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- PDB-7b75: Cryo-EM Structure of Human Thyroglobulin -

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Basic information

Entry
Database: PDB / ID: 7b75
TitleCryo-EM Structure of Human Thyroglobulin
ComponentsThyroglobulin
KeywordsHORMONE / Thyroglobulin / T3 and T4 Hormonogenesis / cryo-EM
Function / homology
Function and homology information


iodide transport / hormone biosynthetic process / thyroid hormone generation / regulation of myelination / thyroid gland development / hormone activity / signal transduction / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Thyroglobulin / Thyroglobulin type-1 repeat signature. / Thyroglobulin type-1 / Thyroglobulin type-1 superfamily / Thyroglobulin type-1 repeat / Thyroglobulin type-1 domain profile. / Thyroglobulin type I repeats. / Tyrosine-protein kinase ephrin type A/B receptor-like / Tyrosine-protein kinase ephrin type A/B receptor-like / Carboxylesterase type B, conserved site ...Thyroglobulin / Thyroglobulin type-1 repeat signature. / Thyroglobulin type-1 / Thyroglobulin type-1 superfamily / Thyroglobulin type-1 repeat / Thyroglobulin type-1 domain profile. / Thyroglobulin type I repeats. / Tyrosine-protein kinase ephrin type A/B receptor-like / Tyrosine-protein kinase ephrin type A/B receptor-like / Carboxylesterase type B, conserved site / Carboxylesterases type-B signature 2. / Carboxylesterase, type B / Carboxylesterase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAdaixo, R. / Righetto, R. / Steiner, E.M. / Taylor, N.M.I. / Stahlberg, H.
Funding support Denmark, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation Denmark
Novo Nordisk FoundationNNF14CC0001 Denmark
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structure of native human thyroglobulin.
Authors: Ricardo Adaixo / Eva M Steiner / Ricardo D Righetto / Alexander Schmidt / Henning Stahlberg / Nicholas M I Taylor /
Abstract: The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its ...The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T) and thyroxine (T) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.
History
DepositionDec 9, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 29, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Thyroglobulin
B: Thyroglobulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)621,04230
Polymers610,1402
Non-polymers10,90228
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area30340 Å2
ΔGint56 kcal/mol
Surface area236890 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1PROTHRA1 - 2483
d_12ens_1NAGNAGB
d_13ens_1NAGNAGC
d_14ens_1NAGNAGD
d_15ens_1NAGNAGE
d_16ens_1NAGNAGF
d_17ens_1NAGNAGG
d_18ens_1NAGNAGH
d_19ens_1NAGNAGI
d_110ens_1NAGNAGJ
d_111ens_1NAGNAGK
d_112ens_1NAGNAGL
d_113ens_1NAGNAGM
d_114ens_1NAGNAGN
d_115ens_1NAGNAGO
d_116ens_1NAGNAGP
d_117ens_1NAGNAGQ
d_118ens_1NAGNAGR
d_119ens_1NAGNAGS
d_120ens_1BMABMAT
d_121ens_1MANMANU
d_122ens_1BMABMAV
d_123ens_1NAGNAGW
d_124ens_1NAGNAGX
d_125ens_1BMABMAY
d_126ens_1MANMANZ
d_127ens_1MANMANAA
d_128ens_1MANMANA
d_21ens_1PROTHRB1 - 2483
d_22ens_1NAGNAGB
d_23ens_1NAGNAGB
d_24ens_1NAGNAGB
d_25ens_1NAGNAGB
d_26ens_1NAGNAGB
d_27ens_1NAGNAGB
d_28ens_1NAGNAGB
d_29ens_1NAGNAGB
d_210ens_1NAGNAGB
d_211ens_1NAGNAGB
d_212ens_1NAGNAGB
d_213ens_1NAGNAGB
d_214ens_1NAGNAGB
d_215ens_1NAGNAGB
d_216ens_1NAGNAGB
d_217ens_1NAGNAGB
d_218ens_1NAGNAGB
d_219ens_1NAGNAGB
d_220ens_1BMABMAB
d_221ens_1MANMANB
d_222ens_1BMABMAB
d_223ens_1NAGNAGB
d_224ens_1NAGNAGB
d_225ens_1BMABMAB
d_226ens_1MANMANB
d_227ens_1MANMANB
d_228ens_1MANMANB

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Thyroglobulin / Tg


Mass: 305069.844 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Organ: Thyroid / Tissue: Thyroid / References: UniProt: P01266

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Sugars , 5 types, 28 molecules

#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: 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.: 2
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
#4: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#5: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d3-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

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: human thyroglobulin / Type: COMPLEX
Details: human thyroglobulin purified from thyroid gland tissue and non in vitro iodinated
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.66 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Organ: Thyroid / Tissue: Thyroid
Buffer solutionpH: 7.5 / Details: 0.22 um filtered and degased
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClC4H11NO31
2150 mMSodium ChlorideNaCl1
31 xSodium AzideNaN31
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: concentrated SEC peak
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

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: BRIGHT FIELD / Nominal magnification: 215000 X / Calibrated magnification: 78125 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8119
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.7image acquisition
3FOCUS10_14_6image acquisition
5CTFFIND4.1CTF correction
10Coot0.9.3model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 593563
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37619 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 123.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00540032
ELECTRON MICROSCOPYf_angle_d1.0754336
ELECTRON MICROSCOPYf_dihedral_angle_d6.8895720
ELECTRON MICROSCOPYf_chiral_restr0.0616040
ELECTRON MICROSCOPYf_plane_restr0.0077122
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.000707201472913 Å

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