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- PDB-5bts: Structural and biophysical characterization of a covalent insulin... -

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Basic information

Entry
Database: PDB / ID: 5bts
TitleStructural and biophysical characterization of a covalent insulin dimer formed during storage of neutral formulation of human insulin
Components
  • Insulin A chain
  • Insulin B chain
KeywordsHORMONE / Insulin / HMWP
Function / homology
Function and homology information


negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior / Signaling by Insulin receptor / IRS activation / Insulin processing / regulation of protein secretion ...negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior / Signaling by Insulin receptor / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of respiratory burst / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / negative regulation of acute inflammatory response / alpha-beta T cell activation / negative regulation of respiratory burst involved in inflammatory response / positive regulation of dendritic spine maintenance / positive regulation of glycogen biosynthetic process / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of protein secretion / regulation of protein localization to plasma membrane / fatty acid homeostasis / Signal attenuation / negative regulation of lipid catabolic process / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / COPI-mediated anterograde transport / positive regulation of lipid biosynthetic 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 / nitric oxide-cGMP-mediated signaling / transport vesicle / positive regulation of protein autophosphorylation / Insulin receptor recycling / neuron projection maintenance / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / positive regulation of brown fat cell differentiation / positive regulation of glycolytic process / activation of protein kinase B activity / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / positive regulation of nitric-oxide synthase activity / positive regulation of cytokine production / positive regulation of long-term synaptic potentiation / acute-phase response / Regulation of insulin secretion / endosome lumen / positive regulation of protein secretion / positive regulation of glucose import / negative regulation of proteolysis / positive regulation of cell differentiation / regulation of transmembrane transporter activity / insulin-like growth factor receptor binding / wound healing / insulin receptor binding / regulation of synaptic plasticity / negative regulation of protein catabolic process / hormone activity / cognition / positive regulation of neuron projection development / positive regulation of protein localization to nucleus / Golgi lumen / vasodilation / glucose metabolic process / regulation of protein localization / insulin receptor signaling pathway / cell-cell signaling / glucose homeostasis / positive regulation of NF-kappaB transcription factor activity / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / secretory granule lumen / protease binding / positive regulation of MAPK cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of cell migration / G protein-coupled receptor signaling pathway / Amyloid fiber formation / endoplasmic reticulum lumen / Golgi membrane / negative regulation of gene expression / positive regulation of cell population proliferation / positive regulation of gene expression / regulation of DNA-templated transcription / extracellular space / extracellular region / identical protein binding
Similarity search - Function
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
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.77 Å
AuthorsNorrman, M. / Hjorth, C.F.
CitationJournal: J.Pharm.Sci. / Year: 2016
Title: Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.
Authors: Hjorth, C.F. / Norrman, M. / Wahlund, P.O. / Benie, A.J. / Petersen, B.O. / Jessen, C.M. / Pedersen, T.A. / Vestergaard, K. / Steensgaard, D.B. / Pedersen, J.S. / Naver, H. / Hubalek, F. / ...Authors: Hjorth, C.F. / Norrman, M. / Wahlund, P.O. / Benie, A.J. / Petersen, B.O. / Jessen, C.M. / Pedersen, T.A. / Vestergaard, K. / Steensgaard, D.B. / Pedersen, J.S. / Naver, H. / Hubalek, F. / Poulsen, C. / Otzen, D.
History
DepositionJun 3, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Mar 9, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Advisory / Data collection / Derived calculations
Category: pdbx_data_processing_status / pdbx_validate_symm_contact ...pdbx_data_processing_status / pdbx_validate_symm_contact / struct_conn / struct_conn_type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)5,8182
Polymers5,8182
Non-polymers00
Water1,18966
1
A: Insulin A chain
B: Insulin B chain

A: Insulin A chain
B: Insulin B chain


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
Unit cell
Length a, b, c (Å)78.392, 78.392, 78.392
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
11B-108-

