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- PDB-6hr1: Crystal structure of the YFPnano fusion protein -

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Basic information

Entry
Database: PDB / ID: 6hr1
TitleCrystal structure of the YFPnano fusion protein
ComponentsMyosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1
KeywordsFusion protein / fluorescent engineered / STRUCTURAL PROTEIN
Function / homology
Function and homology information


plus-end directed microfilament motor activity / regulation of muscle filament sliding / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / filopodium tip / cytoskeleton-dependent intracellular transport / regulation of filopodium assembly / cardiac muscle tissue morphogenesis / filopodium membrane ...plus-end directed microfilament motor activity / regulation of muscle filament sliding / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / filopodium tip / cytoskeleton-dependent intracellular transport / regulation of filopodium assembly / cardiac muscle tissue morphogenesis / filopodium membrane / myosin complex / calcium/calmodulin-dependent protein kinase activity / 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 / microfilament motor activity / autophagosome membrane docking / mitochondrion-endoplasmic reticulum membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / Synthesis of IP3 and IP4 in the cytosol / negative regulation of peptidyl-threonine phosphorylation / Negative regulation of NMDA receptor-mediated neuronal transmission / Phase 0 - rapid depolarisation / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / RHO GTPases activate PAKs / phosphatidylinositol-3,4,5-trisphosphate binding / Ion transport by P-type ATPases / : / Uptake and function of anthrax toxins / Long-term potentiation / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / Smooth Muscle Contraction / regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / calcium channel inhibitor activity / cellular response to interferon-beta / eNOS activation / Protein methylation / voltage-gated potassium channel complex / Activation of AMPK downstream of NMDARs / striated muscle contraction / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / : / Ion homeostasis / titin binding / positive regulation of protein autophosphorylation / regulation of calcium-mediated signaling / sperm midpiece / ruffle / 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 / FCGR3A-mediated IL10 synthesis / protein serine/threonine kinase activator activity / bioluminescence / VEGFR2 mediated vascular permeability / VEGFR2 mediated cell proliferation / regulation of cytokinesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / filopodium / positive regulation of peptidyl-threonine phosphorylation / generation of precursor metabolites and energy / spindle microtubule / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of receptor signaling pathway via JAK-STAT / RAF activation / Transcriptional activation of mitochondrial biogenesis / positive regulation of protein serine/threonine kinase activity / Stimuli-sensing channels / cellular response to type II interferon
Similarity search - Function
Myosin light chain kinase 2, catalytic domain / Unconventional myosin-X, coiled coil domain / Class X myosin, motor domain / Myosin X, N-terminal SH3 domain / Myosin X, FERM domain C-lobe / Unconventional myosin-X coiled coil domain / Myosin X N-terminal SH3 domain / : / MyTH4 domain / MyTH4 domain superfamily ...Myosin light chain kinase 2, catalytic domain / Unconventional myosin-X, coiled coil domain / Class X myosin, motor domain / Myosin X, N-terminal SH3 domain / Myosin X, FERM domain C-lobe / Unconventional myosin-X coiled coil domain / Myosin X N-terminal SH3 domain / : / MyTH4 domain / MyTH4 domain superfamily / MyTH4 domain / MyTH4 domain profile. / Domain in Myosin and Kinesin Tails / RA like domain / Ras-associating (RA) domain / IQ calmodulin-binding motif / FERM central domain / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / FERM/acyl-CoA-binding protein superfamily / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / : / FERM central domain / FERM superfamily, second domain / FERM domain / FERM domain profile. / Band 4.1 domain / Band 4.1 homologues / Kinesin motor domain superfamily / PH domain / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / 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 / PH-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
L(+)-TARTARIC ACID / Myosin light chain kinase 2, skeletal/cardiac muscle / Calmodulin-1 / Green fluorescent protein / Unconventional myosin-X
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Bos taurus (cattle)
Aequorea victoria (jellyfish)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.901 Å
AuthorsBenoit, R.M.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Switzerland
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: Krebs, A.S. / Collu, G. / Bierig, T. / Varma, N. / Benoit, R.M.B.
History
DepositionSep 26, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_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_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 1.2Oct 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 1.3Jan 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
Revision 1.4Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Myosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1
B: Myosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,93527
Polymers98,4002
Non-polymers1,53625
Water6,593366
1
A: Myosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,91111
Polymers49,2001
Non-polymers71110
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Myosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,02516
Polymers49,2001
Non-polymers82515
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)53.700, 117.800, 84.500
Angle α, β, γ (deg.)90.000, 99.900, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Myosin light chain kinase 2, skeletal/cardiac muscle,Unconventional myosin-X,Green fluorescent protein,Calmodulin-1 / MLCK2 / Unconventional myosin-10


