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- PDB-8b6q: X-ray structure of the haloalkane dehalogenase HaloTag7 with an i... -

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Basic information

Entry
Database: PDB / ID: 8b6q
TitleX-ray structure of the haloalkane dehalogenase HaloTag7 with an insertion of Calmodulin-M13 fusion at position 154-156 that mimic the structure of CaProLa, an calcium gated protein labeling technology
ComponentsHaloalkane dehalogenase,Calmodulin-1,Haloalkane dehalogenase,Calmodulin-1,M13 peptide
KeywordsHYDROLASE / haloalkane dehalogenase / HaloTag / HaloTag7 / Self-Labeling Protein / Calmodulin / M13
Function / homology
Function and homology information


haloalkane dehalogenase / haloalkane dehalogenase activity / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 ...haloalkane dehalogenase / haloalkane dehalogenase activity / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / 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) / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / positive regulation of ryanodine-sensitive calcium-release channel activity / Negative regulation of NMDA receptor-mediated neuronal transmission / regulation of cell communication by electrical coupling involved in cardiac conduction / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / Long-term potentiation / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / voltage-gated potassium channel complex / Activation of AMPK downstream of NMDARs / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Ion homeostasis / positive regulation of protein autophosphorylation / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / protein serine/threonine kinase activator activity / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / RAF activation / positive regulation of receptor signaling pathway via JAK-STAT / positive regulation of protein serine/threonine kinase activity / Transcriptional activation of mitochondrial biogenesis / Stimuli-sensing channels / response to toxic substance / spindle pole / cellular response to type II interferon / response to calcium ion / RAS processing / calcium-dependent protein binding / Inactivation, recovery and regulation of the phototransduction cascade / 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 / G2/M transition of mitotic cell cycle / Signaling by BRAF and RAF1 fusions / Platelet degranulation / myelin sheath / Ca2+ pathway / RAF/MAP kinase cascade / vesicle
Similarity search - Function
Haloalkane dehalogenase, subfamily 2 / Epoxide hydrolase-like / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / 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 ...Haloalkane dehalogenase, subfamily 2 / Epoxide hydrolase-like / alpha/beta hydrolase fold / Alpha/beta hydrolase fold-1 / 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 / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Haloalkane dehalogenase / Calmodulin-1
Similarity search - Component
Biological speciesRhodococcus sp. (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsTarnawski, M. / Johnsson, K. / Hiblot, J.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: To Be Published
Title: X-ray structure of the haloalkane dehalogenase HaloTag7 with an insertion of Calmodulin-M13 fusion at position 154-156 that mimic the structure of CaProLa, an calcium gated protein labeling technology
Authors: Tarnawski, M. / Johnsson, K. / Hiblot, J.
History
DepositionSep 27, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Haloalkane dehalogenase,Calmodulin-1,Haloalkane dehalogenase,Calmodulin-1,M13 peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,2168
Polymers52,9411
Non-polymers2767
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area490 Å2
ΔGint-56 kcal/mol
Surface area20460 Å2
Unit cell
Length a, b, c (Å)74.530, 74.530, 177.840
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Space group name HallP4abw2nw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+1/4
#3: y+1/2,-x+1/2,z+3/4
#4: x+1/2,-y+1/2,-z+3/4
#5: -x+1/2,y+1/2,-z+1/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Haloalkane dehalogenase,Calmodulin-1,Haloalkane dehalogenase,Calmodulin-1,M13 peptide


Mass: 52940.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodococcus sp. (in: high G+C Gram-positive bacteria) (bacteria), (gene. exp.) Homo sapiens (human)
Gene: dhaA, CALM1, CALM, CAM, CAM1 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A3G3, UniProt: P0DP23, haloalkane dehalogenase
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / 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: 2.33 Å3/Da / Density % sol: 47.27 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.1 M MES pH 6.0, 0.2 M calcium acetate, 18% (m/v) PEG 8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 0.99989 Å
DetectorType: PSI PILATUS 6M / Detector: PIXEL / Date: May 10, 2019
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.99989 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 16118 / % possible obs: 99.7 % / Redundancy: 9.2 % / Biso Wilson estimate: 65.11 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.106 / Net I/σ(I): 12.78
Reflection shellResolution: 2.6→2.7 Å / Redundancy: 9.9 % / Rmerge(I) obs: 0.851 / Num. unique obs: 1676 / CC1/2: 0.861 / % possible all: 99.3

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5Y2X, 2WEL

5y2x

2wel


Resolution: 2.6→46.39 Å / SU ML: 0.5586 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 36.2607
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3182 806 5 %
Rwork0.2806 15311 -
obs0.2825 16117 99.7 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 89.43 Å2
Refinement stepCycle: LAST / Resolution: 2.6→46.39 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3689 0 7 3 3699
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0023781
X-RAY DIFFRACTIONf_angle_d0.48235131
X-RAY DIFFRACTIONf_chiral_restr0.0412550
X-RAY DIFFRACTIONf_plane_restr0.0043682
X-RAY DIFFRACTIONf_dihedral_angle_d12.83761405
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.760.45371310.3722487X-RAY DIFFRACTION99.43
2.76-2.980.43711310.37392498X-RAY DIFFRACTION99.62
2.98-3.280.41371320.34642503X-RAY DIFFRACTION99.81
3.28-3.750.34151330.3182528X-RAY DIFFRACTION99.7
3.75-4.720.27881350.25222566X-RAY DIFFRACTION99.93
4.73-46.390.26891440.23922729X-RAY DIFFRACTION99.72
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)
15.937791082940.7283454119690.03229366439673.495715500070.2244162864872.36858334968-0.4691380163180.5443299270171.28589461951-0.1831940515610.1297900419620.612525685917-0.313161033587-0.161910338890.2318499823760.467413495127-0.0252065460071-0.2185606530820.479152774424-0.01131164793530.783476172356.0989365234413.939669633-32.548297988
23.183220631243.34506276823-0.8360352328246.8796532857-2.711236693984.90318881925-0.384826657282-0.0882418197098-0.413411359805-0.673877326960.179027651847-0.783098290088-0.2592657146720.6185749595050.1765218369160.718786854387-0.2737589713630.1104138934730.79378756508-0.1567360491550.507878628985-11.529770426-21.8514326138-16.5273708642
33.036530713550.94731106420.4389482328512.637753703340.04641438454871.43924156442-0.144456580794-0.4364699440451.486283295890.172900802568-0.1858783244220.784080003368-0.180063142618-0.3071088261140.2598808648320.49318322844-0.0044998607341-0.04911527563390.512365391516-0.2628260356271.098900996230.77701794228515.082581681-25.6272413434
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 1 through 171 )1 - 1711 - 171
22chain 'A' and (resid 172 through 309 )172 - 309172 - 305
33chain 'A' and (resid 310 through 470 )310 - 470306 - 466

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