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<p>Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (&lt;100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.</p><p></p>

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Covalently constrained ‘Di-Gembodies’ enable parallel structure solutions by cryo-EM

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年8月15日

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  • Gangshun Yi,
  • Dimitrios Mamalis,
  • Mingda Ye,
  • Loic Carrique,
  • Michael Fairhead,
  • Huanyu Li,
  • Katharina L. Duerr,
  • Peijun Zhang,
  • David B. Sauer,
  • Frank von Delft,
  • Benjamin G. Davis,
  • Robert J. C. Gilbert

摘要

Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.