Supplementary Information for

Cryo-EM structure determination of small proteins by nanobody-

binding scaffolds (Legobodies)

Xudong Wu1$ and Tom A. Rapoport1$

1. Howard Hughes Medical Institute and Department of Cell Biology, Harvard

Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

$ Corresponding authors:

Xudong Wu, Howard Hughes Medical Institute and Department of Cell Biology,
Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115,
USA.
Email: xudong_wu2@hms.harvard.edu

Tom Rapoport, Howard Hughes Medical Institute and Department of Cell

Biology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115, USA.
Email: tom_rapoport@hms.harvard.edu

This PDF file includes:

Figures. S1 to S6
Tables. S1 to S4

1
Fig. S1 | Crystal structure of a nanobody/Fab complex (Nb_0/Fab_8D3).
a, Crystals contain both Nb_0 and Fab_8D3. Multiple crystals were pooled and
analyzed by SDS-PAGE followed by Coomassie-blue staining. b, Two views of
the interacting region of Nb_0 and Fab_8D3. Residues of Nb_0 involved in the
interaction are colored in pink and labeled. The surface area buried at the binding
interface is 898 Å2. c, Model of the Nb_0/Fab_8D3 complex in “worms”
representation, colored according to the average B-factor of amino acid residues
(scale on the right).

2
Fig. S2 | Interaction of nanobody with different PrA domains and design of
a “shared helix” between PrAC and MBP.
a, Protein A domains C, D, or E (PrAC, PrAD, PrAE) were fused to MBP via a
flexible linker (L). The fusion proteins were mixed with nanobody at a 1:3 molar
ratio. The samples were subjected to size-exclusion chromatography and
fractions were analyzed by SDS-PAGE and Coomassie-blue staining. b, A
“shared helix” was generated from a helix of PrAC (4NPD) that is not interacting
with the nanobody and the C-terminal helix of MBP (1ANF).

3
Fig. S3 | Cryo-EM analysis of the KDELR/Legobody complex.
a, Representative cryo-EM image with selected particles marked by green
circles. b, Representative 2D class averages of selected particles. c, Cryo-EM
processing workflow. The classes selected for further analysis are shown in pink.
Masks used for classification and refinement are indicated. d, 3D FSC curves
and preferred orientation analysis. The red line shows the global FSC and the
green dotted lines indicate the +1 and – 1 standard deviations around the Mean

4
of Directional FSC curve. The FSC calculations used the mask shown on the
side. A sphericity of 0.965 was estimated, indicating a mostly isotropic
reconstruction without strong orientation bias. e, Euler angle distribution of
refined particles is shown in two different views.

5
Fig. S4 | Fit of models for Legobody components and comparison of the
cryo-EM and crystal structures of the KDELR/nanobody complex.

6
a, Map and fitted models for different parts of the Legobody. b, The cryo-EM
structure of the KDELR/Nb_KR complex was aligned with the structure
determined by X-ray crystallography (PDB code 6I6J). The proteins are shown in
cartoon representation.

7
Fig. S5 | Cryo-EM analysis of the RBD/Legobody complex and comparison
of the cryo-EM and crystal structures of the RBD/nanobody complex.

8
a, Representative cryo-EM image with selected particles marked by green
circles. b, Representative 2D class averages of selected particles. c, Cryo-EM
processing workflow. The classes selected for further analysis are shown in pink.
Masks used for classification and refinement are indicated. d, 3D FSC curves
and preferred orientation analysis. The red line shows the global FSC and the
green dotted lines indicate the +1 and – 1 standard deviations around the Mean
of Directional FSC curve. The FSC calculations used the mask shown on the
side. A sphericity of 0.972 was estimated, indicating a mostly isotropic
reconstruction without strong orientation bias. e, Euler angle distribution of
refined particles is shown in two different views. f, The cryo-EM structure of the
RBD/Nb_RBD complex was aligned with the structure determined by X-ray
crystallography (PDB code 7KGJ). The proteins are shown in cartoon
representation. The red circle highlights loops that are invisible in the cryo-EM
map.

