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- PDB-7rxd: CryoEM structure of RBD domain of COVID-19 in complex with Legobody -

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Basic information

Entry
Database: PDB / ID: 7rxd
TitleCryoEM structure of RBD domain of COVID-19 in complex with Legobody
Components
  • Fab_8D3_2 heavy chain
  • Fab_8D3_2 light chain
  • Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
  • Nb_RBD
  • Spike protein S1
KeywordsSTRUCTURAL PROTEIN / RBD / maltose-binding protein / Fab / nanobody
Function / homology
Function and homology information


IgG binding / carbohydrate transmembrane transporter activity / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / viral life cycle / receptor-mediated endocytosis of virus by host cell ...IgG binding / carbohydrate transmembrane transporter activity / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / viral life cycle / receptor-mediated endocytosis of virus by host cell / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell / periplasmic space / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / extracellular region / integral component of membrane / membrane / identical protein binding
Similarity search - Function
Octapeptide repeat / Octapeptide repeat / B domain / Protein A, Ig-binding domain / IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Lysin motif ...Octapeptide repeat / Octapeptide repeat / B domain / Protein A, Ig-binding domain / IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / Lysin motif / LysM domain superfamily / LysM domain / LysM domain profile. / LysM domain / Immunoglobulin/albumin-binding domain superfamily / YSIRK type signal peptide / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S2 / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Spike glycoprotein / alpha-maltose / Immunoglobulin G-binding protein G / Maltodextrin-binding protein / Immunoglobulin G-binding protein A
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Staphylococcus aureus (bacteria)
Streptococcus sp. (bacteria)
Mus musculus (house mouse)
Vicugna pacos (alpaca)
Severe acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWu, X.D. / Rapoport, T.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM052586 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies).
Authors: Xudong Wu / Tom A Rapoport /
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 ...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 ∼120 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.
History
DepositionAug 22, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
H: Fab_8D3_2 heavy chain
L: Fab_8D3_2 light chain
N: Nb_RBD
R: Spike protein S1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,5806
Polymers149,2385
Non-polymers3421
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 4 types, 4 molecules BHLN

#1: Antibody Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G / IgG-binding protein A / Staphylococcal protein A / SpA / IgG-binding protein G


Mass: 59233.246 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Staphylococcus aureus (bacteria), (gene. exp.) Streptococcus sp. (bacteria)
Gene: DAH37_23060, spa, spg / Production host: Escherichia coli (E. coli)
References: UniProt: A0A4Z0THX4, UniProt: P99134, UniProt: P06654
#2: Antibody Fab_8D3_2 heavy chain


Mass: 25252.217 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#3: Antibody Fab_8D3_2 light chain


Mass: 24095.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Homo sapiens (human)
#4: Antibody Nb_RBD


Mass: 14661.201 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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Protein / Sugars , 2 types, 2 molecules R

#5: Protein Spike protein S1


Mass: 25995.555 Da / Num. of mol.: 1 / Fragment: Receptor Binding Domain (RBD)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#6: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of RBD domain of COVID-19 with Legobody / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.15 MDa / Experimental value: NO
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50.42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282995 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039228
ELECTRON MICROSCOPYf_angle_d0.54712532
ELECTRON MICROSCOPYf_dihedral_angle_d4.3551250
ELECTRON MICROSCOPYf_chiral_restr0.0421367
ELECTRON MICROSCOPYf_plane_restr0.0051605

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