Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

  1. Xudong Wu
  2. Tom A. Rapoport

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Abstract

Structure determination by cryo-EM is difficult or impossible to apply to proteins smaller than ∼100 kDa, excluding many membrane proteins and proteins of pharmaceutical importance from the analysis. Here, we report on a general method that allows structure determination of small proteins. The method is based on the availability of a nanobody to a target protein. The nanobody 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. We call the overall ensemble Legobody. The method is demonstrated for two small proteins that have sizes of ∼22 kDa.

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  1. Version published to 10.1073/pnas.2115001118
  2. ScreenIT

    SciScore for 10.1101/2021.08.09.455715: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Recombinant expression of Fabs: To increase the yield of recombinant expression of the Fabs in HEK293 cells, the constant regions of the light and heavy chains were replaced by sequences from human Fabs (for sequences, see Table. S1).
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Recombinant DNA
    SentencesResources
    Purification of nanobodies: Genes for His-tagged or Strep II-tagged nanobodies were cloned into the pET 26b vector (Novagen).
    pET 26b
    suggested: None
    The genes were cloned into the pET28b vector (Novagen) with either an N- or C-terminal His6 tag.
    pET28b
    suggested: RRID:Addgene_73018)
    The gene for the ALFA peptide was cloned into the pGEX6p1 vector (Cytiva).
    pGEX6p1
    suggested: RRID:Addgene_169015)
    Purification of a complex of KDEL-receptor (KDELR) and Legobody: The codon-optimized gene for the full-length KDELR with a SBP tag at its C-terminus was cloned into the pRS425-Gal1 vector (ATCC® 87331) 35.
    pRS425-Gal1
    suggested: RRID:Addgene_159522)
    Purification of a complex of the SARS-CoV-2 spike RBD domain and Legobody: The codon-optimized gene for the RBD domain (residues 334-526) of SARS-CoV-2 spike protein with an N-terminal Flag tag and a C-terminal His8 tag was cloned into the pCAGEN vector.
    pCAGEN
    suggested: RRID:Addgene_11160)
    Software and Algorithms
    SentencesResources
    All cryo-EM movies were recorded in counting-mode using SerialEM.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Cryo-EM Image Processing: For the KDELR/Legobody complex, dose-fractionated movies were subjected to motion correction using the program MotionCor2 36 with dose-weighting.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    For 3D classification, an initial model was generated ab initio in Relion 3.1.
    Relion
    suggested: (RELION, RRID:SCR_016274)
    Model Building: All model building was done in Coot.
    Coot
    suggested: (Coot, RRID:SCR_014222)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Our Legobody approach thus overcomes current limitations of cryo-EM analysis and greatly expands its use. The new method can be applied to any target protein once a tightly binding nanobody is available. The nanobody is assembled into a Legobody by the binding of two scaffolds, a Fab fragment and a MBP molecule to which domain C of protein A domain has been grafted (MBP_PrAC). All interactions were designed to be rigid. In addition, Fab-interacting domains were fused to MBP_PrAC to further solidify the complex. The Legobody has a characteristic shape, consisting of two lateral arms, formed by the two scaffolds, and a central lobe, contributed by the nanobody. The overall size (∼120 kDa) and shape of the Legobody, and the center of alignment at the position of the nanobody, greatly facilitate all steps of cryo-EM analysis, from particle picking, classifications, to final refinement. We demonstrate the utility of the Legobody method with two examples of small target proteins (KDELR (23kDa) and the RBD (22kDa) of the SARS-CoV-2 spike protein). The membrane protein KDELR poses a particular challenge for cryo-EM analysis, as it is small, has no domains outside membrane, and no symmetry to facilitate particle alignment in EM images. The protein tends to aggregate during purification and on cryo-EM grids at the water-air interface of thin ice. To determine its structure, we not only used the Legobody approach, but also employed two other tricks, which likely are applicable to other ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.08.09.455715v1 on bioRxiv