HOH

21B-142-

HOH

31B-143-

HOH

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Components

#1: Protein/peptide Insulin A chain


Mass: 2383.698 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
#2: Protein/peptide Insulin B chain


Mass: 3433.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 66 / 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: 3.45 Å3/Da / Density % sol: 64.35 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.2 M Ammonium sulfate, 0.1 M Hepes, 25% w/v PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Sep 10, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.77→50 Å / Num. obs: 8003 / % possible obs: 99.8 % / Redundancy: 27 % / Biso Wilson estimate: 21.81 Å2 / Rmerge(I) obs: 0.15 / Rpim(I) all: 0.029 / Rrim(I) all: 0.153 / Χ2: 5.558 / Net I/av σ(I): 58.8 / Net I/σ(I): 9.4 / Num. measured all: 215847
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.77-1.890.4893.33850.8210.1660.5190.71295.8
1.8-1.8323.70.514000.950.1070.5210.733100
1.83-1.8727.20.4943940.9710.0960.5030.74100
1.87-1.9127.10.4953840.9770.0960.5041.44100
1.91-1.9527.60.4744040.9780.0940.4832.127100
1.95-1.9927.40.3383790.9840.0650.3441.464100
1.99-2.0427.70.2634150.9880.0510.2681.578100
2.04-2.127.90.3473850.9880.0670.3533.361100
2.1-2.1627.70.224000.9910.0430.2242.491100
2.16-2.23280.2113890.9940.0410.2153.077100
2.23-2.3128.10.3574020.9930.070.3645.367100
2.31-2.428.40.1774030.9950.0340.183.66100
2.4-2.5128.40.1614010.9940.0310.1643.948100
2.51-2.6428.50.1584070.9960.030.1615.041100
2.64-2.8128.90.1683900.9960.0310.1717.076100
2.81-3.0328.80.1354080.9970.0250.1377.159100
3.03-3.3328.90.1273970.9980.0240.12910.953100
3.33-3.8128.80.1314150.9980.0250.13314.337100
3.81-4.828.90.1134060.9980.0210.11515.718100
4.8-5027.60.1054390.9980.020.10713.21100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data reduction
SCALEPACKdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.15data extraction
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.77→39.196 Å / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2059 810 10.13 %
Rwork0.1573 7185 -
obs0.162 7995 99.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 95.81 Å2 / Biso mean: 29.5848 Å2 / Biso min: 13.27 Å2
Refinement stepCycle: final / Resolution: 1.77→39.196 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms405 0 9 66 480
Biso mean--48.32 39.65 -
Num. residues----51
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01424
X-RAY DIFFRACTIONf_angle_d1.084575
X-RAY DIFFRACTIONf_chiral_restr0.04763
X-RAY DIFFRACTIONf_plane_restr0.00474
X-RAY DIFFRACTIONf_dihedral_angle_d14.523150
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.768-1.87880.22731350.20281188132399
1.8788-2.02380.22961330.183311641297100
2.0238-2.22750.18411350.150111781313100
2.2275-2.54970.20831350.151312061341100
2.5497-3.21220.20231330.146511951328100
3.2122-39.20560.20551390.156412541393100
Refinement TLS params.Method: refined / Details: Chain A / Origin x: -19.3418 Å / Origin y: -0.8681 Å / Origin z: -9.0473 Å
111213212223313233
T0.1428 Å20.001 Å20.021 Å2-0.1791 Å20.0282 Å2--0.136 Å2
L3.2008 °20.5865 °20.0106 °2-3.289 °20.5207 °2--4.4927 °2
S0.0308 Å °0.1092 Å °0.0695 Å °-0.051 Å °0.0666 Å °0.1184 Å °-0.1941 Å °-0.1329 Å °-0.0626 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA1 - 21
2X-RAY DIFFRACTION1allA21
3X-RAY DIFFRACTION1allB1 - 30
4X-RAY DIFFRACTION1allW1 - 66

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