|Entry||Database: PDB / ID: 5n6w|
|Title||Retinoschisin R141H Mutant|
|Keywords||STRUCTURAL PROTEIN / Retinoschisin Discoidin Domain Retinal Structure / structural protein|
|Function / homology||Coagulation factors 5/8 type C domain (FA58C) profile. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / F5/8 type C domain / Galactose-binding-like domain superfamily / Coagulation factor 5/8 C-terminal domain / adaptation of rhodopsin mediated signaling / retina layer formation / phosphatidylinositol-5-phosphate binding / phosphatidylinositol-3,5-bisphosphate binding / oligosaccharide binding ...Coagulation factors 5/8 type C domain (FA58C) profile. / Coagulation factors 5/8 type C domain (FA58C) signature 1. / F5/8 type C domain / Galactose-binding-like domain superfamily / Coagulation factor 5/8 C-terminal domain / adaptation of rhodopsin mediated signaling / retina layer formation / phosphatidylinositol-5-phosphate binding / phosphatidylinositol-3,5-bisphosphate binding / oligosaccharide binding / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3-phosphate binding / extrinsic component of plasma membrane / phosphatidylinositol-4-phosphate binding / phosphatidylinositol-3,4,5-trisphosphate binding / phosphatidylserine binding / visual perception / phosphatidylinositol-4,5-bisphosphate binding / protein homooligomerization / multicellular organism development / cell adhesion / extracellular space / Retinoschisin|
Function and homology information
|Specimen source||Homo sapiens / / human|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.2 Å resolution|
|Authors||Ramsay, E.P. / Collins, R.F. / Owens, T.W. / Siebert, C.A. / Jones, R.P.O. / Roseman, A. / Wang, T. / Baldock, C.|
|Citation||Journal: Hum. Mol. Genet. / Year: 2016|
Title: Structural analysis of X-linked retinoschisis mutations reveals distinct classes which differentially effect retinoschisin function.
Authors: Ewan P Ramsay / Richard F Collins / Thomas W Owens / C Alistair Siebert / Richard P O Jones / Tao Wang / Alan M Roseman / Clair Baldock
Abstract: Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most ...Retinoschisin, an octameric retinal-specific protein, is essential for retinal architecture with mutations causing X-linked retinoschisis (XLRS), a monogenic form of macular degeneration. Most XLRS-associated mutations cause intracellular retention, however a subset are secreted as octamers and the cause of their pathology is ill-defined. Therefore, here we investigated the solution structure of the retinoschisin monomer and the impact of two XLRS-causing mutants using a combinatorial approach of biophysics and cryo-EM. The retinoschisin monomer has an elongated structure which persists in the octameric assembly. Retinoschisin forms a dimer of octamers with each octameric ring adopting a planar propeller structure. Comparison of the octamer with the hexadecamer structure indicated little conformational change in the retinoschisin octamer upon dimerization, suggesting that the octamer provides a stable interface for the construction of the hexadecamer. The H207Q XLRS-associated mutation was found in the interface between octamers and destabilized both monomeric and octameric retinoschisin. Octamer dimerization is consistent with the adhesive function of retinoschisin supporting interactions between retinal cell layers, so disassembly would prevent structural coupling between opposing membranes. In contrast, cryo-EM structural analysis of the R141H mutation at ∼4.2Å resolution was found to only cause a subtle conformational change in the propeller tips, potentially perturbing an interaction site. Together, these findings support distinct mechanisms of pathology for two classes of XLRS-associated mutations in the retinoschisin assembly.
Copyright: The Author 2016. Published by Oxford University Press.
SummaryFull reportAbout validation report
|Date||Deposition: Feb 16, 2017 / Release: Apr 12, 2017|
|Structure viewer||Molecule: |
Downloads & links
Mass: 23041.902 Da / Num. of mol.: 16 / Mutation: R141H Pathogenic Mutation / Source: (gene. exp.) Homo sapiens / / human / Gene: RS1, XLRS1 / Plasmid name: pCEP-Pu/Ac7 / Cell line (production host): HEK293-EBNA / Production host: Homo sapiens / References: UniProt:O15537
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: Retinoschisin / Type: COMPLEX|
Details: Hexadecameric complex of sixteen retinoschisin molecules
Entity ID: 1 / Source: RECOMBINANT
|Molecular weight||Experimental value: NO|
|Source (natural)||Organism: Homo sapiens|
|Source (recombinant)||Cell: HEK293-EBNA / Organism: Homo sapiens / Plasmid: pCEP-Pu/Ac7|
|Buffer solution||pH: 7.4|
|Specimen||Conc.: 0.1 mg/ml / Details: The sample was monodisperse and visible / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 400 / Grid type: C-flat-2/2|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 0.5 sec. / Electron dose: 2.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1200|
|Image scans||Movie frames/image: 14 / Used frames/image: 2-8|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: D8|
|3D reconstruction||Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 7056 / Symmetry type: POINT|
|Atomic model building||Ref protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient|
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