Mass: 49199.820 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: residues -35 to -32 = leftovers from cleaved-off tag res -31 t -11 = Calmodulin-binding peptide (very similar to the peptide in PDB entry 2BBM) res -10 to 0 = ER/K helix (small fragment from ...Details: residues -35 to -32 = leftovers from cleaved-off tag res -31 t -11 = Calmodulin-binding peptide (very similar to the peptide in PDB entry 2BBM) res -10 to 0 = ER/K helix (small fragment from the protein in PDB entry 5HMO, res 818-828) res 1 to 238 = Yellow Fluorescent Protein (nearly identical to pdb entry 3V3D) res 239 - 252 = flexible linker res 253 - 400 = Calmodulin (as in PDB entry 2BBM),residues -35 to -32 = leftovers from cleaved-off tag
Source: (gene. exp.) Oryctolagus cuniculus (rabbit), (gene. exp.) Bos taurus (cattle), (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Homo sapiens (human)
Gene: MYLK2, MYO10, GFP, CALM1, CALM, CAM, CAM1 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta 2
References: UniProt: P07313, UniProt: P79114, UniProt: P42212, UniProt: P0DP23, myosin-light-chain kinase

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Non-polymers , 6 types, 391 molecules

#2: Chemical ChemComp-TLA / L(+)-TARTARIC ACID


Mass: 150.087 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H6O6
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: C3H8O3
#6: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 366 / Source method: isolated from a natural source / Formula: H2O

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Details

Has protein modificationY

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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: 2.67 Å3/Da / Density % sol: 54.01 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 7.8
Details: 24% w/v PEG 3350 0.2 M di-Ammonium tartrate 10% v/v Glycerol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X06DA / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Oct 18, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.9→48.589 Å / Num. obs: 81149 / % possible obs: 99.9 % / Redundancy: 6.491 % / Biso Wilson estimate: 28.71 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.09 / Rrim(I) all: 0.097 / Χ2: 1.032 / Net I/σ(I): 14
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.9-1.955.0340.9541.7659350.6821.06299.4
1.95-25.5980.7582.5258270.810.83599.7
2-2.065.9970.5923.3656990.8930.64899.8
2.06-2.136.1080.4494.5455070.9150.49199.9
2.13-2.26.3330.3386.0753450.9580.36999.8
2.2-2.276.5960.2837.6351580.9720.30799.9
2.27-2.367.1930.2629.2350190.9820.282100
2.36-2.457.210.22410.9448220.9850.241100
2.45-2.567.1380.18112.9146150.9890.196100
2.56-2.697.0810.15114.7443850.9920.163100
2.69-2.836.9520.12217.0541900.9940.13299.9
2.83-3.016.5210.119.1239850.9940.109100
3.01-3.216.3410.07722.8637090.9960.08499.9
3.21-3.476.8970.06626.9834780.9970.07199.9
3.47-3.87.0780.05730.6632250.9970.062100
3.8-4.256.8410.05233.1728810.9980.05699.7
4.25-4.916.7260.04634.3725760.9980.0599.9
4.91-6.016.1150.04831.8521690.9980.05399.8
6.01-8.56.760.04833.9716840.9980.052100
8.5-48.5896.9990.04338.179400.9980.04799.5

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation1.85 Å48.18 Å
Translation1.85 Å48.18 Å

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Processing

Software
NameVersionClassificationNB
PHENIX1.10.1_2155refinement
XDSdata reduction
XSCALEdata scaling
PHASER2.7.16phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3V3D

3v3d


Resolution: 1.901→48.589 Å / SU ML: 0.23 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 22.18
RfactorNum. reflection% reflection
Rfree0.2136 4056 5 %
Rwork0.1806 --
obs0.1823 81142 99.88 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 97.13 Å2 / Biso mean: 36.7914 Å2 / Biso min: 14.33 Å2
Refinement stepCycle: final / Resolution: 1.901→48.589 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6468 0 86 366 6920
Biso mean--46.85 38.64 -
Num. residues----809
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0096729
X-RAY DIFFRACTIONf_angle_d0.9819038
X-RAY DIFFRACTIONf_chiral_restr0.063958
X-RAY DIFFRACTIONf_plane_restr0.0061195
X-RAY DIFFRACTIONf_dihedral_angle_d16.1784040
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 29

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.901-1.92340.35021390.28952642278199
1.9234-1.94680.28671380.256726092747100
1.9468-1.97150.30151400.246626722812100
1.9715-1.99740.24771400.236926662806100
1.9974-2.02480.27791380.243626142752100
2.0248-2.05370.32111390.230826662805100
2.0537-2.08440.26591390.21926392778100
2.0844-2.11690.22181390.199826372776100
2.1169-2.15160.2151410.197926682809100
2.1516-2.18870.25921370.193326382775100
2.1887-2.22850.24181420.193226822824100
2.2285-2.27140.2441370.190726202757100
2.2714-2.31780.2561410.187126652806100
2.3178-2.36820.24451380.189426342772100
2.3682-2.42320.25681410.191126822823100
2.4232-2.48380.24091400.18626542794100
2.4838-2.5510.20741410.17526792820100
2.551-2.62610.22561390.177626372776100
2.6261-2.71080.19221400.17726522792100
2.7108-2.80770.22131410.183126812822100
2.8077-2.92010.21761370.185326142751100
2.9201-3.0530.23391410.186626802821100
3.053-3.21390.16631410.166826652806100
3.2139-3.41520.20211400.174626602800100
3.4152-3.67880.18971410.167726792820100
3.6788-4.04890.22081400.157426672807100
4.0489-4.63440.16871410.146926842825100
4.6344-5.83720.19461410.170926692810100
5.8372-48.60480.18351440.184227312875100

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