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- PDB-5en9: High resolution x-ray crystal structure of isotope-labeled ester-... -

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Basic information

Entry
Database: PDB / ID: 5en9
TitleHigh resolution x-ray crystal structure of isotope-labeled ester-insulin
Components
  • Insulin chain A
  • Insulin chain B
KeywordsHORMONE / Isotope-labeled / ester-insulin / chemical protein synthesis / spontaneous resolution
Function / homology
Function and homology information


Signaling by Insulin receptor / negative regulation of glycogen catabolic process / alpha-beta T cell activation / Insulin processing / IRS activation / Insulin receptor recycling / negative regulation of NAD(P)H oxidase activity / nitric oxide-cGMP-mediated signaling pathway / negative regulation of feeding behavior / negative regulation of fatty acid metabolic process ...Signaling by Insulin receptor / negative regulation of glycogen catabolic process / alpha-beta T cell activation / Insulin processing / IRS activation / Insulin receptor recycling / negative regulation of NAD(P)H oxidase activity / nitric oxide-cGMP-mediated signaling pathway / negative regulation of feeding behavior / negative regulation of fatty acid metabolic process / 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 / negative regulation of reactive oxygen species biosynthetic process / positive regulation of cellular protein metabolic process / regulation of protein localization to plasma membrane / COPI-mediated anterograde transport / Regulation of insulin secretion / positive regulation of dendritic spine maintenance / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / regulation of cellular amino acid metabolic process / 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 lipid catabolic process / negative regulation of protein secretion / positive regulation of lipid biosynthetic process / fatty acid homeostasis / endosome lumen / transport vesicle / positive regulation of insulin receptor signaling pathway / endoplasmic reticulum-Golgi intermediate compartment membrane / neuron projection maintenance / insulin-like growth factor receptor binding / positive regulation of protein autophosphorylation / positive regulation of glycolytic process / negative regulation of acute inflammatory response / regulation of transmembrane transporter activity / positive regulation of cell differentiation / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / regulation of synaptic plasticity / positive regulation of brown fat cell differentiation / cognition / regulation of protein localization / positive regulation of long-term synaptic potentiation / positive regulation of cytokine production / acute-phase response / activation of protein kinase B activity / positive regulation of glucose import / vasodilation / hormone activity / negative regulation of proteolysis / negative regulation of protein catabolic process / insulin receptor binding / positive regulation of protein localization to nucleus / insulin receptor signaling pathway / glucose metabolic process / Golgi lumen / positive regulation of nitric-oxide synthase activity / cell-cell signaling / glucose homeostasis / wound healing / endoplasmic reticulum to Golgi vesicle-mediated transport / positive regulation of MAPK cascade / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of phosphatidylinositol 3-kinase signaling / positive regulation of cell growth / secretory granule lumen / protease binding / positive regulation of NF-kappaB transcription factor activity / positive regulation of cell migration / positive regulation of protein kinase B signaling / Amyloid fiber formation / Golgi membrane / G protein-coupled receptor signaling pathway / amyloid fibril formation / endoplasmic reticulum lumen / regulation of transcription, DNA-templated / positive regulation of gene expression / positive regulation of cell population proliferation / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Insulin / Insulin family / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin/IGF/Relaxin family / Insulin, conserved site / Insulin-like superfamily / Insulin family signature.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.5 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 1.1Jan 20, 2016Group: Data collection
Revision 1.2Mar 16, 2016Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Dec 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-16 kcal/mol
Surface area3330 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 operation15_455-x-1/2,y,-z1
Buried area4240 Å2
ΔGint-39 kcal/mol
Surface area5460 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)77.981, 77.981, 77.981
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number199
Space group name H-MI213

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Components

#1: Protein/peptide Insulin chain A


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


Mass: 3433.953 Da / Num. of mol.: 1 / 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: 3.4 Å3/Da / Density % sol: 63.78 %
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.1 M HEPES, 0.2 M sodium citrate tribasic dihydrate, 13% 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 wavelength
IDWavelength (Å)Relative weight
10.97921
20.981
ReflectionResolution: 1.5→50 Å / Num. obs: 12858 / % possible obs: 100 % / Redundancy: 19.5 % / Biso Wilson estimate: 19.62 Å2 / Rmerge(I) obs: 0.065 / Rpim(I) all: 0.015 / Rrim(I) all: 0.067 / Χ2: 0.769 / Net I/av σ(I): 56.116 / Net I/σ(I): 6.8 / Num. measured all: 251212
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.5-1.5319.10.6776170.960.1590.6950.452100
1.53-1.5518.30.6516330.9570.1560.6690.454100
1.55-1.5816.30.4816490.970.1220.4960.471100
1.58-1.6219.60.4116380.980.0950.4220.476100
1.62-1.6520.70.346440.9880.0760.3480.501100
1.65-1.6920.60.2946280.9890.0660.3010.489100
1.69-1.7320.50.2456530.9920.0550.2520.522100
1.73-1.7820.40.1866330.9940.0420.1910.556100
1.78-1.8320.40.1666180.9960.0380.170.58100
1.83-1.8920.30.1416260.9970.0320.1440.618100
1.89-1.9619.80.1176580.9970.0270.120.679100
1.96-2.0419.50.0986290.9980.0230.10.695100
2.04-2.1318.10.0866670.9980.0210.0890.785100
2.13-2.2416.90.076200.9990.0180.0720.862100
2.24-2.3821.10.0676470.9990.0150.0680.877100
2.38-2.5620.90.066400.9990.0140.0620.895100
2.56-2.8220.60.0556650.9990.0130.0571.014100
2.82-3.2320.20.056500.9990.0110.0511.12100
3.23-4.0717.70.0486530.9990.0120.0491.364100
4.07-5019.80.0556900.9980.0130.0571.84899.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data collection
SCALEPACKdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.5→27.57 Å / SU ML: 0.09 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 15.08 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1766 1295 10.07 %
Rwork0.1427 11563 -
obs0.1461 12858 100 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 61.56 Å2 / Biso mean: 28.0461 Å2 / Biso min: 12.62 Å2
Refinement stepCycle: final / Resolution: 1.5→27.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms404 0 0 48 452
Biso mean---43.98 -
Num. residues----51
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006490
X-RAY DIFFRACTIONf_angle_d1.029676
X-RAY DIFFRACTIONf_chiral_restr0.0972
X-RAY DIFFRACTIONf_plane_restr0.00692
X-RAY DIFFRACTIONf_dihedral_angle_d10.26357
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 9 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
1.498-1.55790.18171430.121512721415
1.5579-1.62880.14971400.117412691409
1.6288-1.71470.16631350.111812671402
1.7147-1.82210.17241440.108312861430
1.8221-1.96280.1541450.118212591404
1.9628-2.16020.14611470.119312841431
2.1602-2.47260.17941380.136912841422
2.4726-3.11460.16651450.147712981443
3.1146-27.57510.1991580.165113441502

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