9
Fig. S6 | Generation of a Legobody-compatible ALFAtag nanobody.
a, Structure of the complex of the original ALFA nanobody with the ALFA
peptide. The model shows the polypeptides in cartoon representation. Residues
shown as red sticks deviate from those seen in the common nanobody
framework (in yellow) and would interfere with the assembly into a Legobody.
They were therefore mutated to generate Nb_ALFA. b, To demonstrate that
Nb_ALFA can be assembled into the Legobody without loss of antigen binding,
purified Legobody containing Nb_ALFA was incubated with or without a GST
fusion of ALFA peptide (GST_ALFA peptide). The samples were incubated with
glutathione beads and the bound material analyzed by SDS-PAGE and
Coomassie-blue staining. The input corresponds to 50% of the material used for
the pull-down.

10
Table S1. Sequences of all Proteins

Sequences shown in yellow are cleavable signal sequences.

Fab_8D3_H-H6
MDWTWRVFCLLAVAPGAHSDVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHW
VRQAPEMGLEWVAYISSGSTTIYYGDTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMY
YCARRPLYDGDYGYPMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCGSHHHHHH

Fab_8D3_2_H-H6
MDWTWRVFCLLAVAPGAHSDVQLVESGGGLVQPGKSLRLSCAASGFTFSNFGMHW
VRQAPEMGLEWVAYISSGSTTKYYGDTVKGRFTISRDNPKNTLYLQMNSLRSEDTAM
YYCARRPLYDGDYGYPMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCGSHHHHHH

Fab_8D3_L & Fab_8D3_2_L

MVLQTQVFISLLLWISGAYGNIMLTQSPSSLAVSAGERVTMSCKSTQSILYNSNQKTYL
AWYQQKPGQSPKLLIYWASTRASGVPDRFTGSGSGTDFTLTINSVQPEDLAVYYCHQ
YLSAWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC

Nb_N0-H6 (CDRs are shown in cyan)

MKYLLPTAAAGLLLLAAQPAMAQVQLVEYGGGSVQAGGYLRLSCVASGSISLSSGMG
WYRQAPGKERELVASISGGSSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAV
YYCAASEQLTSGHAYWGQGTQVTVSSLEHHHHHH

Nb_MBP-Strep2 (derived from Sb_MBP#1)

MKYLLPTAAAGLLLLAAQPAMAQVQLVEYGGGSVQAGGSLRLSCVASGDIKYISYLG
WFRQAPGKEREGVAALYTSTGRTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDT
AVYYCAAAEWGSQSPLTQWFYRYWGQGTQVTVSSGGENLYFQSGSSAWSHPQFE
KGGGSGGGSGGSAWSHPQFEK
Nb_KR-H6 (derived from Syb37)
MKYLLPTAAAGLLLLAAQPAMAQVQLVESGGGLVQAGGSLRLSCAASGFPVKRWSM
TWYRQAPGKEREWVAAIRSAGHWTHYADSVKGRFTISRDNAKNTVYLQMNSLKPED
TAVYYCNVKDEGDFSYWYDYWGQGTQVTVSSLEHHHHHH

Nb_ALFA-H6
MKYLLPTAAAGLLLLAAQPAMAEVQLQESGGGLVQAGGSLRLSCTASGVTISALNAM
AMGWYRQAPGKRRVMVAAVSERGNTMYRESVKGRFTVSRDFTNKTVYLQMNSLKP
EDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSSLEHHHHHH

Nb_RBD-H6 (derived from Sb#45)

MKYLLPTAAAGLLLLAAQPAMAQVQLVESGGGLVQAGGSLRLSCAASGFPVYRDRM
AWYRQAPGKEREWVAAIYSAGQQTRYADSVKGRFTISRDNAKNTVYLQMNSLKPED
TAVYYCNVKDVGHHYEYYDYWGQGTQVTVSSLEHHHHHH

