Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).
Proc Natl Acad Sci U S A
; 118(41)2021 10 12.
Article
in English
| MEDLINE | ID: covidwho-1462069
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
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This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
ABSTRACT
We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of â¼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Cryoelectron Microscopy
/
Single-Domain Antibodies
/
Spike Glycoprotein, Coronavirus
/
SARS-CoV-2
Limits:
Humans
Language:
English
Year:
2021
Document Type:
Article
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