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- PDB-6xyr: Structure of the T4Lnano fusion protein -

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Basic information

Entry
Database: PDB / ID: 6xyr
TitleStructure of the T4Lnano fusion protein
ComponentsT4Lnano,Endolysin,Calmodulin,Endolysin,Calmodulin-1
KeywordsSTRUCTURAL PROTEIN / Fusion protein / crystal engineering / rigid helix / molecular biomimetics
Function / homology
Function and homology information


CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Activation of Ca-permeable Kainate Receptor / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation ...CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Activation of Ca-permeable Kainate Receptor / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / negative regulation of high voltage-gated calcium channel activity / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / positive regulation of cyclic-nucleotide phosphodiesterase activity / organelle localization by membrane tethering / negative regulation of calcium ion export across plasma membrane / CLEC7A (Dectin-1) induces NFAT activation / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / regulation of cardiac muscle cell action potential / Activation of RAC1 downstream of NMDARs / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Unblocking of NMDA receptors, glutamate binding and activation / Phase 0 - rapid depolarisation / negative regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / RHO GTPases activate PAKs / : / Ion transport by P-type ATPases / Long-term potentiation / Uptake and function of anthrax toxins / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / cellular response to interferon-beta / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / Protein methylation / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Activation of AMPK downstream of NMDARs / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / : / Ion homeostasis / titin binding / viral release from host cell by cytolysis / regulation of calcium-mediated signaling / positive regulation of protein autophosphorylation / voltage-gated potassium channel complex / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / sarcomere / FCERI mediated Ca+2 mobilization / peptidoglycan catabolic process / protein serine/threonine kinase activator activity / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / positive regulation of peptidyl-threonine phosphorylation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / RAF activation / positive regulation of receptor signaling pathway via JAK-STAT / response to calcium ion / Transcriptional activation of mitochondrial biogenesis / positive regulation of protein serine/threonine kinase activity / Stimuli-sensing channels / cellular response to type II interferon / G2/M transition of mitotic cell cycle / spindle pole / RAS processing / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / calcium-dependent protein binding / Inactivation, recovery and regulation of the phototransduction cascade / Signaling by BRAF and RAF1 fusions / cell wall macromolecule catabolic process / Platelet degranulation / lysozyme / myelin sheath / lysozyme activity / Ca2+ pathway
Similarity search - Function
: / Endolysin T4 type / : / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site ...: / Endolysin T4 type / : / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Endolysin / Calmodulin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Enterobacteria phage T4 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / molecular replacement / Resolution: 2.079 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
DepositionJan 31, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Advisory / Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / pdbx_database_proc / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Jan 19, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.year
Revision 1.3Jun 19, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation / Item: _citation.journal_id_ISSN

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: T4Lnano,Endolysin,Calmodulin,Endolysin,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,58813
Polymers40,8561
Non-polymers73212
Water2,432135
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1910 Å2
ΔGint-81 kcal/mol
Surface area18400 Å2
Unit cell
Length a, b, c (Å)86.380, 104.260, 63.950
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein T4Lnano,Endolysin,Calmodulin,Endolysin,Calmodulin-1 / Lysis protein / Lysozyme / Muramidase


Mass: 40855.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry ...Details: aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM),aa 6 - 25 = Calmodulin-binding peptide aa 26 - 36 = single alpha-helical domain (from pdb entry 5HMO) aa 37 - 199 = T4 Lysozyme aa 200 - 213 = linker aa 214 - 361 = Calmodulin (pdb entry 2BBM)
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Enterobacteria phage T4 (virus)
Gene: CALM1, CALM, CAM, CAM1 / Production host: Escherichia coli (E. coli) / References: UniProt: P00720, UniProt: P0DP23, lysozyme
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 135 / 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

CrystalDensity Matthews: 3.52 Å3/Da / Density % sol: 65.1 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop
Details: 8% PEG 8000, 200 mM LiCl2, 100 mM Tris pH 8.0, 15% Glycerol

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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: Mar 3, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.079→46.1 Å / Num. obs: 35404 / % possible obs: 99.84 % / Redundancy: 13.1 % / Biso Wilson estimate: 41.68 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.1276 / Rpim(I) all: 0.03666 / Net I/σ(I): 14.53
Reflection shellResolution: 2.079→2.154 Å / Rmerge(I) obs: 2.043 / Mean I/σ(I) obs: 1.31 / Num. unique obs: 3434 / CC1/2: 0.6 / Rpim(I) all: 0.5821

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Phasing

Phasing
Method
SAD
molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
XSCALEdata scaling
PHASERphasing
SHELXDEphasing
PHENIX1.11.1_2575refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: SAD / Resolution: 2.079→46.1 Å / SU ML: 0.27 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 25.21
RfactorNum. reflection% reflection
Rfree0.2357 1798 5.08 %
Rwork0.1905 --
obs0.1928 35391 99.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 137.74 Å2 / Biso mean: 55.491 Å2 / Biso min: 30.92 Å2
Refinement stepCycle: final / Resolution: 2.079→46.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2783 0 76 139 2998
Biso mean--73.38 50.18 -
Num. residues----348
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0112846
X-RAY DIFFRACTIONf_angle_d1.0383812
X-RAY DIFFRACTIONf_chiral_restr0.047411
X-RAY DIFFRACTIONf_plane_restr0.006500
X-RAY DIFFRACTIONf_dihedral_angle_d16.4841739
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.0792-2.13550.35621500.3367247099
2.1355-2.19830.31081260.30252571100
2.1983-2.26920.30811230.2622570100
2.2692-2.35030.28291440.23492534100
2.3503-2.44440.27711230.21862566100
2.4444-2.55570.26031500.19952569100
2.5557-2.69040.26741340.19172548100
2.6904-2.85890.23771330.21252568100
2.8589-3.07960.30341440.20542593100
3.0796-3.38950.25581360.20392589100
3.3895-3.87970.25281330.17532611100
3.8797-4.88720.17751510.14422652100
4.8872-46.10.19081510.17572752100

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