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- EMDB-22474: Interphotoreceptor retinoid-binding protein (IRBP) in complex wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-22474
TitleInterphotoreceptor retinoid-binding protein (IRBP) in complex with a monoclonal antibody (F3F5 mAb5)
Map data
Sample
  • Complex: Complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody
    • Complex: monoclonal antibody F3F5 mAb5
      • Protein or peptide: mAb5 Fab light chain
      • Protein or peptide: mAb5 Fab heavy chain
    • Complex: Interphotoreceptor retinoid-binding protein (IRBP)
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3
Function / homology
Function and homology information


cone matrix sheath / The retinoid cycle in cones (daylight vision) / The canonical retinoid cycle in rods (twilight vision) / all-trans-retinol binding / oleic acid binding / retinal binding / retinol binding / serine-type peptidase activity / proteolysis / extracellular region
Similarity search - Function
N-terminal domain of Peptidase_S41 in eukaryotic IRBP / tail specific protease / Tail specific protease / Peptidase family S41 / ClpP/crotonase-like domain superfamily
Similarity search - Domain/homology
Retinol-binding protein 3 / Retinol-binding protein 3
Similarity search - Component
Biological speciesMus musculus (house mouse) / Bos taurus (cattle) / Bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsSears AE / Albiez S / Gulati S / Wang B / Kiser P / Kovacik L / Engel A / Stahlberg H / Palczewski K
Funding support United States, Switzerland, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY024864 United States
National Institutes of Health/National Eye Institute (NIH/NEI)T32 GM002750 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY027283 United States
Swiss National Science FoundationNCCR TransCure Switzerland
CitationJournal: FASEB J / Year: 2020
Title: Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.
Authors: Avery E Sears / Stefan Albiez / Sahil Gulati / Benlian Wang / Philip Kiser / Lubomir Kovacik / Andreas Engel / Henning Stahlberg / Krzysztof Palczewski /
Abstract: Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and ...Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
History
DepositionAug 17, 2020-
Header (metadata) releaseOct 7, 2020-
Map releaseOct 7, 2020-
UpdateDec 16, 2020-
Current statusDec 16, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.73
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.73
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jti
  • Surface level: 0.73
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22474.png

Downloads & links

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Map

FileDownload / File: emd_22474.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.662 Å
Density
Contour LevelBy AUTHOR: 0.73 / Movie #1: 0.73
Minimum - Maximum-1.1136531 - 4.32778
Average (Standard dev.)0.0038234561 (±0.060296994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 398.87997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.6621.6621.662
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z398.880398.880398.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ510510510
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-1.1144.3280.004

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Supplemental data

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Mask #1

Fileemd_22474_msk_1.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 : Complex between interphotoreceptor retinoid-binding protein and a...

EntireName: Complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody
Components
  • Complex: Complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody
    • Complex: monoclonal antibody F3F5 mAb5
      • Protein or peptide: mAb5 Fab light chain
      • Protein or peptide: mAb5 Fab heavy chain
    • Complex: Interphotoreceptor retinoid-binding protein (IRBP)
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3
      • Protein or peptide: Retinol-binding protein 3

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Supramolecule #1: Complex between interphotoreceptor retinoid-binding protein and a...

SupramoleculeName: Complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 193 KDa

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Supramolecule #2: monoclonal antibody F3F5 mAb5

SupramoleculeName: monoclonal antibody F3F5 mAb5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse)

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Supramolecule #3: Interphotoreceptor retinoid-binding protein (IRBP)

SupramoleculeName: Interphotoreceptor retinoid-binding protein (IRBP) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#6
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: mAb5 Fab light chain

MacromoleculeName: mAb5 Fab light chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 22.596021 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA QTEDEAIYF CALWYSNHWV FGGGTKLTVL GQPKSSPSVT LFPPSSEELE TNKATLVCTI TDFYPGVVTV DWKVDGTPVT Q GMETTQPS ...String:
QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA QTEDEAIYF CALWYSNHWV FGGGTKLTVL GQPKSSPSVT LFPPSSEELE TNKATLVCTI TDFYPGVVTV DWKVDGTPVT Q GMETTQPS KQSNNKYMAS SYLTLTARAW ERHSSYSCQV THEGHTVEKS LS

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Macromolecule #2: mAb5 Fab heavy chain

MacromoleculeName: mAb5 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.777783 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: QVQLKESGPG LVAPSQSLSI TCTVSGFLLI SNGVHWVRQP PGKGLEWLGV IWAGGNTNYN SALMSRVSIS KDNSKSQVFL KMKSLQTDD TAMYYCARDF YDYDVFYYAM DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL ...String:
QVQLKESGPG LVAPSQSLSI TCTVSGFLLI SNGVHWVRQP PGKGLEWLGV IWAGGNTNYN SALMSRVSIS KDNSKSQVFL KMKSLQTDD TAMYYCARDF YDYDVFYYAM DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVP

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Macromolecule #3: Retinol-binding protein 3

