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- PDB-5ena: Xray crystal structure of isotope-labeled human insulin -

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Basic information

Entry
Database: PDB / ID: 5ena
TitleXray crystal structure of isotope-labeled human insulin
Components
  • Insulin chain A
  • Insulin chain B
KeywordsHORMONE / Isotope-labeled / Human insulin / Chemical protein synthesis / Spontaneous resolution
Function / homology
Function and homology information


Signaling by Insulin receptor / alpha-beta T cell activation / negative regulation of glycogen catabolic process / Insulin processing / IRS activation / Insulin receptor recycling / negative regulation of NAD(P)H oxidase activity / nitric oxide-cGMP-mediated signaling pathway / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior ...Signaling by Insulin receptor / alpha-beta T cell activation / negative regulation of glycogen catabolic process / Insulin processing / IRS activation / Insulin receptor recycling / negative regulation of NAD(P)H oxidase activity / nitric oxide-cGMP-mediated signaling pathway / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior / regulation of protein secretion / Regulation of gene expression in beta cells / positive regulation of peptide hormone secretion / positive regulation of respiratory burst / negative regulation of respiratory burst involved in inflammatory response / negative regulation of gluconeogenesis / positive regulation of cellular protein metabolic process / regulation of cellular amino acid metabolic process / negative regulation of acute inflammatory response / negative regulation of reactive oxygen species biosynthetic process / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / positive regulation of dendritic spine maintenance / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / Regulation of insulin secretion / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of nitric oxide mediated signal transduction / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / Signal attenuation / negative regulation of protein secretion / positive regulation of lipid biosynthetic process / negative regulation of lipid catabolic process / fatty acid homeostasis / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of insulin receptor signaling pathway / endosome lumen / neuron projection maintenance / insulin-like growth factor receptor binding / positive regulation of protein autophosphorylation / positive regulation of glycolytic process / positive regulation of cell differentiation / regulation of transmembrane transporter activity / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / positive regulation of brown fat cell differentiation / regulation of synaptic plasticity / cognition / positive regulation of long-term synaptic potentiation / regulation of protein localization / positive regulation of cytokine production / acute-phase response / activation of protein kinase B activity / positive regulation of glucose import / hormone activity / negative regulation of proteolysis / negative regulation of protein catabolic process / insulin receptor binding / positive regulation of protein localization to nucleus / vasodilation / insulin receptor signaling pathway / Golgi lumen / glucose metabolic process / positive regulation of nitric-oxide synthase activity / cell-cell signaling / glucose homeostasis / wound healing / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase signaling / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / secretory granule lumen / protease binding / positive regulation of NF-kappaB transcription factor activity / positive regulation of protein kinase B signaling / positive regulation of cell migration / G protein-coupled receptor signaling pathway / Golgi membrane / Amyloid fiber formation / endoplasmic reticulum lumen / regulation of transcription, DNA-templated / positive regulation of cell population proliferation / positive regulation of gene expression / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Insulin / Insulin family / Insulin/IGF/Relaxin family / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin family signature. / Insulin, conserved site / Insulin-like superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.35 Å
AuthorsMandal, K. / Dhayalan, B. / Avital-Shmilovici, M. / Tokmakoff, A. / Kent, S.B.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK08993 United States
CitationJournal: Chembiochem / Year: 2016
Title: Crystallization of Enantiomerically Pure Proteins from Quasi-Racemic Mixtures: Structure Determination by X-Ray Diffraction of Isotope-Labeled Ester Insulin and Human Insulin.
Authors: Mandal, K. / Dhayalan, B. / Avital-Shmilovici, M. / Tokmakoff, A. / Kent, S.B.
History
DepositionNov 9, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2016Group: Database references
Revision 1.2Sep 20, 2017Group: Author supporting evidence / Derived calculations / Refinement description
Category: pdbx_audit_support / pdbx_struct_oper_list / software
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation / _software.classification
Revision 1.3Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insulin chain A
B: Insulin chain B


