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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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsRamsay, E.P. / Collins, R.F. / Owens, T.W. / Siebert, C.A. / Jones, R.P.O. / Roseman, A. / Wang, T. / Baldock, C.
Funding support United Kingdom, 1items
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, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 12, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Experimental preparation / Refinement description
Category: em_3d_fitting / em_sample_support / em_software
Item: _em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name
Revision 1.2Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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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
Polymers368,67016
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29630 Å2
ΔGint-130 kcal/mol
Surface area131310 Å2
MethodPISA

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Components

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


Mass: 23041.902 Da / Num. of mol.: 16 / Mutation: R141H Pathogenic Mutation
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RS1, XLRS1 / Plasmid: pCEP-Pu/Ac7 / Cell line (production host): HEK293-EBNA / Production host: Homo sapiens (human) / References: UniProt: O15537
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

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

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / 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) / Num. of grids imaged: 1 / Num. of real images: 1200
Image scansMovie frames/image: 14 / Used frames/image: 2-8

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
4CTFFIND4CTF correction
7DockEM2model fitting
9EMAN2initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13Flex-EMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D8 (2x8 fold dihedral)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7056 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient

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