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- EMDB-24728: CryoEM structure of KDELR with Legobody -

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Basic information

Entry
Database: EMDB / ID: EMD-24728
TitleCryoEM structure of KDELR with Legobody
Map datasharpened map for the complex
Sample
  • Complex: The complex of KDELR with Legobody
    • Protein or peptide: Fab_8D3_2 heavy chain
    • Protein or peptide: Fab_8D3_2 light chain
    • Protein or peptide: Nb_KR
    • Protein or peptide: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
    • Protein or peptide: ER lumen protein-retaining receptor 2
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
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
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) / Streptococcus sp. (bacteria) / Gallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWu XD / Rapoport TA
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateOct 20, 2021-
Current statusOct 20, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rxc
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24728.png

Downloads & links

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Map

FileDownload / File: emd_24728.map.gz / Format: CCP4 / Size: 46.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map for the complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.14933106 - 0.23163602
Average (Standard dev.)0.00016516831 (±0.005520515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions230230230
Spacing230230230
CellA=B=C: 243.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z230230230
origin x/y/z0.0000.0000.000
length x/y/z243.800243.800243.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS230230230
D min/max/mean-0.1490.2320.000

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Supplemental data

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Additional map: unsharpened map

Fileemd_24728_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The complex of KDELR with Legobody

EntireName: The complex of KDELR with Legobody
Components
  • Complex: The complex of KDELR with Legobody
    • Protein or peptide: Fab_8D3_2 heavy chain
    • Protein or peptide: Fab_8D3_2 light chain
    • Protein or peptide: Nb_KR
    • Protein or peptide: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
    • Protein or peptide: ER lumen protein-retaining receptor 2
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate

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Supramolecule #1: The complex of KDELR with Legobody

SupramoleculeName: The complex of KDELR with Legobody / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Fab_8D3_2 heavy chain

MacromoleculeName: Fab_8D3_2 heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.252217 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DVQLVESGGG LVQPGKSLRL SCAASGFTFS NFGMHWVRQA PEMGLEWVAY ISSGSTTKYY GDTVKGRFTI SRDNPKNTLY LQMNSLRSE DTAMYYCARR PLYDGDYGYP MDYWGQGTSV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA ...String:
DVQLVESGGG LVQPGKSLRL SCAASGFTFS NFGMHWVRQA PEMGLEWVAY ISSGSTTKYY GDTVKGRFTI SRDNPKNTLY LQMNSLRSE DTAMYYCARR PLYDGDYGYP MDYWGQGTSV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCGS HHHHHH

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Macromolecule #2: Fab_8D3_2 light chain

MacromoleculeName: Fab_8D3_2 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.095852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NIMLTQSPSS LAVSAGERVT MSCKSTQSIL YNSNQKTYLA WYQQKPGQSP KLLIYWASTR ASGVPDRFTG SGSGTDFTLT INSVQPEDL AVYYCHQYLS AWTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV ...String:
NIMLTQSPSS LAVSAGERVT MSCKSTQSIL YNSNQKTYLA WYQQKPGQSP KLLIYWASTR ASGVPDRFTG SGSGTDFTLT INSVQPEDL AVYYCHQYLS AWTFGGGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

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Macromolecule #3: Nb_KR

MacromoleculeName: Nb_KR / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 14.69826 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGFPVK RWSMTWYRQA PGKEREWVAA IRSAGHWTHY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCNVK DEGDFSYWYD YWGQGTQVTV SSLEHHHHHH

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Macromolecule #4: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,I...

MacromoleculeName: Maltodextrin-binding protein,Immunoglobulin G-binding protein A,Immunoglobulin G-binding protein G
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus sp. (bacteria)
Molecular weightTheoretical: 59.233246 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY ...String:
MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY AFKYENGKYD IKDVGVDNAG AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NKGETAMTIN GPWAWSNIDT SK VNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT DEGLEAVNKD KPLGAVALKS YEEELAKDPR IAA TMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDQALAFAQ ILIMPNLTEE QRNGFIQSLK DDPSVSKEIL AEAK KLNEH QAPKGGSGGA GSGDQQSAFY EILNMPNLNE AQRNGFIQSL KDDPSQSTNV LGEAKKLNES QAGGGSGGGS GGSAV TTYK LVINGKTLKG ETTTKAVDAE TAEKAFKQYA NDNGVDGVWT YDDATKTFTV TEGSGHHHHH H

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Macromolecule #5: ER lumen protein-retaining receptor 2

MacromoleculeName: ER lumen protein-retaining receptor 2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 30.083959 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MNIFRLTGDL SHLAAIIILL LKIWKSRSCA GISGKSQLLF ALVFTTRYLD LFTSFISLYN TSMKLIYIAC SYATVYLIYM KFKATYDGN HDTFRVEFLI VPVGGLSFLV NHDFSPLEIL WTFSIYLESV AILPQLFMIS KTGEAETITT HYLFFLGLYR A LYLVNWIW ...String:
MNIFRLTGDL SHLAAIIILL LKIWKSRSCA GISGKSQLLF ALVFTTRYLD LFTSFISLYN TSMKLIYIAC SYATVYLIYM KFKATYDGN HDTFRVEFLI VPVGGLSFLV NHDFSPLEIL WTFSIYLESV AILPQLFMIS KTGEAETITT HYLFFLGLYR A LYLVNWIW RYYFEGFFDL IAVVAGVVQT VLYCDFFYLY VTKVLKGKKL SLPAGSGGEN LYFQSGGGMD EKTTGWRGGH VV EGLAGEL EQLRARLEHH PQGQREP

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Macromolecule #7: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 7 / Number of copies: 4 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.44 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 246878

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