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- PDB-7jti: Interphotoreceptor retinoid-binding protein (IRBP) in complex wit... -

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Basic information

Entry
Database: PDB / ID: 7jti
TitleInterphotoreceptor retinoid-binding protein (IRBP) in complex with a monoclonal antibody (F3F5 mAb5)
Components
  • (Retinol-binding protein ...) x 4
  • mAb5 Fab heavy chain
  • mAb5 Fab light chain
KeywordsTRANSPORT PROTEIN/IMMUNE SYSTEM / IRBP / retinoid / Interphotoreceptor retinoid-binding protein / Antibody / TRANSPORT PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


cone matrix sheath / 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 (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsSears, A.E. / Albiez, S. / Gulati, S. / Wang, B. / Kiser, P. / Kovacik, L. / Engel, A. / Stahlberg, H. / Palczewski, K.
Funding support United States, Switzerland, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY024864 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY027283 United States
National Institutes of Health/National Eye Institute (NIH/NEI)T32 GM002750 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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
L: mAb5 Fab light chain
H: mAb5 Fab heavy chain
A: Retinol-binding protein 3
B: Retinol-binding protein 3
C: Retinol-binding protein 3
D: Retinol-binding protein 3


Theoretical massNumber of molelcules
Total (without water)177,6206
Polymers177,6206
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: surface plasmon resonance, ELISA also shown in manuscript to provide evidence.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Retinol-binding protein ... , 4 types, 4 molecules ABCD

#3: Protein Retinol-binding protein 3 / Interphotoreceptor retinoid-binding protein / IRBP / Interstitial retinol-binding protein / Protein 7S


Mass: 33267.301 Da / Num. of mol.: 1 / Fragment: Module 1 (UNP residues 329-631) / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / Organ: eye / Tissue: retina / References: UniProt: P12661
#4: Protein Retinol-binding protein 3


Mass: 33386.637 Da / Num. of mol.: 1 / Fragment: Module 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: F1Q9N9*PLUS
#5: Protein Retinol-binding protein 3 / Interphotoreceptor retinoid-binding protein / IRBP / Interstitial retinol-binding protein / Protein 7S


Mass: 32399.768 Da / Num. of mol.: 1 / Fragment: Module 3 (UNP residues 633-932) / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P12661
#6: Protein Retinol-binding protein 3 / Interphotoreceptor retinoid-binding protein / IRBP / Interstitial retinol-binding protein / Protein 7S


Mass: 32192.508 Da / Num. of mol.: 1 / Fragment: Module 4 (UNP residues 936-1231) / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P12661

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Antibody , 2 types, 2 molecules LH

#1: Antibody mAb5 Fab light chain


Mass: 22596.021 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#2: Antibody mAb5 Fab heavy chain


Mass: 23777.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Details

Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex between interphotoreceptor retinoid-binding protein and a monoclonal antibodyCOMPLEXall0MULTIPLE SOURCES
2monoclonal antibody F3F5 mAb5COMPLEX#1-#21RECOMBINANT
3Interphotoreceptor retinoid-binding protein (IRBP)COMPLEX#3-#61NATURAL
Molecular weightValue: 0.193 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Bos taurus (domestic cattle)9913
Source (recombinant)Organism: Mus musculus (house mouse)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2300 mMsodium chlorideNaCl1
30.01 %DDM1
41.0 mMSTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample had minor aggregation but was overall monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: Blot for 3 seconds before plunging.

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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 FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 2649
Details: Images were collected in movie mode at 35 frames per second.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 1000 / Height: 1000 / Movie frames/image: 35

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cisTEMCTF correction
7PHENIXmodel fitting
9cryoSPARC2initial Euler assignment
12PHENIX3D reconstruction
13UCSF Chimera3D reconstruction
14UCSF Chimeramodel refinement
Image processingDetails: The selected images were low-pass filtered and normalized.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 281804
Details: Ab initio, cisTEM processing using low-pass filtered reconstruction
3D reconstructionResolution: 7.4 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 17900 / Symmetry type: POINT
Atomic model buildingB value: 156 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 1J7X

1j7x


Pdb chain-ID: A / Accession code: 1J7X / Source name: PDB / Type: experimental model

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