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- PDB-5ljw: MamK non-polymerising A278D mutant bound to AMPPNP -

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Basic information

Entry
Database: PDB / ID: 5ljw
TitleMamK non-polymerising A278D mutant bound to AMPPNP
ComponentsActin-like ATPase
KeywordsHYDROLASE / bacterial cytoskeleton / filamentous protein / actin-like / magnetosomes
Function / homology
Function and homology information


biomineralization / magnetosome membrane / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / hydrolase activity / cytoskeleton / ATP hydrolysis activity / GTP binding / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
MreB/Mbl protein / Actin / Actin family / ATPase, nucleotide binding domain / ATPase, nucleotide binding domain / Nucleotidyltransferase; domain 5 / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Actin-like protein MamK
Similarity search - Component
Biological speciesMagnetospirillum magneticum AMB-1 (magnetotactic)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / Resolution: 1.8 Å
AuthorsLowe, J.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)U105184326 United Kingdom
Wellcome Trust095514/Z/11/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization.
Authors: Jan Löwe / Shaoda He / Sjors H W Scheres / Christos G Savva /
Abstract: Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In ...Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type.
History
DepositionJul 20, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2016Group: Database references
Revision 1.2Aug 30, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Actin-like ATPase
B: Actin-like ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,4336
Polymers75,3722
Non-polymers1,0614
Water9,656536
1
A: Actin-like ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,2173
Polymers37,6861
Non-polymers5312
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Actin-like ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,2173
Polymers37,6861
Non-polymers5312
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
γ
α
β
Length a, b, c (Å)72.572, 71.803, 79.761
Angle α, β, γ (deg.)90.00, 98.41, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Actin-like ATPase / MamK


Mass: 37686.160 Da / Num. of mol.: 2 / Mutation: A278D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Magnetospirillum magneticum AMB-1 (magnetotactic)
Gene: amb0965 / Production host: Escherichia coli (E. coli) / References: UniProt: Q2W8Q6
#2: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 536 / Source method: isolated from a natural source / Formula: H2O

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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: 2.73 Å3/Da / Density % sol: 54.9 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / Details: 50 mM HEPES pH 7.3, 48 % (w/v) PEG 1000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.97941 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Feb 3, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97941 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. obs: 138054 / % possible obs: 95.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.4 % / CC1/2: 0.994 / Rmerge(I) obs: 0.083 / Net I/σ(I): 6.9
Reflection shellResolution: 1.8→1.9 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.499 / Mean I/σ(I) obs: 1.8 / CC1/2: 0.81 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIXdev_1810refinement
SCALAdata scaling
autoSHARPphasing
RefinementMethod to determine structure: SIRAS / Resolution: 1.8→44.858 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 0.71 / Phase error: 21.1
RfactorNum. reflection% reflection
Rfree0.2064 6665 4.95 %
Rwork0.1781 --
obs0.1795 134653 91.21 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.8→44.858 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4989 0 64 536 5589
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0135138
X-RAY DIFFRACTIONf_angle_d1.4066981
X-RAY DIFFRACTIONf_dihedral_angle_d13.341917
X-RAY DIFFRACTIONf_chiral_restr0.055819
X-RAY DIFFRACTIONf_plane_restr0.007901
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.82050.34132180.32524520X-RAY DIFFRACTION96
1.8205-1.84190.32342420.30854393X-RAY DIFFRACTION94
1.8419-1.86440.33732330.28734457X-RAY DIFFRACTION96
1.8644-1.8880.31812410.26764469X-RAY DIFFRACTION95
1.888-1.91280.2822590.25314395X-RAY DIFFRACTION96
1.9128-1.9390.24292610.23444428X-RAY DIFFRACTION95
1.939-1.96670.271840.2244512X-RAY DIFFRACTION96
1.9667-1.99610.25272530.21644484X-RAY DIFFRACTION95
1.9961-2.02720.272470.21774437X-RAY DIFFRACTION95
2.0272-2.06050.2052520.2014390X-RAY DIFFRACTION95
2.0605-2.0960.2452290.20244440X-RAY DIFFRACTION95
2.096-2.13410.2472300.20454454X-RAY DIFFRACTION95
2.1341-2.17520.19382220.1914430X-RAY DIFFRACTION94
2.1752-2.21960.20732260.18714507X-RAY DIFFRACTION96
2.2196-2.26780.20442050.18394475X-RAY DIFFRACTION94
2.2678-2.32060.24742190.17484296X-RAY DIFFRACTION92
2.3206-2.37860.21822220.17394253X-RAY DIFFRACTION92
2.3786-2.44290.21471990.1754299X-RAY DIFFRACTION91
2.4429-2.51480.19672210.17414247X-RAY DIFFRACTION91
2.5148-2.5960.22012340.17264182X-RAY DIFFRACTION90
2.596-2.68870.22681890.17614242X-RAY DIFFRACTION89
2.6887-2.79640.20712250.17063998X-RAY DIFFRACTION87
2.7964-2.92360.20862160.17834026X-RAY DIFFRACTION86
2.9236-3.07770.23352220.18193980X-RAY DIFFRACTION86
3.0777-3.27050.21451970.18273764X-RAY DIFFRACTION81
3.2705-3.52290.19182300.17593807X-RAY DIFFRACTION82
3.5229-3.87730.18082120.15813734X-RAY DIFFRACTION81
3.8773-4.43790.14981880.14253928X-RAY DIFFRACTION83
4.4379-5.58960.15481910.14974131X-RAY DIFFRACTION88
5.5896-44.87230.20251980.17044310X-RAY DIFFRACTION92

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