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- PDB-7uai: Meprin alpha helix in complex with fetuin-B -

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Basic information

Entry
Database: PDB / ID: 7uai
TitleMeprin alpha helix in complex with fetuin-B
Components
  • Fetuin-B
  • Meprin A subunit alpha
KeywordsONCOPROTEIN / Metalloprotease / complex / helical / extracellular
Function / homology
Function and homology information


meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metalloendopeptidase inhibitor activity / metallodipeptidase activity / negative regulation of endopeptidase activity / binding of sperm to zona pellucida / cysteine-type endopeptidase inhibitor activity / endopeptidase inhibitor activity / signaling receptor ligand precursor processing ...meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metalloendopeptidase inhibitor activity / metallodipeptidase activity / negative regulation of endopeptidase activity / binding of sperm to zona pellucida / cysteine-type endopeptidase inhibitor activity / endopeptidase inhibitor activity / signaling receptor ligand precursor processing / single fertilization / metalloendopeptidase activity / metallopeptidase activity / extracellular space / extracellular exosome / zinc ion binding / extracellular region / plasma membrane
Similarity search - Function
Proteinase inhibitor I25C, fetuin, conserved site / Fetuin-B-type cystatin domain / Fetuin family signature 1. / Fetuin family signature 2. / Fetuin-B-type cystatin domain profile. / Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) ...Proteinase inhibitor I25C, fetuin, conserved site / Fetuin-B-type cystatin domain / Fetuin family signature 1. / Fetuin family signature 2. / Fetuin-B-type cystatin domain profile. / Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / : / TRAF/meprin, MATH domain / MAM domain signature. / Cystatin domain / Cystatin-like domain / Cystatin domain / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / Cystatin superfamily / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / TRAF-like / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / EGF-like domain / Metallopeptidase, catalytic domain superfamily / EGF-like domain profile. / EGF-like domain / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Meprin A subunit alpha / Fetuin-B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBayly-Jones, C. / Lupton, C.J. / Fritz, C. / Schlenzig, D. / Whisstock, J.C.
Funding support Germany, Australia, 2items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research Germany
Australian Research Council (ARC) Australia
CitationJournal: Nat Commun / Year: 2022
Title: Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.
Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar ...Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar Schlenzig / James C Whisstock /
Abstract: The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal ...The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly.
History
DepositionMar 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Meprin A subunit alpha
A: Fetuin-B
B: Meprin A subunit alpha
C: Meprin A subunit alpha
D: Meprin A subunit alpha
H: Fetuin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)364,67237
Polymers354,9976
Non-polymers9,67631
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 6 molecules EBCDAH

#1: Protein
Meprin A subunit alpha / / Endopeptidase-2 / N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha / PABA peptide ...Endopeptidase-2 / N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha / PABA peptide hydrolase / PPH alpha


Mass: 67285.820 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MEP1A / Plasmid: pMT/BiP/V5 / Cell line (production host): Schneider-2 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q16819, meprin A
#2: Protein Fetuin-B / 16G2 / Fetuin-like protein IRL685 / Gugu


Mass: 42926.691 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FETUB / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9UGM5

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Sugars , 4 types, 19 molecules

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


Type: oligosaccharide / Mass: 627.594 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1b_1-5_2*NCC/3=O]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}}LINUCSPDB-CARE
#4: 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.: 8
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 732.682 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1221m-1a_1-5]/1-1-2-3/a4-b1_a6-d1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#8: 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.: 4 / 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

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Non-polymers , 2 types, 12 molecules

#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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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: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tetrameric portion of meprin alpha helix in complex with fetuin-B [subparticle localised reconstruction]
Type: COMPLEX
Details: Subparticle localised reconstruction of tetrameric region of recombinant, secreted helical meprin alpha in complex with fetuin-B
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 85 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Drosophila (fruit flies) / Cell: Schneider-2 / Plasmid: pMT/BiP/V5
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMTRIS1
2100 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco EasyGlow / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: 3 s blot, -3 force

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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: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4068
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansMovie frames/image: 25 / Used frames/image: 1-25

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.8.1particle selectionFilament mode
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
5Warp1.0.7CTF correction
8UCSF ChimeraX1.3model fitting
9UCSF Chimera1.13model fitting
10ISOLDE1.1.0model fitting
11Coot0.9.2model fitting
13cryoSPARC3.3.1initial Euler assignmentGlobal alignment consensus map
14RELION3.1initial Euler assignmentGlobal alignment localised reconstruction
15RELION3.1final Euler assignmentGlobal alignment localised reconstruction
16cryoSPARC3.3.1final Euler assignmentLocal refinement of localised reconstruction (non-uniform)
17RELION3.1classification
19ISOLDE1.1.0model refinement
20Coot0.9.2model refinement
Image processingDetails: Compressed to LZW TIFF. Motion corrected by MotionCor.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 519760
Details: Localised subparticles extrated after expansion of pseudo-helical symmetry
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115951 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
17UAB

7uab

1
27AUW

7auw

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01322456
ELECTRON MICROSCOPYf_angle_d1.77730504
ELECTRON MICROSCOPYf_dihedral_angle_d22.3448115
ELECTRON MICROSCOPYf_chiral_restr0.1043439
ELECTRON MICROSCOPYf_plane_restr0.0123906

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