblies using rigid three-body fusion. Vulovic I, Yao Q, Park YJ, Courbet A, Norris A, Busch F, Sahasrabuddhe A, Merten H, Sahtoe DD, Ueda G, Fallas JA, Weaver SJ, Hsia Y, Langan RA, Plückthun A, Wysocki VH, Veesler D, Jensen GJ, Baker D. Proc Natl Acad Sci U S A 118 e2015037118 (2021)
  5. Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM. Yao Q, Weaver SJ, Mock JY, Jensen GJ. Structure 27 1148-1155.e3 (2019)
  6. Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold. Castells-Graells R, Meador K, Arbing MA, Sawaya MR, Gee M, Cascio D, Gleave E, Debreczeni JÉ, Breed J, Leopold K, Patel A, Jahagirdar D, Lyons B, Subramaniam S, Phillips C, Yeates TO. Proc Natl Acad Sci U S A 120 e2305494120 (2023)
  7. Crystals of TELSAM-target protein fusions that exhibit minimal crystal contacts and lack direct inter-TELSAM contacts. Nawarathnage S, Soleimani S, Mathis MH, Bezzant BD, Ramírez DT, Gajjar P, Bunn DR, Stewart C, Smith T, Pedroza Romo MJ, Brown S, Doukov T, Moody JD. Open Biol 12 210271 (2022)
  8. Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria. Travis BA, Peck JV, Salinas R, Dopkins B, Lent N, Nguyen VD, Borgnia MJ, Brennan RG, Schumacher MA. Nat Commun 13 3793 (2022)
  9. Insights into the oligomeric structure of the HIV-1 Vpu protein. Majeed S, Adetuyi O, Borbat PP, Majharul Islam M, Ishola O, Zhao B, Georgieva ER. J Struct Biol 215 107943 (2023)
  10. Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia. Zhang K, Horikoshi N, Li S, Powers AS, Hameedi MA, Pintilie GD, Chae HD, Khan YA, Suomivuori CM, Dror RO, Sakamoto KM, Chiu W, Wakatsuki S. ACS Cent Sci 8 214-222 (2022)
  11. HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self-association. Majeed S, Dang L, Islam MM, Ishola O, Borbat PP, Ludtke SJ, Georgieva ER. Sci Rep 13 14691 (2023)
  12. Structural basis for the helical filament formation of Escherichia coli glutamine synthetase. Huang PC, Chen SK, Chiang WH, Ho MR, Wu KP. Protein Sci 31 e4304 (2022)
  13. A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM. Langeberg CJ, Kieft JS. Nucleic Acids Res 51 e100 (2023)
  14. Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples. Gonen S. Methods Mol Biol 2215 115-123 (2021)
  15. Sample Preparation for Electron Cryo-Microscopy of Macromolecular Machines. Deniaud A, Kabasakal BV, Bufton JC, Schaffitzel C. Adv Exp Med Biol 3234 173-190 (2024)


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