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- PDB-5n6w: Retinoschisin R141H Mutant -

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Basic information

Entry
Database: PDB / ID: 5n6w
TitleRetinoschisin R141H Mutant
ComponentsRetinoschisin
KeywordsSTRUCTURAL PROTEIN / Retinoschisin Discoidin Domain Retinal Structure / structural protein
Function / homologyCoagulation 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 sourceHomo sapiens / / human
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsRamsay, E.P. / Collins, R.F. / Owens, T.W. / Siebert, C.A. / Jones, R.P.O. / Roseman, A. / Wang, T. / Baldock, C.
CitationJournal: 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 16, 2017 / Release: Apr 12, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Apr 12, 2017Structure modelrepositoryInitial release
1.1Aug 30, 2017Structure modelData collection / Experimental preparation / Refinement descriptionem_3d_fitting / em_sample_support / em_software_em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name

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Assembly

Deposited unit
A: Retinoschisin
B: Retinoschisin
C: Retinoschisin
D: Retinoschisin
E: Retinoschisin
F: Retinoschisin
G: Retinoschisin
H: Retinoschisin
I: Retinoschisin
J: Retinoschisin
K: Retinoschisin
L: Retinoschisin
M: Retinoschisin
N: Retinoschisin
O: Retinoschisin
P: Retinoschisin


Theoretical massNumber of molelcules
Total (without water)368,67016
Polyers368,67016
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)29630
ΔGint (kcal/M)-130
Surface area (Å2)131310
MethodPISA

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Components

#1: Protein/peptide
Retinoschisin / / X-linked juvenile retinoschisis protein


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Retinoschisin / Type: COMPLEX
Details: Hexadecameric complex of sixteen retinoschisin molecules
Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens
Source (recombinant)Cell: HEK293-EBNA / Organism: Homo sapiens / Plasmid: pCEP-Pu/Ac7
Buffer solutionpH: 7.4
Buffer component
IDConc.UnitsNameFormulaBuffer ID
110mMTrisC4H11NO31
2150mMSodium ChlorideNaCl1
SpecimenConc.: 0.1 mg/ml / Details: The sample was monodisperse and visible / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansMovie frames/image: 14 / Used frames/image: 2-8

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Processing

EM software
IDNameVersionCategory
1EMAN2.0particle selection
4CTFFIND4CTF correction
7DockEM2model fitting
9EMAN2.0initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13Flex-EMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D8
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 7056 / Symmetry type: POINT
Atomic model buildingRef protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient

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