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TitleDevelopment of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 118, Issue 47, Year 2021
Publish dateNov 23, 2021
AuthorsJoël S Bloch / Somnath Mukherjee / Julia Kowal / Ekaterina V Filippova / Martina Niederer / Els Pardon / Jan Steyaert / Anthony A Kossiakoff / Kaspar P Locher /
PubMed AbstractWith conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that ...With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ∼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in in large amounts, it may be useful not only for structural biology but also for biomedical applications.
External linksProc Natl Acad Sci U S A / PubMed:34782475
MethodsEM (single particle)
Resolution3.47 - 8.45 Å
Structure data

EMDB-13424, PDB-7php:
Structure of Multidrug and Toxin Compound Extrusion (MATE) transporter NorM by NabFab-fiducial assisted cryo-EM
Method: EM (single particle) / Resolution: 3.47 Å

EMDB-13425:
Map of complex of Vibrio cholerae MATE transporter NorM, chimeric nanobody NorM-Nb17_4, NabFab, and anti-Fab nanobody with fulcrum in center of micelle
Method: EM (single particle) / Resolution: 3.68 Å

EMDB-13426, PDB-7phq:
Structure of homo-dimeric Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM
Method: EM (single particle) / Resolution: 8.45 Å

EMDB-13438, PDB-7pij:
Structure of Staphylococcus capitis divalent metal ion transporter (DMT) by NabFab-fiducial assisted cryo-EM
Method: EM (single particle) / Resolution: 3.78 Å

Chemicals

ChemComp-HOH:
WATER / Water

Source
  • vibrio cholerae rc385 (bacteria)
  • synthetic construct (others)
  • lama glama (llama)
  • staphylococcus capitis (bacteria)
KeywordsMEMBRANE PROTEIN / NabFab / anti-nanobody Fab / fiducial / NorM / MATE / DMT

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