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- PDB-6zhb: 3D electron diffraction structure of bovine insulin -

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Basic information

Entry
Database: PDB / ID: 6zhb
Title3D electron diffraction structure of bovine insulin
Components(Insulin) x 2
KeywordsHORMONE / INSULIN FAMILY / CARBOHYDRATE METABOLISM / HORMONE-GROWTH
Function / homology
Function and homology information


estradiol secretion / positive regulation of blood circulation / negative regulation of lactation / glucose import in response to insulin stimulus / positive regulation of cell maturation / positive regulation of lactation / response to L-arginine / positive regulation of mammary gland epithelial cell proliferation / response to butyrate / negative regulation of appetite ...estradiol secretion / positive regulation of blood circulation / negative regulation of lactation / glucose import in response to insulin stimulus / positive regulation of cell maturation / positive regulation of lactation / response to L-arginine / positive regulation of mammary gland epithelial cell proliferation / response to butyrate / negative regulation of appetite / feeding behavior / response to growth hormone / response to food / positive regulation of Rho protein signal transduction / positive regulation of peptide hormone secretion / protein secretion / negative regulation of lipid catabolic process / response to glucose / positive regulation of protein secretion / insulin receptor binding / response to nutrient levels / positive regulation of insulin secretion / hormone activity / glucose metabolic process / glucose homeostasis / response to heat / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of gene expression / negative regulation of apoptotic process / extracellular space / identical protein binding
Similarity search - Function
Insulin / Insulin family / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily
Similarity search - Domain/homology
Biological speciesBos taurus (domestic cattle)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 3.25 Å
AuthorsBlum, T. / Housset, D. / Clabbers, M.T.B. / van Genderen, E. / Bacia-Verloop, M. / Zander, U. / McCarthy, A.A. / Schoehn, G. / Ling, W.L. / Abrahams, J.P.
Funding support France, Switzerland, 4items
OrganizationGrant numberCountry
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
Swiss National Science Foundation31003A_17002 Switzerland
Swiss National Science Foundation200021_165669 Switzerland
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2021
Title: Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.
Authors: Thorsten B Blum / Dominique Housset / Max T B Clabbers / Eric van Genderen / Maria Bacia-Verloop / Ulrich Zander / Andrew A McCarthy / Guy Schoehn / Wai Li Ling / Jan Pieter Abrahams /
Abstract: Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron ...Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.
History
DepositionJun 22, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Jan 24, 2024Group: Refinement description / Category: pdbx_initial_refinement_model
Revision 1.3Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)11,5535
Polymers11,4874
Non-polymers651
Water00
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)34,65815
Polymers34,46112
Non-polymers1963
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area17920 Å2
ΔGint-200 kcal/mol
Surface area12130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.400, 82.400, 33.460
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number146
Space group name H-MH3
Components on special symmetry positions
IDModelComponents
11D-101-

ZN

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Components

#1: Protein/peptide Insulin


Mass: 2339.645 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: purchased from Sigma (I1882) / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P01317
#2: Protein/peptide Insulin


Mass: 3403.927 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: purchased from Sigma (I1882) / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P01317
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Insulin Zn complex / Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.0057 MDa / Experimental value: NO
Source (natural)Organism: Bos taurus (domestic cattle)
Buffer solutionpH: 6.5 / Details: MES buffer, 50 mM
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMZinc ChlorideZnCl21
210 mMSodium ChlorideNaCl1
SpecimenConc.: 26 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3D nano-sized crystals (about 200 nm)
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 295 K / Details: Manual plunger

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN / Temperature (max): 90 K / Temperature (min): 90 K
Image recordingAverage exposure time: 0.5 sec. / Electron dose: 5 e/Å2 / Film or detector model: OTHER / Num. of diffraction images: 475 / Num. of grids imaged: 1
EM diffractionCamera length: 900 mm
EM diffraction shellResolution: 3.25→30.3 Å / Fourier space coverage: 84.4 % / Multiplicity: 2.7 / Num. of structure factors: 1127 / Phase residual: 0.001 °
EM diffraction statsDetails: No experimental phase error information available (diffraction experiment)
Fourier space coverage: 84.4 % / High resolution: 3.25 Å / Num. of intensities measured: 1201 / Num. of structure factors: 1127 / Phase error: 0 ° / Phase residual: 0.001 ° / Phase error rejection criteria: 0 / Rmerge: 0.272 / Rsym: 0.272

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0230refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
EM softwareName: CCP4 package / Category: molecular replacement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 120 ° / A: 82.4 Å / B: 82.4 Å / C: 33.46 Å / Space group name: R3 / Space group num: 146
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2A3G

2a3g


Resolution: 3.25→30.3 Å / Cor.coef. Fo:Fc: 0.934 / Cor.coef. Fo:Fc free: 0.754 / SU B: 58.278 / SU ML: 0.899 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.989 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.3189 108 9.6 %RANDOM
Rwork0.1809 ---
obs0.1942 1019 84.36 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 107.08 Å2 / Biso mean: 49.104 Å2 / Biso min: 19.39 Å2
Baniso -1Baniso -2Baniso -3
1--0.19 Å2-0.09 Å2-0 Å2
2---0.19 Å20 Å2
3---0.6 Å2
Refinement stepCycle: final / Resolution: 3.25→30.3 Å
LigandSolventTotal
Num. atoms1 0 775
Biso mean19.39 --
Num. residues--99
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0060.014796
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0010.017669
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.0341.6461083
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg0.7741.641562
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg7.43597
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg33.0423.90241
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg19.30215118
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg18.484152
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0480.296
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0030.02903
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02161
LS refinement shellResolution: 3.251→3.334 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.501 4 -
Rwork0.349 64 -
all-68 -
obs--70.83 %

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