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TitleMegabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.
Journal, issue, pagesNat Methods, Vol. 18, Issue 1, Page 60-68, Year 2021
Publish dateJan 6, 2021
AuthorsTomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre Wohlkönig / Thomas Zögg / Han Remaut / James H Naismith / Hugues Nury / Wim Vranken / A Radu Aricescu / Els Pardon / Jan Steyaert /
PubMed AbstractNanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their ...Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
External linksNat Methods / PubMed:33408403 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution1.90000643078 - 3.15 Å
Structure data

EMDB-11610:
CryoEM structure of a beta3K279T GABA(A)R homomer in complex with megabody MbNbF3c7HopQ
Method: EM (single particle) / Resolution: 3.0 Å

4542

6qfa

EMDB-4542, PDB-6qfa:
CryoEM structure of a beta3K279T GABA(A)R homomer in complex with histamine and megabody Mb25
Method: EM (single particle) / Resolution: 2.49 Å

PDB-6xux:
Crystal structure of Megabody Mb-Nb207-cYgjK_NO
Method: X-RAY DIFFRACTION / Resolution: 1.90000643078 Å

PDB-6xv8:
Crystal structure of Megabody Mb-Nb207-c7HopQ_G10
Method: X-RAY DIFFRACTION / Resolution: 3.15 Å

PDB-6xvi:
Crystal structure of Megabody Mb-Nb207-c7HopQ_A12
Method: X-RAY DIFFRACTION / Resolution: 2.59599994893 Å

Chemicals

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

ChemComp-HSM:
HISTAMINE / neurotransmitter, hormone*YM

ChemComp-CA:
Unknown entry

ChemComp-HOH:
WATER

Source
  • homo sapiens (human)
  • lama glama (llama)
  • helicobacter pylori (strain g27) (bacteria)
  • escherichia coli k-12 (bacteria)
  • helicobacter pylori g27 (bacteria)
KeywordsMEMBRANE PROTEIN / Megabody / protein engineering / cryo-EM / STRUCTURAL PROTEIN / Scaffold

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