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- PDB-9pse: In situ MicroED structure of IL-5 activated human eosinophil majo... -

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Basic information

Entry
Database: PDB / ID: 9pse
TitleIn situ MicroED structure of IL-5 activated human eosinophil major basic protein-1
ComponentsBone marrow proteoglycan
KeywordsIMMUNE SYSTEM / Effector / Nanocrystal / In-situ / Intracellular
Function / homology
Function and homology information


extracellular matrix structural constituent conferring compression resistance / defense response to nematode / negative regulation of macrophage cytokine production / negative regulation of interleukin-10 production / positive regulation of interleukin-4 production / transport vesicle / heparin binding / : / carbohydrate binding / ficolin-1-rich granule lumen ...extracellular matrix structural constituent conferring compression resistance / defense response to nematode / negative regulation of macrophage cytokine production / negative regulation of interleukin-10 production / positive regulation of interleukin-4 production / transport vesicle / heparin binding / : / carbohydrate binding / ficolin-1-rich granule lumen / defense response to bacterium / immune response / Neutrophil degranulation / extracellular exosome / extracellular region
Similarity search - Function
Eosinophil major basic protein / Eosinophil major basic protein, C-type lectin-like domain / : / C-type lectin, conserved site / C-type lectin domain signature. / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / C-type lectin fold
Similarity search - Domain/homology
Bone marrow proteoglycan
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3.18 Å
AuthorsYang, J.E. / Bingman, C.A. / Mitchell, J. / Mosher, D. / Wright, E.R.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM114561 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM139168 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)P01 HL088594 United States
Department of Energy (DOE, United States)DE-SC0023013 United States
Department of Energy (DOE, United States)DE-SC0018409 United States
CitationJournal: To Be Published
Title: In situ microED structure of the Eosinophil major basic protein-1
Authors: Yang, J.E. / Bingman, C.A. / Mitchell, J. / Mosher, D. / Wright, E.R.
History
DepositionJul 25, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Bone marrow proteoglycan
A: Bone marrow proteoglycan
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,4604
Polymers27,3902
Non-polymers712
Water543
1
B: Bone marrow proteoglycan
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,7302
Polymers13,6951
Non-polymers351
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
A: Bone marrow proteoglycan
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,7302
Polymers13,6951
Non-polymers351
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)30.200, 57.800, 58.600
Angle α, β, γ (deg.)90.000, 91.200, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Bone marrow proteoglycan / BMPG / Proteoglycan 2


Mass: 13694.769 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human MBP-1 derives from a precursor or proform proMBP coded by gene PRG2 in eosinophils. Protein MBP-1 naturally forms intragranular nanocrystals that get disassembled upon activation, including cytokine IL-5.
Source: (gene. exp.) Homo sapiens (human) / Gene: PRG2, MBP / Production host: Homo sapiens (human) / References: UniProt: P13727
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY
Nonpolymer detailsAuthors state that the chloride ions A301 and B301 modeled could be bromide ions.

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: In situ MicroED structure of the IL-5 activated human eosinophil major basic protein-1
Type: CELL
Details: Mature human eosinophil cells were collected and purified from human donor blood. This was followed by eosinophil resting and deposition on EM grids. The cells were then activated by IL-5 ...Details: Mature human eosinophil cells were collected and purified from human donor blood. This was followed by eosinophil resting and deposition on EM grids. The cells were then activated by IL-5 for 1 hour prior to grid plunge-freezing. Cryo-FIB milling of individual eosinophil cells was then performed to expose unperturbed cytosolic secretory granules, inside which nanocrystals of the major basic protein-1 were located. MicroED data were collected on these crystals for in situ structure determination.
Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human) / Cellular location: cytoplasm / Organ: blood / Organelle: secretory granule
Buffer solutionpH: 7.4
Details: Harvested from human donor peripheral blood and rested in 1640 RPMI medium supplemented with 0.1% human serum albumin (HSA)
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pre-vacuum was 1 x 10-3 Pa and the process pressure was 1 Pa
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 %

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 0.1 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 40
Details: Ten datasets, each with 40 images, were scaled together using XSCALE. The electron dose per image was 0.1 e/A^2.
EM diffraction shellResolution: 3→30.69 Å / Fourier space coverage: 85.7 % / Multiplicity: 4.45 / Num. of structure factors: 6805 / Phase residual: 38.78 °
EM diffraction statsFourier space coverage: 85.7 % / High resolution: 3 Å / Num. of intensities measured: 35354 / Num. of structure factors: 6805
Phase error rejection criteria: no rejections based on phase error
Rmerge: 63.1
ReflectionResolution: 3→30.69 Å / Num. obs: 6805 / % possible obs: 85.7 % / Biso Wilson estimate: 47.9 Å2 / CC1/2: 0.682 / Rmerge(I) obs: 0.66 / Rrim(I) all: 0.724 / Net I/σ(I): 2.47 / Num. measured all: 35354
Reflection shell

Diffraction-ID: 1

Resolution (Å)% possible obs (%)Rmerge(I) obsNum. measured obsNum. unique obsCC1/2Rrim(I) allNet I/σ(I) obs
3-3.4470.21.376633518780.2751.5851.16
3.44-4.3389.50.7711308023460.7830.8372.43
4.33-30.6997.80.5521593925810.6630.6043.47

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286refinement
XDSdata reduction
XSCALEdata scaling
PDB_EXTRACTdata extraction
PHENIXphasing
EM software
IDNameVersionCategory
6Coot0.9.8.92model fitting
9PHENIX1.21.1_5286model refinement
11PHENIX2.8.3molecular replacement
16PHENIX1.21.1-5286-0003D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 91.2 ° / ∠γ: 90 ° / A: 30.2 Å / B: 57.8 Å / C: 58.6 Å / Space group name: P1211 / Space group num: 3
CTF correctionType: NONE
3D reconstructionResolution: 3.18 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 40 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood
Details: Iterative refinement using phenix.refine and model building in COOT
Atomic model building

3D fitting-ID: 1 / Chain-ID: A / Chain residue range: 107-222

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue rangeSource nameType
11h8u

1h8u

A1h8u1107-222PDBexperimental model
2AF-P13727-F1-model_v42AlphaFoldin silico model
RefinementResolution: 3.18→3.18 Å / SU ML: 0.5783 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.2491
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3195 363 10.38 %
Rwork0.2776 3133 -
obs0.2815 3496 85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 40 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0021990
ELECTRON CRYSTALLOGRAPHYf_angle_d0.42372692
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0388258
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0059344
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.1875698
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.430.3834930.3305848ELECTRON CRYSTALLOGRAPHY69.24
3.43-4.320.31831170.29021092ELECTRON CRYSTALLOGRAPHY88.64
4.33-30.190.30031530.24971193ELECTRON CRYSTALLOGRAPHY96.83

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