MacromoleculeName: Retinol-binding protein 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle) / Organ: eye / Tissue: retina
Molecular weightTheoretical: 33.267301 KDa
SequenceString: VIQRLQEALR EYYTLVDRVP ALLSHLAAMD LSSVVSEDDL VTKLNAGLQA VSEDPRLQVQ VVRPKEASSG PEEEAEEPPE AVPEVPEDE AVRRALVDSV FQVSVLPGNV GYLRFDSFAD ASVLEVLGPY ILHQVWEPLQ DTEHLIMDLR QNPGGPSSAV P LLLSYFQS ...String:
VIQRLQEALR EYYTLVDRVP ALLSHLAAMD LSSVVSEDDL VTKLNAGLQA VSEDPRLQVQ VVRPKEASSG PEEEAEEPPE AVPEVPEDE AVRRALVDSV FQVSVLPGNV GYLRFDSFAD ASVLEVLGPY ILHQVWEPLQ DTEHLIMDLR QNPGGPSSAV P LLLSYFQS PDASPVRLFS TYDRRTNITR EHFSQTELLG RPYGTQRGVY LLTSHRTATA AEELAFLMQS LGWATLVGEI TA GSLLHTH TVSLLETPEG GLALTVPVLT FIDNHGECWL GGGVVPDAIV LAEEALDRAQ EVLEFH

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Macromolecule #4: Retinol-binding protein 3

MacromoleculeName: Retinol-binding protein 3 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 33.386637 KDa
SequenceString: FSPTLIADMA KIFMDNYCSP EKLTGMEEAI DAASSNTEIL SISDPTMLAN VLTDGVKKTI SDSRVKVTYE PDLILAAPPA MPDIPLEHL AAMIKGTVKV EILEGNIGYL KIQHIIGEEM AQKVGPLLLE YIWDKILPTS AMILDFRSTV TGELSGIPYI V SYFTDPEP ...String:
FSPTLIADMA KIFMDNYCSP EKLTGMEEAI DAASSNTEIL SISDPTMLAN VLTDGVKKTI SDSRVKVTYE PDLILAAPPA MPDIPLEHL AAMIKGTVKV EILEGNIGYL KIQHIIGEEM AQKVGPLLLE YIWDKILPTS AMILDFRSTV TGELSGIPYI V SYFTDPEP LIHIDSVYDR TADLTIELWS MPTLLGKRYG TSKPLIILTS KDTLGIAEDV AYCLKNLKRA TIVGENTAGG TV KMSKMKV GDTDFYVTVP VAKSINPITG KSWEINGVAP DVDVAAEDAL DAAIAIIKLR AEIPALAQAA AT

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Macromolecule #5: Retinol-binding protein 3

MacromoleculeName: Retinol-binding protein 3 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 32.399768 KDa
SequenceString: SLGELVEGTG RLLEAHYARP EVVGQMGALL RAKLAQGAYR TAVDLESLAS QLTADLQEMS GDHRLLVFHS PGEMVAEEAP PPPPVVPSP EELSYLIEAL FKTEVLPGQL GYLRFDAMAE LETVKAVGPQ LVQLVWQKLV DTAALVVDLR YNPGSYSTAV P LLCSYFFE ...String:
SLGELVEGTG RLLEAHYARP EVVGQMGALL RAKLAQGAYR TAVDLESLAS QLTADLQEMS GDHRLLVFHS PGEMVAEEAP PPPPVVPSP EELSYLIEAL FKTEVLPGQL GYLRFDAMAE LETVKAVGPQ LVQLVWQKLV DTAALVVDLR YNPGSYSTAV P LLCSYFFE AEPRRHLYSV FDRATSRVTE VWTLPHVTGQ RYGSHKDLYV LVSHTSGSAA EAFAHTMQDL QRATIIGEPT AG GALSVGI YQVGSSALYA SMPTQMAMSA STGEAWDLAG VEPDITVPMS VALSTARDIV TLR

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Macromolecule #6: Retinol-binding protein 3

MacromoleculeName: Retinol-binding protein 3 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 32.192508 KDa
SequenceString: PTVLQTAGKL VADNYASPEL GVKMAAELSG LQSRYARVTS EAALAELLQA DLQVLSGDPH LKTAHIPEDA KDRIPGIVPM QIPSPEVFE DLIKFSFHTN VLEGNVGYLR FDMFGDCELL TQVSELLVEH VWKKIVHTDA LIVDMRFNIG GPTSSISALC S YFFDEGPP ...String:
PTVLQTAGKL VADNYASPEL GVKMAAELSG LQSRYARVTS EAALAELLQA DLQVLSGDPH LKTAHIPEDA KDRIPGIVPM QIPSPEVFE DLIKFSFHTN VLEGNVGYLR FDMFGDCELL TQVSELLVEH VWKKIVHTDA LIVDMRFNIG GPTSSISALC S YFFDEGPP ILLDKIYNRP NNSVSELWTL SQLEGERYGS KKSMVILTST LTAGAAEEFT YIMKRLGRAL VIGEVTSGGC QP PQTYHVD DTDLYLTIPT ARSVGAADGS SWEGVGVVPD VAVPAEAALT RAQEMLQHT

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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

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHEPES
300.0 mMsodium chlorideNaClSodium chloride
0.01 %DDM
1.0 mMSTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 1.12 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 3 seconds before plunging..
DetailsThe sample had minor aggregation but was overall monodisperse.

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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 / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 1000 pixel / Digitization - Dimensions - Height: 1000 pixel / Number grids imaged: 4 / Number real images: 2649 / Average exposure time: 1.0 sec. / Average electron dose: 80.0 e/Å2
Details: Images were collected in movie mode at 35 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 281804
Details: Ab initio, cisTEM processing using low-pass filtered reconstruction
CTF correctionSoftware - Name: cisTEM
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software: (Name: PHENIX, UCSF Chimera) / Number images used: 17900
DetailsThe selected images were low-pass filtered and normalized.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1j7x


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 156 / Target criteria: Correlation coefficient
Output model

PDB-7jti:
Interphotoreceptor retinoid-binding protein (IRBP) in complex with a monoclonal antibody (F3F5 mAb5)

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