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- PDB-6yt3: Structure of the MoStoNano fusion protein -

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Basic information

Entry
Database: PDB / ID: 6yt3
TitleStructure of the MoStoNano fusion protein
Components
  • Molybdenum storage protein subunit alpha
  • Thioredoxin,Molybdenum storage protein subunit beta
KeywordsSTRUCTURAL PROTEIN / Fusion protein / crystal engineering / rigid helix / molecular biomimetics
Function / homology
Function and homology information


glycerol ether metabolic process / nutrient reservoir activity / molybdenum ion binding / protein-disulfide reductase activity / cytoplasm
Similarity search - Function
Molybdenum storage protein subunit alpha/beta / Aspartate/glutamate/uridylate kinase / Amino acid kinase family / Acetylglutamate kinase-like superfamily / Thioredoxin / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Thioredoxin / Molybdenum storage protein subunit beta / Molybdenum storage protein subunit alpha
Similarity search - Component
Biological speciesAzotobacter vinelandii (bacteria)
Salmonella enterica subsp. enterica serovar Bovismorbificans (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.85 Å
AuthorsBenoit, R.M. / Bierig, T. / Collu, C. / Engilberge, S. / Olieric, V.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Novartis FreeNovation Switzerland
Promedica Siftung Switzerland
Citation
Journal: Structure / Year: 2022
Title: Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.
Authors: Gabriella Collu / Tobias Bierig / Anna-Sophia Krebs / Sylvain Engilberge / Niveditha Varma / Ramon Guixà-González / Timothy Sharpe / Xavier Deupi / Vincent Olieric / Emiliya Poghosyan / Roger M Benoit /
Abstract: Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we ...Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix. This enables the connection of two domains at a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano. Analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example, to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact on construct flexibility (for the study of protein dynamics), and to design novel biomaterials.
#1: Journal: Biorxiv / Year: 2020
Title: Chimeric single alpha-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology
Authors: Collu, G. / Bierig, T. / Krebs, A.-S. / Engilberge, S. / Varma, N. / Guixa-Gonzalez, R. / Deupi, X. / Olieric, V. / Poghosyan, E. / Benoit, R.M.
History
DepositionApr 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Advisory / Data collection ...Advisory / Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / database_2 / database_PDB_rev / database_PDB_rev_record / entity / pdbx_database_proc / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.details
Revision 1.2Jan 19, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Molybdenum storage protein subunit alpha
B: Thioredoxin,Molybdenum storage protein subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,9045
Polymers71,8662
Non-polymers1,0393
Water3,657203
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6540 Å2
ΔGint-41 kcal/mol
Surface area25970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)165.697, 165.697, 352.162
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
Components on special symmetry positions
IDModelComponents
11A-405-

HOH

21A-476-

HOH

31A-482-

HOH

41A-485-

HOH

51A-494-

HOH

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Components

#1: Protein Molybdenum storage protein subunit alpha / Mo storage protein subunit alpha / MoSto subunit alpha


Mass: 31428.973 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Strain: DJ / ATCC BAA-1303 / Gene: mosA, Avin_43200 / Production host: Escherichia coli (E. coli) / References: UniProt: P84308
#2: Protein Thioredoxin,Molybdenum storage protein subunit beta / MoSto subunit beta


Mass: 40436.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: chain B has three components that are fused together: Thioredoxin at the N-terminus, then a short EEEKRKREEE rigid helix, then the beta-subunit of MoSto.
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Bovismorbificans (bacteria), (gene. exp.) Azotobacter vinelandii (strain DJ / ATCC BAA-1303) (bacteria)
Gene: trxA_2, trxA, A3789_21210, AL561_14185, B7N00_20260, B7N01_17730, B7N34_19660, B7N35_19470, B7N60_21445, B7N72_16575, B7N73_22720, B7N78_20580, B7N79_19980, B7N80_16005, B7N84_20570, B7N95_ ...Gene: trxA_2, trxA, A3789_21210, AL561_14185, B7N00_20260, B7N01_17730, B7N34_19660, B7N35_19470, B7N60_21445, B7N72_16575, B7N73_22720, B7N78_20580, B7N79_19980, B7N80_16005, B7N84_20570, B7N95_20865, CAD68_20095, CBK57_22150, DK062_18215, DOI63_23750, DP779_24195, DPA33_18890, DPK57_04940, DPS13_18845, DPZ52_04610, DRU31_19690, E0916_20730, EJV93_21005, ERS008198_02131, ERS008202_02904, ERS008207_01732, EWC73_19305, EXP31_20825, NCTC5754_04586, mosB, Avin_43210
Strain: DJ / ATCC BAA-1303 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0U0X1R7, UniProt: P84253
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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

Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.1 M tri-Sodium citrate pH 5.6, 10% PEG 4000, 10% Isopropanol

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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 16, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.85→47.89 Å / Num. obs: 33352 / % possible obs: 99.9 % / Redundancy: 10.6 % / CC1/2: 0.998 / Rpim(I) all: 0.071 / Net I/σ(I): 8.2
Reflection shellResolution: 2.85→2.98 Å / Mean I/σ(I) obs: 2.3 / Num. unique obs: 1670 / CC1/2: 0.8 / Rpim(I) all: 0.351

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4F6T

4f6t


Resolution: 2.85→47.89 Å / Cor.coef. Fo:Fc: 0.871 / Cor.coef. Fo:Fc free: 0.851 / SU R Cruickshank DPI: 0.436 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.44 / SU Rfree Blow DPI: 0.312 / SU Rfree Cruickshank DPI: 0.315
RfactorNum. reflection% reflectionSelection details
Rfree0.263 1695 5.08 %RANDOM
Rwork0.233 ---
obs0.234 33352 76.3 %-
Displacement parametersBiso max: 160.57 Å2 / Biso mean: 65.14 Å2 / Biso min: 3 Å2
Baniso -1Baniso -2Baniso -3
1-1.8279 Å20 Å20 Å2
2--1.8279 Å20 Å2
3----3.6558 Å2
Refine analyzeLuzzati coordinate error obs: 0.47 Å
Refinement stepCycle: final / Resolution: 2.85→47.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4628 0 63 211 4902
Biso mean--81.81 39.92 -
Num. residues----619
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1647SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes843HARMONIC5
X-RAY DIFFRACTIONt_it4810HARMONIC20
X-RAY DIFFRACTIONt_nbd1SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion635SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact5610SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d4810HARMONIC20.009
X-RAY DIFFRACTIONt_angle_deg6562HARMONIC21.14
X-RAY DIFFRACTIONt_omega_torsion2.43
X-RAY DIFFRACTIONt_other_torsion19.28
LS refinement shellResolution: 2.85→2.9 Å / Rfactor Rfree error: 0 / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0.3457 29 4.34 %
Rwork0.2377 639 -
all0.243 668 -
obs--28.45 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.47850.27380.00521.9294-0.60082.55840.00650.23790.3217-0.2351-0.00080.1065-0.4712-0.1815-0.0057-0.01380.032-0.063-0.16990.0596-0.120875.7404-25.100241.3063
20.01480.3388-0.18491.2739-0.90750.77520.07050.42340.029-0.3379-0.11750.1756-0.272-0.11810.04710.09760.0559-0.12410.15110.1742-0.278570.6845-22.642114.9528
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* }A34 - 276
2X-RAY DIFFRACTION2{ B|* }B2 - 377

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