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- PDB-8ghz: Cryo-EM structure of fish immunogloblin M-Fc -

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Basic information

Entry
Database: PDB / ID: 8ghz
TitleCryo-EM structure of fish immunogloblin M-Fc
ComponentsTeleost immunoglobulin M protein
KeywordsIMMUNE SYSTEM / Immunoglobulin M / IgM
Biological speciesOncorhynchus mykiss (rainbow trout)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsLyu, M. / Stadtmueller, B.M. / Malyutin, A.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165570 United States
CitationJournal: Nat Commun / Year: 2023
Title: The structure of the teleost Immunoglobulin M core provides insights on polymeric antibody evolution, assembly, and function.
Authors: Mengfan Lyu / Andrey G Malyutin / Beth M Stadtmueller /
Abstract: Polymeric (p) immunoglobulins (Igs) serve broad functions during vertebrate immune responses. Typically, pIgs contain between two and six Ig monomers, each with two antigen binding fragments and one ...Polymeric (p) immunoglobulins (Igs) serve broad functions during vertebrate immune responses. Typically, pIgs contain between two and six Ig monomers, each with two antigen binding fragments and one fragment crystallization (Fc). In addition, many pIgs assemble with a joining-chain (JC); however, the number of monomers and potential to include JC vary with species and heavy chain class. Here, we report the cryo-electron microscopy structure of IgM from a teleost (t) species, which does not encode JC. The structure reveals four tIgM Fcs linked through eight C-terminal tailpieces (Tps), which adopt a single β-sandwich-like domain (Tp assembly) located between two Fcs. Specifically, two of eight heavy chains fold uniquely, resulting in a structure distinct from mammalian IgM, which typically contains five IgM monomers, one JC and a centrally-located Tp assembly. Together with mutational analysis, structural data indicate that pIgs have evolved a range of assembly mechanisms and structures, each likely to support unique antibody effector functions.
History
DepositionMar 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 27, 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.journal_volume / _citation.page_first / _citation.page_last / _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

Downloads & links

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Assembly

Deposited unit
A: Teleost immunoglobulin M protein
B: Teleost immunoglobulin M protein
C: Teleost immunoglobulin M protein
D: Teleost immunoglobulin M protein
E: Teleost immunoglobulin M protein
F: Teleost immunoglobulin M protein
G: Teleost immunoglobulin M protein
H: Teleost immunoglobulin M protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)220,68016
Polymers215,9888
Non-polymers4,6928
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Teleost immunoglobulin M protein


Mass: 26998.514 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oncorhynchus mykiss (rainbow trout) / Plasmid: pD2610-v1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
Has ligand of interestY

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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: rainbow trout (Oncorhynchus mykiss) immunoglobulin M Fc
Type: COMPLEX
Details: Heavy chain constant domains 3 and 4 including C-terminal tailpiece
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Oncorhynchus mykiss (rainbow trout)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTrisC4H11NO31
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco easiGlow at 25 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 600 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
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3618
Details: Images were collected with SerialEM using beam image-shift in a 3x3x3 pattern. A total of ~60 e/A2 was fractionated into 40 frames movies.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selectionBlob picker, followed by templated picker after 2D classification
2SerialEMimage acquisition
4cryoSPARC4CTF correction
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
13cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1300000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 169.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314587
ELECTRON MICROSCOPYf_angle_d0.67419869
ELECTRON MICROSCOPYf_dihedral_angle_d4.7472006
ELECTRON MICROSCOPYf_chiral_restr0.0482363
ELECTRON MICROSCOPYf_plane_restr0.0052513

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