MBP_PrA/G-H6
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP
DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIY
NKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGK
YDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWS
NIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA
VNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA
ASGRQTVDQALAFAQILIMPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNEHQAPKG
GSGGAGSGDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQ
AGGGSGGGSGGSAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGV
WTYDDATKTFTVTEGSGHHHHHH

H6-MBP_PrAC
MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV
AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIA
VEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA
FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI
NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL
LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDQALAFAQILIMPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLN
EAQAPK

H6-MBP_L_PrAC
MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV
AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIA
VEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA
FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI
NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL
LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGEGSEQQNAF
YEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK

H6-MBP_L_PrAD
MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV
AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIA
VEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA
FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI
NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL
LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGEGSDQQSAF
YEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK

H6-MBP_L_PrAE
MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV
AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIA
VEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA
FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI
NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL
LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA
VRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGENLYFQGEGSAQQNAF
YQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK

KDELR-SBP
MNIFRLTGDLSHLAAIIILLLKIWKSRSCAGISGKSQLLFALVFTTRYLDLFTSFISLYNTS
MKLIYIACSYATVYLIYMKFKATYDGNHDTFRVEFLIVPVGGLSFLVNHDFSPLEILWTF
SIYLESVAILPQLFMISKTGEAETITTHYLFFLGLYRALYLVNWIWRYYFEGFFDLIAVVA
GVVQTVLYCDFFYLYVTKVLKGKKLSLPAGSGGENLYFQSGGGMDEKTTGWRGGHV
VEGLAGELEQLRARLEHHPQGQREP

FLAG-19RBD-H8
MDWTWRVFCLLAVAPGAHSGDYKDDDDKGGENLYFQGGSGDSTGSSNLCPFGEVF
NATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS
FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFR
KSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLS
FELLHAPATVCGGGGSGSGHHHHHHHH

GST_ALFA peptide
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYI
DGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETL
KVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPK
LVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSGS
GGSPSRLEEELRRRLTEP
Table S2. X-Ray data collection, and refinement statistics

Nb_0/Fab_8D3
Data Accession
PDB 7R9D
Data collection
Space group P 1 21 1
Cell dimensions
a, b, c (Å) 64.51, 60.287, 77.244
α, β, γ (°) 90.00, 109.341, 90.00
Resolution (Å) 1.83 (1.895-1.83) *
Rmerge 0.068 (0.55)
I / σI 16.46 (2.95)
Completeness (%) 99.35 (99.15)
Redundancy 7.6 (7.1)

Model Refinement
Software Phenix
Resolution (Å) 1.83
No. Reflections 49251
Rwork / Rfree 0.18/0.21
Number of non-hydrogen atoms 4705
Protein 4211
Water 494
Protein Residues 554
2
Average B factors (Å )
Protein 36.13
R.m.s. deviations
Bond lengths (Å) 0.01
Bond angles (°) 0.91
Ramachandran statistics (%)
Outliers 0
Allowed 2.38
Favored 97.62
MolProbity score 1.37
All-atom clashscore 5.43
Poor rotamers (%) 0.64

*Values in parentheses are for highest-resolution shell.

