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- PDB-9dkz: In situ microED structure of the Eosinophil major basic protein-1 -

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Basic information

Entry
Database: PDB / ID: 9dkz
TitleIn situ microED structure of the 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: 3.2 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: bioRxiv / Year: 2024
Title: In situ crystalline structure of the human eosinophil major basic protein-1.
Authors: Jie E Yang / Joshua M Mitchell / Craig A Bingman / Deane F Mosher / Elizabeth R Wright /
Abstract: Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils ...Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of macromolecular structure determination.
History
DepositionSep 10, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature
Revision 1.2Nov 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bone marrow proteoglycan


Theoretical massNumber of molelcules
Total (without water)13,6951
Polymers13,6951
Non-polymers00
Water181
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.240, 57.870, 59.060
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Space group name HallP22ab(z,x,y)
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y+1/2,-z+1/2
#4: -x,-y+1/2,z+1/2

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Components

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


Mass: 13694.769 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The major basic protein-1 structure was directly determined from intragranular nanocrystals inside unperturbed human eosinophil cells obtained from donors.
Source: (gene. exp.) Homo sapiens (human) / Tissue: blood / Cell: eosinophil / Gene: PRG2, MBP / Production host: Homo sapiens (human) / References: UniProt: P13727
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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 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 cell deposition on EM grids, grid plunge-freezing, and cryo-FIB milling of ...Details: Mature human eosinophil cells were collected and purified from human donor blood. This was followed by eosinophil cell deposition on EM grids, grid plunge-freezing, and cryo-FIB milling of individual eosinophil cells. Cryo-FIB milling of the cells exposed unperturbed cytosolic secretory granules, inside which nanocrystals of the major basic protein-1 were located. Micro-ED data was collected on these crystals for in-situ structure determination.
Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human) / Cellular location: cytoplasm / Organelle: mature eosinophil secretory granule / Tissue: blood
Buffer solutionpH: 7 / Details: secretory granule matrix
SpecimenConc.: 1000000 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Human eosinophil cells were collected from human donor blood. The cells were rested in 1640 RPMI medium supplemented with 0.1% human serum albumin, before direct deposition onto gold EM ...Details: Human eosinophil cells were collected from human donor blood. The cells were rested in 1640 RPMI medium supplemented with 0.1% human serum albumin, before direct deposition onto gold EM grids and plunge freezing. Subsequently, cryo-FIB milling was used to expose unperturbed intragranular major basic protien-1 nanocrystals present within the cytosolic secretory granules.
Specimen supportDetails: 10 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 310.15 K / Details: Blot time of 6~8 sec, single-side back blotting

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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 / Calibrated defocus min: 0 nm / Calibrated defocus max: 0 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 85 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 0.15 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 40 / Num. of grids imaged: 3
Details: The final dataset was merged from six crystals, each with 40 images. Tilt was -20 to +20 degrees, 1 degree per frame, 1 second per frame
EM diffraction shellResolution: 3.2→28.93 Å / Fourier space coverage: 95.6 % / Multiplicity: 7.5 / Num. of structure factors: 1912 / Phase residual: 26.33 °
EM diffraction statsFourier space coverage: 95.6 % / High resolution: 3.2 Å / Num. of intensities measured: 24722 / Num. of structure factors: 3303 / Phase error: 26.33 ° / Phase error rejection criteria: 0 / Rmerge: 67.29
ReflectionHighest resolution: 3.2 Å / Num. obs: 1912 / % possible obs: 96.1 % / Biso Wilson estimate: 85.01 Å2 / CC1/2: 0.931 / Rmerge(I) obs: 0.69 / Rrim(I) all: 0.719 / Net I/σ(I): 3.2 / Num. measured all: 24817
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)% possible obs (%)Rmerge(I) obsNum. measured obsNum. unique obsCC1/2Rrim(I) allNet I/σ(I) obs
3.2-3.29984.1320311500.5594.290.81
3.29-3.3895.63.53118191300.3643.6630.91
3.38-3.4995.32.21718431410.6182.3031.53
3.49-3.61001.68118411300.6991.7421.9
3.6-3.7396.21.53717361270.8041.5941.78
3.73-3.8794.81.03717121270.9181.0762.71
3.87-4.0395.90.87315841160.8610.9063.16
4.03-4.2199.20.6515401170.9540.6743.57
4.21-4.4194.20.67814851130.9530.7043.7
4.41-4.6597.20.53814101060.9290.564.81
4.65-4.93980.4841255980.9180.5024.94
4.93-5.2792.10.4981200930.9720.5185.07
5.27-5.691000.5571167910.9310.584.25
5.69-6.2493.50.5441064860.9760.5664.6
6.24-6.9798.70.602948740.920.6264.26
6.97-8.05920.477834690.9130.4975.14
8.05-9.8698.50.419676650.9850.4395.08
9.86-13.9596.10.454465490.8340.4785.61
13.9581.10.326207300.9690.3534.14

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286refinement
PHASER2.8.3phasing
XDSJun 30, 2023 BUILT=20230630data reduction
XSCALEJun 30, 2023 BUILT=20230630data scaling
EM software
IDNameVersionCategory
1EPU1.13image acquisition
6Coot0.9.8.92model fitting
8PHENIX1.21.1-5286molecular replacement
12PHENIX1.21.1-52863D reconstruction
13PHENIX1.21.1_5286model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 31.24 Å / B: 57.87 Å / C: 59.06 Å / Space group name: P22121 / Space group num: 18
CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Details: Alternating rounds of phenix.refine and map filling in Coot
Atomic model building

3D fitting-ID: 1 / Chain-ID: A / Chain residue range: 106-222

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

1h8u

A1h8u1106-222PDBexperimental model
2AF-P13727-F1-model_v42AlphaFoldin silico model
RefinementResolution: 3.2→28.93 Å / SU ML: 0.2682 / Cross valid method: FREE R-VALUE / Phase error: 26.171
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.311 199 10.57 %Random selection
Rwork0.2679 1683 --
obs0.2727 1882 95.58 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 91.79 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0055995
ELECTRON CRYSTALLOGRAPHYf_angle_d1.04331346
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0573129
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.007172
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.6272349
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY

Resolution (Å)Rfactor RfreeNum. reflection Rfree% reflection Rfree (%)Rfactor RworkRfactor Rfree error% reflection obs (%)
3.2-3.450.478337100.381740.9994.6
3.45-3.790.353943100.272428.5996.5
3.79-4.340.288237100.265322.7795.4
4.34-5.460.303939100.231519.1996.2
5.47-28.930.27094390.26520.9995.3

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