Theoretical massNumber of molelcules
Total (without water)5,8182
Polymers5,8182
Non-polymers00
Water86548
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1520 Å2
ΔGint-15 kcal/mol
Surface area3420 Å2
MethodPISA
2
A: Insulin chain A
B: Insulin chain B

A: Insulin chain A
B: Insulin chain B


Theoretical massNumber of molelcules
Total (without water)11,6354
Polymers11,6354
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation16_554x,-y,-z-1/21
Buried area4330 Å2
ΔGint-38 kcal/mol
Surface area5540 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)77.935, 77.935, 77.935
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number199
Space group name H-MI213
Components on special symmetry positions
IDModelComponents
11A-102-

HOH

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Components

#1: Protein/peptide Insulin chain A


Mass: 2383.698 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308
#2: Protein/peptide Insulin chain B


Mass: 3433.953 Da / Num. of mol.: 1 / Mutation: 1-(13C=18O)PheB24 / Source method: obtained synthetically / Details: chemical synthesis / Source: (synth.) Homo sapiens (human) / References: UniProt: P01308
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 48 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.39 Å3/Da / Density % sol: 63.72 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1 M HEPES, 0.2 M sodium citrate tribasic dihydrate, 11% MPD

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.98 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Oct 23, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.35→50 Å / Num. obs: 17473 / % possible obs: 100 % / Redundancy: 19.6 % / Rmerge(I) obs: 0.069 / Rpim(I) all: 0.016 / Rrim(I) all: 0.071 / Χ2: 0.785 / Net I/σ(I): 59.3 / Num. measured all: 342077
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.35-1.37200.7153.88630.9440.1640.7340.431100
1.37-1.419.90.5628640.9590.1290.5770.454100
1.4-1.4319.80.4778640.9720.110.4890.458100
1.43-1.4519.60.4048410.9790.0930.4150.456100
1.45-1.4919.40.3068880.9890.0710.3140.469100
1.49-1.5219.20.278640.990.0630.2780.49100
1.52-1.5618.10.2218650.9910.0530.2280.503100
1.56-1.617.30.1868670.9920.0460.1920.522100
1.6-1.6520.70.1568660.9960.0350.160.544100
1.65-1.720.70.1368670.9970.0310.140.575100
1.7-1.7620.50.1118570.9980.0250.1130.599100
1.76-1.8320.50.0978740.9980.0220.0990.679100
1.83-1.9220.10.0848690.9990.0190.0860.721100
1.92-2.0219.70.0748650.9990.0170.0760.785100
2.02-2.1418.10.0658820.9990.0160.0670.857100
2.14-2.3118.70.0578850.9990.0130.0590.923100
2.31-2.54210.0568800.9990.0120.0571.004100
2.54-2.9120.50.0538840.9990.0120.0541.097100
2.91-3.6619.70.0568930.9990.0130.0581.494100
3.66-50180.079350.9970.0170.0732.59599.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data scaling
SCALEPACKdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1B2A
Resolution: 1.35→27.554 Å / SU ML: 0.1 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 13.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1639 1745 9.99 %
Rwork0.1491 15727 -
obs0.1505 17472 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 69.14 Å2 / Biso mean: 27.5288 Å2 / Biso min: 14.04 Å2
Refinement stepCycle: final / Resolution: 1.35→27.554 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms405 0 0 48 453
Biso mean---40.79 -
Num. residues----51
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.015433
X-RAY DIFFRACTIONf_angle_d1.454591
X-RAY DIFFRACTIONf_chiral_restr0.11264
X-RAY DIFFRACTIONf_plane_restr0.00977
X-RAY DIFFRACTIONf_dihedral_angle_d14.773283
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 12 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
1.3497-1.38950.17221420.132612831425
1.3895-1.43430.1631490.120913161465
1.4343-1.48560.14991440.114912861430
1.4856-1.5450.16531460.113713011447
1.545-1.61530.14851440.109913031447
1.6153-1.70050.13881410.114312941435
1.7005-1.8070.13681510.122312921443
1.807-1.94650.12771390.12213131452
1.9465-2.14230.15441440.13113081452
2.1423-2.45220.14761440.141213201464
2.4522-3.08880.14071540.156913311485
3.0888-27.55990.20571470.178713801527

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