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- EMDB-3595: Retinoschisin R141H Mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-3595
TitleRetinoschisin R141H Mutant
Map dataRetinoschisin R141H D8 Hexadecameric Complex
Sample
  • Complex: Retinoschisin
    • Protein or peptide: Retinoschisin
KeywordsRetinoschisin Discoidin Domain Retinal Structure / structural protein
Function / homology
Function and homology information


neuron to neuron synapse / retina layer formation / eye development / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylserine binding / photoreceptor inner segment / visual perception / protein homooligomerization / cell adhesion / external side of plasma membrane ...neuron to neuron synapse / retina layer formation / eye development / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylserine binding / photoreceptor inner segment / visual perception / protein homooligomerization / cell adhesion / external side of plasma membrane / protein-containing complex binding / protein-containing complex / extracellular space
Similarity search - Function
Coagulation factors 5/8 type C domain (FA58C) signature 1. / Coagulation factor 5/8 C-terminal domain, discoidin domain / Coagulation factors 5/8 type C domain (FA58C) profile. / F5/8 type C domain / Coagulation factor 5/8 C-terminal domain / Galactose-binding-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsRamsay EP / Collins RF / Owens TW / Siebert CA / Jones RPO / Roseman A / Wang T / Baldock C
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust099735/Z/12/Z United Kingdom
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 XLRS- ...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.
History
DepositionFeb 16, 2017-
Header (metadata) releaseMar 29, 2017-
Map releaseApr 12, 2017-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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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

emd_3595.png

Downloads & links

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Map

FileDownload / File: emd_3595.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRetinoschisin R141H D8 Hexadecameric Complex
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.04040661 - 0.07888982
Average (Standard dev.)0.00025765845 (±0.0039747357)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: Retinoschisin
    • Protein or peptide: Retinoschisin

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Supramolecule #1: Retinoschisin

SupramoleculeName: Retinoschisin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Hexadecameric complex of sixteen retinoschisin molecules
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Retinoschisin

MacromoleculeName: Retinoschisin / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.041902 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: STEDEGEDPW YQKACKCDCQ GGPNALWSAG ATSLDCIPEC PYHKPLGFES GEVTPDQITC SNPEQYVGWY SSWTANKARL NSQGFGCAW LSKFQDSSQW LQIDLKEIKV ISGILTQGHC DIDEWMTKYS VQYRTDERLN WIYYKDQTGN NRVFYGNSDR T STVQNLLR ...String:
STEDEGEDPW YQKACKCDCQ GGPNALWSAG ATSLDCIPEC PYHKPLGFES GEVTPDQITC SNPEQYVGWY SSWTANKARL NSQGFGCAW LSKFQDSSQW LQIDLKEIKV ISGILTQGHC DIDEWMTKYS VQYRTDERLN WIYYKDQTGN NRVFYGNSDR T STVQNLLR PPIISRFIRL IPLGWHVRIA IRMELLECVS KCA

UniProtKB: Retinoschisin

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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

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
150.0 mMNaClSodium Chloride
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsThe sample was monodisperse and visible

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-8 / Number grids imaged: 1 / Number real images: 1200 / Average exposure time: 0.5 sec. / Average electron dose: 2.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: EMAN Generated Initial Model
Final reconstructionApplied symmetry - Point group: D8 (2x8 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 7056
Initial angle assignmentType: COMMON LINE / Software - Name: EMAN (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 2 / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-5n6w:
Retinoschisin R141H Mutant

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