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- PDB-6s4j: Crystal structure of zinc free A14E, B25H, B29K(N(eps)-[2-(2-[2-(... -

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Basic information

Entry
Database: PDB / ID: 6s4j
TitleCrystal structure of zinc free A14E, B25H, B29K(N(eps)-[2-(2-[2-(2-[2-(Octadecandioyl-gamma-Glu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]), desB27, desB30 human insulin
Components(Insulin) x 2
KeywordsHORMONE / insulin / oral / zinc free
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 / 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 ...negative regulation of NAD(P)H oxidase activity / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic 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 / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / positive regulation of respiratory burst / 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 / 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 / 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 / positive regulation of cell differentiation / positive regulation of glucose import / negative regulation of proteolysis / regulation of synaptic plasticity / wound healing / insulin receptor binding / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / cognition / vasodilation / Golgi lumen / positive regulation of protein localization to nucleus / glucose metabolic process / regulation of protein localization / glucose homeostasis / cell-cell signaling / insulin receptor signaling pathway / 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
IMIDAZOLE / Chem-KUT / Insulin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsJohansson, E.
CitationJournal: Nat Commun / Year: 2020
Title: Molecular engineering of safe and efficacious oral basal insulin.
Authors: Hubalek, F. / Refsgaard, H.H.F. / Gram-Nielsen, S. / Madsen, P. / Nishimura, E. / Munzel, M. / Brand, C.L. / Stidsen, C.E. / Claussen, C.H. / Wulff, E.M. / Pridal, L. / Ribel, U. / ...Authors: Hubalek, F. / Refsgaard, H.H.F. / Gram-Nielsen, S. / Madsen, P. / Nishimura, E. / Munzel, M. / Brand, C.L. / Stidsen, C.E. / Claussen, C.H. / Wulff, E.M. / Pridal, L. / Ribel, U. / Kildegaard, J. / Porsgaard, T. / Johansson, E. / Steensgaard, D.B. / Hovgaard, L. / Glendorf, T. / Hansen, B.F. / Jensen, M.K. / Nielsen, P.K. / Ludvigsen, S. / Rugh, S. / Garibay, P.W. / Moore, M.C. / Cherrington, A.D. / Kjeldsen, T.
History
DepositionJun 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 24, 2024Group: Advisory / Data collection ...Advisory / 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 / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Insulin
B: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)6,3594
Polymers5,5722
Non-polymers7872
Water64936
1
A: Insulin
B: Insulin
hetero molecules

A: Insulin
B: Insulin
hetero molecules

A: Insulin
B: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,07812
Polymers16,7176
Non-polymers2,3616
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_575-y,x-y+2,z1
crystal symmetry operation3_355-x+y-2,-x,z1
Unit cell
Length a, b, c (Å)44.986, 44.986, 45.884
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number150
Space group name H-MP321
Components on special symmetry positions
IDModelComponents
11A-108-

HOH

21B-220-

HOH

31B-225-

HOH

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Components

#1: Protein/peptide Insulin /


Mass: 2349.638 Da / Num. of mol.: 1 / Mutation: Y14E
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 /


Mass: 3222.718 Da / Num. of mol.: 1 / Mutation: F25H,des27,des30
Source method: isolated from a genetically manipulated source
Details: LYS B30 covalently attached to [2-(2-[2-(2-[2-(Octadecandioyl-gamma-Glu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01308
#3: Chemical ChemComp-IMD / IMIDAZOLE / Imidazole


Mass: 69.085 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5N2
#4: Chemical ChemComp-KUT / KUT [2-(2-[2-(2-[2-(Octadecandioyl-gamma-Glu)amino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]


Mass: 717.888 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C35H63N3O12 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.41 Å3/Da / Density % sol: 48.86 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.12M monosaccharides, 0.1M buffer system 1 pH 6.5, 20% (v/v) PEG500MME, 10% (w/v) Peg 20000 (F1 from Marpheus screen, Molecular Dimensions)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: May 11, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.5→45.884 Å / Num. obs: 8938 / % possible obs: 99.85 % / Redundancy: 9.3 % / Biso Wilson estimate: 21.61 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.05618 / Rpim(I) all: 0.01939 / Rrim(I) all: 0.05951 / Net I/σ(I): 21.02
Reflection shellResolution: 1.5→1.554 Å / Redundancy: 7.8 % / Rmerge(I) obs: 1.635 / Mean I/σ(I) obs: 1.26 / Num. unique obs: 861 / CC1/2: 0.6 / Rpim(I) all: 0.6144 / Rrim(I) all: 1.75 / % possible all: 98.85

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Processing

Software
NameVersionClassification
PHENIX(1.15_3448: ???)refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6S4I

6s4i


Resolution: 1.5→45.88 Å / SU ML: 0.15 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 31.25
RfactorNum. reflection% reflection
Rfree0.2097 447 5 %
Rwork0.1873 --
obs0.1885 8934 99.85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 33.65 Å2
Refinement stepCycle: LAST / Resolution: 1.5→45.88 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms387 0 55 36 478
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.012471
X-RAY DIFFRACTIONf_angle_d1.401629
X-RAY DIFFRACTIONf_dihedral_angle_d7.256370
X-RAY DIFFRACTIONf_chiral_restr0.07264
X-RAY DIFFRACTIONf_plane_restr0.00781
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5004-1.71750.26051440.25722750X-RAY DIFFRACTION100
1.7175-2.16380.20691490.21072821X-RAY DIFFRACTION100
2.1638-45.90540.20411540.17242916X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: -34.6332 Å / Origin y: 19.4109 Å / Origin z: 55.916 Å
111213212223313233
T0.0362 Å20.0344 Å20.0167 Å2-0.068 Å20.0016 Å2--0.2481 Å2
L4.5453 °2-0.1655 °20.0468 °2-2.5133 °20.3658 °2--2.2046 °2
S-0.0277 Å °-0.2407 Å °-0.2054 Å °0.1547 Å °-0.0978 Å °-0.0581 Å °0.1896 Å °0.0528 Å °0.0089 Å °
Refinement TLS groupSelection details: all

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