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- PDB-5cjo: Crystal Structure Analysis of Elbow-Engineered-Fab-Bound Human In... -

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Basic information

Entry
Database: PDB / ID: 5cjo
TitleCrystal Structure Analysis of Elbow-Engineered-Fab-Bound Human Insulin Degrading Enzyme (IDE) in Complex with Insulin
Components
  • FAB Heavy chain with engineered elbow
  • FAB light chainFragment antigen-binding
  • Insulin-degrading enzyme
  • Insulin
KeywordsHYDROLASE/HYDROLASE INHIBITOR / HYDROLASE / FAB / Elbow-engineer / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / ubiquitin-modified protein reader activity / insulin binding / negative regulation of NAD(P)H oxidase activity ...insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / ubiquitin-modified protein reader activity / insulin binding / negative regulation of NAD(P)H oxidase activity / regulation of aerobic respiration / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / peptide catabolic process / Signaling by Insulin receptor / IRS activation / negative regulation of fatty acid metabolic process / nitric oxide-cGMP-mediated signaling / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / amyloid-beta clearance / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / positive regulation of respiratory burst / peroxisomal matrix / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / positive regulation of nitric oxide mediated signal transduction / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / amyloid-beta metabolic process / negative regulation of lipid catabolic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / negative regulation of reactive oxygen species biosynthetic process / positive regulation of insulin receptor signaling pathway / transport vesicle / positive regulation of protein autophosphorylation / insulin-like growth factor receptor binding / Insulin receptor recycling / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / endoplasmic reticulum-Golgi intermediate compartment membrane / neuron projection maintenance / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / proteolysis involved in protein catabolic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / endosome lumen / positive regulation of cytokine production / acute-phase response / positive regulation of protein secretion / regulation of transmembrane transporter activity / Peroxisomal protein import / positive regulation of cell differentiation / peptide binding / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / protein catabolic process / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / metalloendopeptidase activity / antigen processing and presentation of endogenous peptide antigen via MHC class I / cognition / peroxisome / vasodilation / Golgi lumen / positive regulation of protein localization to nucleus / positive regulation of protein catabolic process / glucose metabolic process / regulation of protein localization / glucose homeostasis / virus receptor activity / cell-cell signaling / insulin receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / positive regulation of protein binding / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / basolateral plasma membrane / secretory granule lumen
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) ...Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / Insulin / Insulin family / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Insulin / Insulin-degrading enzyme
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.287 Å
Authorsliang, w.g. / bailey, L. / tang, w.j.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117372 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM087519 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM094588 United States
CitationJournal: J. Mol. Biol. / Year: 2017
Title: Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.
Authors: Bailey, L.J. / Sheehy, K.M. / Dominik, P.K. / Liang, W.G. / Rui, H. / Clark, M. / Jaskolowski, M. / Kim, Y. / Deneka, D. / Tang, W.J. / Kossiakoff, A.A.
History
DepositionJul 14, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 20, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2017Group: Author supporting evidence / Derived calculations
Category: pdbx_struct_assembly_auth_evidence / pdbx_struct_oper_list
Item: _pdbx_struct_oper_list.symmetry_operation
Revision 1.2Jan 10, 2018Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.4Sep 27, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insulin-degrading enzyme
H: FAB Heavy chain with engineered elbow
L: FAB light chain
a: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,3156
Polymers166,0114
Non-polymers3042
Water0
1
A: Insulin-degrading enzyme
H: FAB Heavy chain with engineered elbow
L: FAB light chain
a: Insulin
hetero molecules

A: Insulin-degrading enzyme
H: FAB Heavy chain with engineered elbow
L: FAB light chain
a: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)332,62912
Polymers332,0228
Non-polymers6074
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_585x,-y+3,-z1
Unit cell
Length a, b, c (Å)75.689, 109.218, 263.405
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP22121
DetailsGel filtration and SAXS

