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- PDB-5e7w: X-ray Structure of Human Recombinant 2Zn insulin at 0.92 Angstrom -

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Basic information

Entry
Database: PDB / ID: 5e7w
TitleX-ray Structure of Human Recombinant 2Zn insulin at 0.92 Angstrom
Components(Insulin) x 2
KeywordsIMMUNE SYSTEM / Insulin / human / recombinant / high-resolution
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 / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / 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 / nitric oxide-cGMP-mediated signaling / negative regulation of fatty acid metabolic process / 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 / positive regulation of insulin receptor signaling pathway / negative regulation of reactive oxygen species biosynthetic process / transport vesicle / positive regulation of protein autophosphorylation / Insulin receptor recycling / insulin-like growth factor receptor binding / NPAS4 regulates expression of target genes / positive regulation of protein metabolic process / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / 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 / Golgi lumen / vasodilation / 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
ACETATE ION / N-PROPANOL / Insulin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 0.9519 Å
AuthorsLisgarten, D.R. / Naylor, C.E. / Palmer, R.A. / Lobley, C.M.C.
CitationJournal: Chem Cent J / Year: 2017
Title: Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K.
Authors: Lisgarten, D.R. / Palmer, R.A. / Lobley, C.M.C. / Naylor, C.E. / Chowdhry, B.Z. / Al-Kurdi, Z.I. / Badwan, A.A. / Howlin, B.J. / Gibbons, N.C.J. / Saldanha, J.W. / Lisgarten, J.N. / Basak, A.K.
History
DepositionOct 13, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Oct 12, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI
Revision 1.2Dec 6, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.3Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Insulin
B: Insulin
C: Insulin
D: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)11,8858
Polymers11,6354
Non-polymers2504
Water3,963220
1
A: Insulin
B: Insulin
C: Insulin
D: Insulin
hetero molecules

A: Insulin
B: Insulin
C: Insulin
D: Insulin
hetero molecules

A: Insulin
B: Insulin
C: Insulin
D: Insulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,65624
Polymers34,90612
Non-polymers75012
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area20360 Å2
ΔGint-288 kcal/mol
Surface area13410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.820, 81.820, 33.850
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number146
Space group name H-MH3
Components on special symmetry positions
IDModelComponents
11B-100-

ZN

21D-202-

ZN

31B-255-

HOH

41B-266-

HOH

51D-351-

HOH

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Components

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Protein/peptide , 2 types, 4 molecules ACBD

#1: Protein/peptide Insulin /


Mass: 2383.698 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human Insulin A and C Chain Insulin was purchased from Insugen
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Komagataella pastoris (fungus) / References: UniProt: P01308
#2: Protein/peptide Insulin /


Mass: 3433.953 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Human Insulin B and D chain Insulin was purchased from Insugen
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Komagataella pastoris (fungus) / References: UniProt: P01308

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Non-polymers , 4 types, 224 molecules

#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#5: Chemical ChemComp-POL / N-PROPANOL / 1-PROPONOL / Propan-1-ol


Mass: 60.095 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 220 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION

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Sample preparation

CrystalDensity Matthews: 1.89 Å3/Da / Density % sol: 35 % / Description: needle
Crystal growTemperature: 293 K / Method: batch mode / pH: 6.3
Details: The crystals were prepared by a batch method similar to that of Baker et al, 1988 [1], modified as follows: 0.01g of insulin as a fine powder was placed in a clean test tube; 0.02M HCl was ...Details: The crystals were prepared by a batch method similar to that of Baker et al, 1988 [1], modified as follows: 0.01g of insulin as a fine powder was placed in a clean test tube; 0.02M HCl was added to dissolve the protein; on addition of 0.15 mL of 0.15 M zinc acetate the solution became cloudy due to precipitation of the protein; 0.3 mL of acetone and then 0.5 mL of trisodium citrate together with 0.8 mL of water were added and the solution went clear; the pH was checked and increased with NaOH to a pH between 8 and 9 for different batches, thus ensuring complete dissolution. It was then adjusted to the required value of pH 6.3. If any slight turbidity occurred, it was removed by warming the solution. The solution was then filtered using a Millipore membrane/acetate cellulose acetate filter. This removes any nuclei which will encourage precipitation or formation of masses of small crystals. The solution was then warmed to 50 deg C by surrounding the test tube with preheated water in a Dewar. This allowed the solution to cool slowly to room temperature. The test tube was lightly sealed with cling film; crystals formed within a few days and were of suitable size for X-ray diffraction within two weeks; the test tube containing crystals was kept at 4 degC prior to data collection. The crystal used for data collection was about 0.2 mm3.
PH range: 6.2 - 6.4 / Temp details: Room Temperature

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.77 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 27, 2012
Details: Data were collected at 16000keV (0.77 Angstrom) and 100 deg K with the Pilatus 6M detector as close to the sample as possible (179.5mm). The EDNA strategy was used to obtain a start angle ...Details: Data were collected at 16000keV (0.77 Angstrom) and 100 deg K with the Pilatus 6M detector as close to the sample as possible (179.5mm). The EDNA strategy was used to obtain a start angle and 180 deg of data were collected with 0.1 deg oscillation and 0.1s exposure. The resolution of useful diffraction data achieved and used for structure analysis was 0.92 Angstrom.
RadiationMonochromator: Beamline fixed at 16000keV / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.77 Å / Relative weight: 1
ReflectionResolution: 0.92→40.91 Å / Num. obs: 58647 / % possible obs: 100 % / Redundancy: 5 % / Rmerge(I) obs: 0.034 / Net I/σ(I): 20.2
Reflection shellResolution: 0.92→0.94 Å / Redundancy: 4.7 % / Rmerge(I) obs: 0.967 / Mean I/σ(I) obs: 1.6 / % possible all: 100

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Processing

Software
NameVersionClassification
SHELXL2014/7refinement
XDSFast dp Xia 2data reduction
AimlessFast dp Xia 2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3W7Y

3w7y


Resolution: 0.9519→10 Å / Cross valid method: FREE R-VALUE / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.1444 2692 5 %thin shells
Rwork0.1111 ---
obs-50178 94.9 %-
Displacement parametersBiso mean: 18.45 Å2
Refinement stepCycle: LAST / Resolution: 0.9519→10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms810 0 10 220 1040

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