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- PDB-8un7: Single particle analysis of recombinant human MFAP4 -

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Basic information

Entry
Database: PDB / ID: 8un7
TitleSingle particle analysis of recombinant human MFAP4
ComponentsMicrofibril-associated glycoprotein 4
KeywordsCELL ADHESION / MFAP4 Octamer Extracellular Matrix
Function / homology
Function and homology information


regulation of collagen metabolic process / elastic fiber / microfibril / elastic fiber assembly / UV protection / Molecules associated with elastic fibres / cellular response to UV-B / supramolecular fiber organization / collagen-containing extracellular matrix / cell adhesion ...regulation of collagen metabolic process / elastic fiber / microfibril / elastic fiber assembly / UV protection / Molecules associated with elastic fibres / cellular response to UV-B / supramolecular fiber organization / collagen-containing extracellular matrix / cell adhesion / extracellular space / extracellular region
Similarity search - Function
Fibrinogen-related domains (FReDs) / Fibrinogen, alpha/beta/gamma chain, C-terminal globular, subdomain 1 / Fibrinogen beta and gamma chains, C-terminal globular domain / Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain / Fibrinogen-like, C-terminal / Fibrinogen C-terminal domain profile.
Similarity search - Domain/homology
Microfibril-associated glycoprotein 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsWozny, M.W. / Nelea, V.
Funding support Canada, 2items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
CitationJournal: Nat Commun / Year: 2024
Title: Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.
Authors: Michael R Wozny / Valentin Nelea / Iram Fatima S Siddiqui / Shaynah Wanga / Vivian de Waard / Mike Strauss / Dieter P Reinhardt /
Abstract: Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular ...Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.
History
DepositionOct 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Microfibril-associated glycoprotein 4
D: Microfibril-associated glycoprotein 4
B: Microfibril-associated glycoprotein 4
E: Microfibril-associated glycoprotein 4
C: Microfibril-associated glycoprotein 4
F: Microfibril-associated glycoprotein 4
G: Microfibril-associated glycoprotein 4
H: Microfibril-associated glycoprotein 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)239,38532
Polymers235,5258
Non-polymers3,86024
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Microfibril-associated glycoprotein 4


Mass: 29440.645 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MFAP4 / Cell line (production host): HEK293-EBNA / Production host: Homo sapiens (human) / References: UniProt: P55083
#2: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C8H15NO6
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
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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: Octamer assembly of MFAP4 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 280 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293-EBNA / Plasmid: pCEP4
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 455190 / Symmetry type: POINT

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