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Components

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Antibody , 2 types, 2 molecules HL

#2: Antibody FAB Heavy chain with engineered elbow


Mass: 25734.756 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)
#3: Antibody FAB light chain / Fragment antigen-binding


Mass: 23446.021 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Escherichia coli (E. coli)

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Protein / Protein/peptide , 2 types, 2 molecules Aa

#1: Protein Insulin-degrading enzyme / / Abeta-degrading protease / Insulin protease / Insulinase / Insulysin


Mass: 114560.578 Da / Num. of mol.: 1
Mutation: C110L, E111Q, C171S, C178A, C257V, C414L, C573N, C590S, C789S, C812A, C819A, C904S, C966N, C974A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / References: UniProt: P14735, insulysin
#4: Protein/peptide Insulin /


Mass: 2269.595 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 3.42 Å3/Da / Density % sol: 64 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 6% v/v Tacsimate pH 7.0, 0.1 M HEPES pH 7.0, 8% w/v Polyethylene glycol monomethyl ether 5,000, and 8% v/v tert-butanol as additive.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.9788 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jun 23, 2015
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9788 Å / Relative weight: 1
ReflectionResolution: 3.28→50 Å / Num. obs: 34037 / % possible obs: 99.8 % / Redundancy: 15 % / Rmerge(I) obs: 0.1 / Rpim(I) all: 0.043 / Rrim(I) all: 0.167 / Net I/σ(I): 18.1
Reflection shellResolution: 3.28→3.34 Å / Rmerge(I) all: 0.662 / Mean I/σ(I) all: 2.03 / CC1/2: 0.887 / Rpim(I) all: 0.253 / Rrim(I) all: 0.937 / Χ2: 0.741

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4NXO

4nxo


Resolution: 3.287→47.45 Å / SU ML: 0.38 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.01 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2453 1995 5.87 %
Rwork0.1956 --
obs0.1985 33959 99.5 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.287→47.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10950 0 16 0 10966
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00311238
X-RAY DIFFRACTIONf_angle_d0.7315215
X-RAY DIFFRACTIONf_dihedral_angle_d13.4314143
X-RAY DIFFRACTIONf_chiral_restr0.0281662
X-RAY DIFFRACTIONf_plane_restr0.0041951
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2866-3.36870.32171310.28692102X-RAY DIFFRACTION95
3.3687-3.45980.30931420.2642266X-RAY DIFFRACTION99
3.4598-3.56160.37271410.25082240X-RAY DIFFRACTION100
3.5616-3.67650.28361390.2322263X-RAY DIFFRACTION100
3.6765-3.80780.29731420.2242276X-RAY DIFFRACTION100
3.8078-3.96020.28311400.22072249X-RAY DIFFRACTION100
3.9602-4.14040.29791430.19862275X-RAY DIFFRACTION100
4.1404-4.35850.20611420.18232272X-RAY DIFFRACTION100
4.3585-4.63140.20971420.16632280X-RAY DIFFRACTION100
4.6314-4.98860.19531430.1532295X-RAY DIFFRACTION100
4.9886-5.48990.23811450.16952322X-RAY DIFFRACTION100
5.4899-6.28280.2241440.19212310X-RAY DIFFRACTION100
6.2828-7.90970.22451470.20262349X-RAY DIFFRACTION100
7.9097-47.45450.22251540.17622465X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 38.1772 Å / Origin y: 145.2737 Å / Origin z: 46.0362 Å
111213212223313233
T0.5428 Å20.0285 Å20.0032 Å2-0.585 Å2-0.0285 Å2--0.5984 Å2
L0.4228 °20.1032 °20.6341 °2-0.5768 °2-0.0314 °2--1.2982 °2
S0.1132 Å °0.0417 Å °-0.1177 Å °0.0711 Å °-0.0085 Å °-0.1339 Å °0.1731 Å °0.1243 Å °-0.0975 Å °
Refinement TLS groupSelection details: all

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