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- PDB-7rxc: CryoEM structure of KDELR with Legobody -

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Basic information

Entry
Database: PDB / ID: 7rxc
TitleCryoEM structure of KDELR with Legobody
Components
  • ER lumen protein-retaining receptor 2
  • Fab_8D3_2 heavy chain
  • Fab_8D3_2 light chain
  • Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
  • Nb_KR
KeywordsSTRUCTURAL PROTEIN / protein A / maltose-binding protein / Fab / nanobody
Function / homology
Function and homology information


KDEL sequence binding / COPI-dependent Golgi-to-ER retrograde traffic / ER retention sequence binding / COPI-coated vesicle membrane / protein retention in ER lumen / COPI-mediated anterograde transport / maintenance of protein localization in endoplasmic reticulum / cis-Golgi network / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / IgG binding ...KDEL sequence binding / COPI-dependent Golgi-to-ER retrograde traffic / ER retention sequence binding / COPI-coated vesicle membrane / protein retention in ER lumen / COPI-mediated anterograde transport / maintenance of protein localization in endoplasmic reticulum / cis-Golgi network / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / IgG binding / carbohydrate transmembrane transporter activity / endoplasmic reticulum to Golgi vesicle-mediated transport / protein transport / periplasmic space / Golgi membrane / endoplasmic reticulum membrane / endoplasmic reticulum / extracellular region / membrane
Similarity search - Function
ER lumen protein retaining receptor / ER lumen protein retaining receptor / ER lumen protein retaining receptor signature 1. / ER lumen protein retaining receptor signature 2. / Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / IgG-binding B / B domain ...ER lumen protein retaining receptor / ER lumen protein retaining receptor / ER lumen protein retaining receptor signature 1. / ER lumen protein retaining receptor signature 2. / Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B 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 / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
alpha-maltose / Chem-POV / Maltodextrin-binding protein / Immunoglobulin G-binding protein G / Immunoglobulin G-binding protein A / ER lumen protein-retaining receptor 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Vicugna pacos (alpaca)
Escherichia coli (E. coli)
Staphylococcus aureus (bacteria)
Streptococcus sp. (bacteria)
Gallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-24728
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
H: Fab_8D3_2 heavy chain
L: Fab_8D3_2 light chain
N: Nb_KR
B: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
K: ER lumen protein-retaining receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,74610
Polymers153,3645
Non-polymers3,3835
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 HLNB

#1: 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) / Cell line (production host): HEK293 freestyle / Production host: Homo sapiens (human)
#2: 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) / Cell line (production host): hek293 freestyle / Production host: Homo sapiens (human)
#3: Antibody Nb_KR


Mass: 14698.260 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)
#4: 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

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Protein / Sugars / Non-polymers , 3 types, 6 molecules K

#5: Protein ER lumen protein-retaining receptor 2 / KDEL endoplasmic reticulum protein retention receptor 2 / KDEL receptor 2


Mass: 30083.959 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: KDELR2, RCJMB04_8l23 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q5ZKX9
#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
#7: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM

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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 KDELR 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: 51.44 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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 246878 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039592
ELECTRON MICROSCOPYf_angle_d0.56413010
ELECTRON MICROSCOPYf_dihedral_angle_d6.2981327
ELECTRON MICROSCOPYf_chiral_restr0.0411440
ELECTRON MICROSCOPYf_plane_restr0.0061627

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