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- PDB-9cbv: MicroED structure of the human MP20 protein -

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Basic information

Entry
Database: PDB / ID: 9cbv
TitleMicroED structure of the human MP20 protein
ComponentsLens fiber membrane intrinsic protein
KeywordsCELL ADHESION / Lens / Cataract / MicroED / Tetraspanin
Function / homology
Function and homology information


structural constituent of eye lens / cell-cell junction assembly / lens development in camera-type eye / cell junction / vesicle / plasma membrane
Similarity search - Function
Lens fibre membrane intrinsic protein / PMP-22/EMP/MP20 / PMP-22 / EMP / MP20 family signature 2. / : / PMP-22/EMP/MP20/Claudin family / PMP-22/EMP/MP20/Claudin superfamily
Similarity search - Domain/homology
Lens fiber membrane intrinsic protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3.5 Å
AuthorsNicolas, W.J. / Shiriaeva, A. / Martynowycz, M.W. / Grey, A.C. / Ruma, Y. / Donaldson, P.J. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)P41GM136508 United States
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Nat Commun / Year: 2025
Title: Structure of the lens MP20 mediated adhesive junction.
Authors: William J Nicolas / Anna Shiriaeva / Michael W Martynowycz / Angus C Grey / Yasmeen N Ruma / Paul J Donaldson / Tamir Gonen /
Abstract: Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we ...Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. We find that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. Investigation of MP20 localization in human lenses indicate that in young fiber cells MP20 is initially localized to the cytoplasm in differentiating fiber cells but upon fiber cell maturation is inserted into the plasma membrane, correlating with the restriction of the diffusion of extracellular tracers into the lens. Together these results suggest that MP20 forms lens thin junctions in vivo, confirming its role as a structural protein in the human eye lens essential for its optical transparency.
#1: Journal: bioRxiv / Year: 2024
Title: Structure of the lens MP20 mediated adhesive junction.
Authors: William J Nicolas / Anna Shiriaeva / Michael W Martynowycz / Angus C Grey / Yasmeen Ruma / Paul J Donaldson / Tamir Gonen /
Abstract: Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we ...Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C- termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency.
History
DepositionJun 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lens fiber membrane intrinsic protein


Theoretical massNumber of molelcules
Total (without water)25,1611
Polymers25,1611
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)56.180, 56.180, 142.500
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number90
Space group name H-MP4212
Space group name HallP4ab2ab
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z
#3: y+1/2,-x+1/2,z
#4: x+1/2,-y+1/2,-z
#5: -x+1/2,y+1/2,-z
#6: -x,-y,z
#7: y,x,-z
#8: -y,-x,-z

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Components

#1: Protein Lens fiber membrane intrinsic protein / MP18 / MP19 / MP20


Mass: 25160.506 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LIM2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P55344
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: MP20 crystals grown in lipidic cubic phase / Type: ORGANELLE OR CELLULAR COMPONENT
Details: MP20 constructs were expressed in SF9 insect (Spodoptera frugiperda) cells using the Bac-to-bac 251 baculovirus expression system (Invitrogen).
Entity ID: all / Source: NATURAL
Molecular weightValue: 0.022 MDa / Experimental value: NO
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)
EM crystal formationInstrument: syringe coupling system (Hamilton)
Lipid mixture: Molten lipid mix (90% w/w monoolein and 10% w/w cholesterol)
Lipid protein ratio: 3.2
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: OTHER / Details: Frozen in LN2 at ambiant humidity (~40%)

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Data collection

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: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal magnification: 2500 X / Calibrated magnification: 2941 X / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.00325 sec. / Electron dose: 0.00361 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of diffraction images: 130000 / Num. of real images: 130000
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1202 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 3.5→52.26 Å / Fourier space coverage: 86.6 % / Multiplicity: 20.7 / Num. of structure factors: 2785 / Phase residual: 28.84 °
EM diffraction statsFourier space coverage: 86.6 % / High resolution: 3.5 Å / Num. of intensities measured: 58259 / Num. of structure factors: 2785 / Phase error: 28.84 ° / Phase error rejection criteria: NONE / Rmerge: 0.3316
ReflectionBiso Wilson estimate: 100.39 Å2

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Processing

SoftwareName: XSCALE / Classification: data scaling
EM software
IDNameVersionCategoryDetails
1SerialEM`image acquisitionCustom script used to collect data
6Cootmodel fitting
8PHENIXmodel refinement
9PHENIXmolecular replacement
13PHENIX3D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 56.18 Å / B: 56.18 Å / C: 142.5 Å / Space group name: P4212 / Space group num: 90
CTF correctionType: NONE
3D reconstructionResolution: 3.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 88.55 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingDetails: Initially truncated to Poly-A chain / Source name: AlphaFold / Type: in silico model
RefinementResolution: 3.5→52.26 Å / SU ML: 0.4707 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.8438
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.351 151 5.4 %
Rwork0.3316 2644 -
obs0.333 2795 86.27 %
Solvent computationShrinkage radii: 0 Å / VDW probe radii: 0.6 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 88.55 Å2
Refinement stepCycle: LAST / Resolution: 3.5→52.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1323 0 0 0 1323
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00151367
ELECTRON CRYSTALLOGRAPHYf_angle_d0.34011853
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0307197
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0029222
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d4.2035183
LS refinement shellResolution: 3.5→52.26 Å
RfactorNum. reflection% reflection
Rfree0.351 151 -
Rwork0.3316 2644 -
obs--86.27 %

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