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- EMDB-3595: Retinoschisin R141H Mutant -

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Entry
Database: EMDB / ID: 3595
TitleRetinoschisin R141H Mutant
Map dataRetinoschisin R141H D8 Hexadecameric Complex
SampleRetinoschisin
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
SourceHomo sapiens / / human
Methodsingle particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsRamsay EP / Collins RF / Owens TW / Siebert CA / Jones RPO / 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 ReportPDB-ID: 5n6w

SummaryFull reportAbout validation report
DateDeposition: Feb 16, 2017 / Header (metadata) release: Mar 29, 2017 / Map release: Apr 12, 2017 / Last update: Aug 30, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5n6w
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3595.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.29 Å/pix.
= 330.24 Å
256 pix
1.29 Å/pix.
= 330.24 Å
256 pix
1.29 Å/pix.
= 330.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.29 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.04040661 - 0.07888982
Average (Standard dev.)0.00025765845 (0.0039747357)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin000
Limit255255255
Spacing256256256
CellA=B=C: 330.24 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.291.291.29
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z330.240330.240330.240
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0400.0790.000

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Supplemental data

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Sample components

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Entire Retinoschisin

EntireName: Retinoschisin
Details: Hexadecameric complex of sixteen retinoschisin molecules
Number of components: 2

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Component #1: protein, Retinoschisin

ProteinName: Retinoschisin
Details: Hexadecameric complex of sixteen retinoschisin molecules
Recombinant expression: No
SourceSpecies: Homo sapiens / / human
Source (engineered)Expression System: Homo sapiens / / human / Vector: pCEP-Pu/Ac7 / Cell of expression system: HEK293-EBNA

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Component #2: protein, Retinoschisin

ProteinName: Retinoschisin / Recombinant expression: No
MassTheoretical: 23.041902 kDa
Source (engineered)Expression System: Homo sapiens / / human / Vector: pCEP-Pu/Ac7

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/ml / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2.8 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 105000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 4500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1200

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D8 (2*8 fold dihedral) / Number of projections: 7056
3D reconstructionSoftware: RELION / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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