Table S3. Cryo-EM data collection, refinement, and validation statistics

KDELR/ RBD/
Structure
Legobody Legobody

Data Accession
PDB 7RXC 7RXD
EMDB EMD-24728 EMD-24729
Data Collection
Microscope Titan Krios Titan Krios
Voltage (kV) 300 300
Stage Movement Stage Movement
Exposure navigation
&beamshift &beamshift
Automation software SerialEM SerialEM
Detector Gatan K3 (Counting) Gatan K3 (Counting)
Energy filter 25 eV 25 eV
Nominal magnification 81k 81k
Pixel Size (Å/pixel) 1.06 1.06
Exposure time (s), frames 2.2, 50 2.16, 50
- -1 -1
Exposure rate (e pixel s ) 26.31 26.18
Electron exposure (e-/Å2) 51.44 50.42
Defocus range (µm) -1.0 to -2.6 -1.0 to -2.6
Micrographs collected 4,863 5795
Reconstruction
Software Relion 3.1 Relion 3.1
Micrographs used 4,803 5095
Particles used in refinement 246,878 282,995
Symmetry C1 C1
Overall resolution (Å)
3.2 3.6
FSC=0.143 (masked)
Map sharpening B-factor (Å2) -100 -130
Local resolution range (Å) 3.0 ~ 5.0 3.4 ~ 5.4
Model Refinement
Software Phenix Phenix
Non-hydrogen atoms 9361 9008
Protein residues 1178 1148
Ligands 5 1
Average B factors (Å2)
Protein 42.8 40.7
Ligands 33.8 40.1
R.M.S. deviations
Bond length (Å) 0.003 0.003
Bond angle (˚) 0.581 0.568
Ramachandran statistics (%)
Outliers 0 0
Allowed 2.26 2.59
Favored 97.74 97.41
MolProbity score 1.56 1.61
Clashscore 9.46 9.3
Poor rotamers (%) 0 0
Model vs. Map FSC
FSC=0.5 (masked, Å) 3.3 3.8
Table S4. Analysis of clashes between Legobody domains and targets

Crystal structures of target/nanobody complexes were aligned with the structure of the Legobody on the basis of the nanobody.
Steric clashes with the different Legobody domains are listed. The Table shows only one entry for a target protein if several similar
structures have been reported. In some cases, the existences of clashes are uncertain (labeled as “possible”).

Small membrane proteins (<100 kDa)

Clash with Clash with Clash with

Target Name PDB code No Clash Fab_8D3
PrAD MBP_PrAC

beta2 adrenoceptor 3P0G X

M2 muscarinic acetylcholine receptor 4MQS X

succinate receptor 6IBB Possible

Viral GPCR US28 5WB1 X

opioid receptor 5C1M X

KDEL receptor 6I6J X

lactose permease 5GXB X

L-amino acid transporter 6F2G X

ADP/ATP Carrier 6GCI X

dipeptide and tripeptide permease 6GS4 X

glycine transporter 6ZBV X

divalent metal ion transporter 5M94 X

MraY translocase 6OYH X

insertase BamA 6QGX X

 Small soluble Proteins (<100 kDa)

Clash with Clash with Clash with

Target Name PDB code No Clash
PrAD MBP_PrAC Fab_8D3

RBD of SARS-CoV-2 7KGJ X

ALFA 6I2G X

GFP 3K1K X

BC2 peptide tag 5IVN X

D3 domain of MTIP 4GFT X

Complement C3 6XZU X

Vsig4 5IML X

PorM 6EY0 X

Lysozyme 3EBA X

Nucleoporin-98 7NOW Possible

Gelsolin 6H1F X

CD38 5F1O X

fimbrial adhesin AC 4W6X X

Surface protein P12p 7KJI X

 MAGEB1 6R7T X

Capsid protein of norovirus 5O02 X

Nucleoporin NIC96 6X07 X

Complement C1q 6Z6V X

PD-L1 5JDS X

CTLA-4 6RQM X

Nup120 6X06 X

DHFR 4EIZ X

GAK kinase 4C59 X

Synaptojanin1 7A17 X

Human prion protein 4N9O X

CD9 6Z1V X

Nup54 5C2U X

Urokinase-type plasminogen activator 5LHP X

EDS1 6I8H X

Octarellin V.1 5BOP X

 Capsid protein p24 5O2U X

Ricin 4LGP X

Cyclin-G-associated kinase 5Y7Z X

CD1d 6V7Y X

Toxin HigB-2 5JA8 X

RhoB 6SGE X

human serum albumin 5VNW X