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- PDB-5ljv: MamK double helical filament -

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Basic information

Entry
Database: PDB / ID: 5ljv
TitleMamK double helical filament
ComponentsActin-like ATPase
KeywordsSTRUCTURAL PROTEIN / bacterial cytoskeleton / filamentous protein / actin-like / magnetosomes
Function / homology
Function and homology information


magnetosome assembly / magnetosome membrane / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / cytoskeleton / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
: / MreB/Mbl protein / ATPase, nucleotide binding domain / Actin family / Actin / ATPase, nucleotide binding domain / Nucleotidyltransferase; domain 5 / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin-like protein MamK
Similarity search - Component
Biological speciesMagnetospirillum magneticum AMB-1 (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.64 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 21, 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 / Data collection / Derived calculations
Category: em_software / pdbx_audit_support ...em_software / pdbx_audit_support / pdbx_struct_conn_angle / struct_conn
Item: _em_software.name / _pdbx_audit_support.funding_organization ..._em_software.name / _pdbx_audit_support.funding_organization / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_label_atom_id
Revision 1.3Oct 10, 2018Group: Data collection / Refinement description / Structure summary
Category: entity / refine ...entity / refine / refine_hist / refine_ls_restr / refine_ls_shell
Item: _entity.formula_weight / _refine_hist.pdbx_refine_id ..._entity.formula_weight / _refine_hist.pdbx_refine_id / _refine_ls_restr.pdbx_refine_id / _refine_ls_shell.pdbx_refine_id
Revision 1.4Oct 23, 2019Group: Data collection / Other / Structure summary / Category: atom_sites / cell / entity
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _entity.formula_weight
Revision 1.5May 15, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine / struct_conn / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine.ls_d_res_high / _refine.ls_d_res_low / _struct_conn.pdbx_dist_value / _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_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Actin-like ATPase
B: Actin-like ATPase
C: Actin-like ATPase
D: Actin-like ATPase
E: Actin-like ATPase
F: Actin-like ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,56218
Polymers225,8536
Non-polymers2,70912
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18420 Å2
ΔGint-140 kcal/mol
Surface area73420 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16B
26C
17B
27D
18B
28E
19B
29F
110C
210D
111C
211E
112C
212F
113D
213E
114D
214F
115E
215F

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: ASN / Beg label comp-ID: ASN / End auth comp-ID: PRO / End label comp-ID: PRO / Refine code: _ / Auth seq-ID: 10 - 334 / Label seq-ID: 10 - 334

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11AA
21BB
12AA
22CC
13AA
23DD
14AA
24EE
15AA
25FF
16BB
26CC
17BB
27DD
18BB
28EE
19BB
29FF
110CC
210DD
111CC
211EE
112CC
212FF
113DD
213EE
114DD
214FF
115EE
215FF

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

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Components

#1: Protein
Actin-like ATPase


Mass: 37642.152 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Magnetospirillum magneticum AMB-1 (bacteria)
Gene: amb0965 / Production host: Escherichia coli (E. coli) / References: UniProt: Q2W8Q6
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: MamK / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Magnetospirillum magneticum AMB-1 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pHis17
Buffer solutionpH: 7.4
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated defocus min: 1100 nm / Calibrated defocus max: 3000 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 40 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 1665
Image scansWidth: 4000 / Height: 4000 / Movie frames/image: 46

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Processing

SoftwareName: REFMAC / Version: 5.8.0137 / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 23.77 ° / Axial rise/subunit: 52.15 Å / Axial symmetry: C2
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 596427 / Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: R-factor
RefinementResolution: 3.64→3.64 Å / Cor.coef. Fo:Fc: 0.844 / SU B: 16.854 / SU ML: 0.242 / ESU R: 0.976
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.25066 --
obs0.25066 73184 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 108.731 Å2
Baniso -1Baniso -2Baniso -3
1-6.05 Å21.19 Å20 Å2
2--5.56 Å20.02 Å2
3----11.62 Å2
Refinement stepCycle: 1 / Total: 15000
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.030.01915258
ELECTRON MICROSCOPYr_bond_other_d0.010.0214976
ELECTRON MICROSCOPYr_angle_refined_deg2.865220736
ELECTRON MICROSCOPYr_angle_other_deg2.061334476
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.7451944
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.78224.078618
ELECTRON MICROSCOPYr_dihedral_angle_3_deg20.593152592
ELECTRON MICROSCOPYr_dihedral_angle_4_deg23.52415108
ELECTRON MICROSCOPYr_chiral_restr0.1550.22436
ELECTRON MICROSCOPYr_gen_planes_refined0.0140.02117070
ELECTRON MICROSCOPYr_gen_planes_other0.0070.023204
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it10.8038.8457794
ELECTRON MICROSCOPYr_mcbond_other10.7938.8457793
ELECTRON MICROSCOPYr_mcangle_it17.35613.2749732
ELECTRON MICROSCOPYr_mcangle_other17.35613.2759733
ELECTRON MICROSCOPYr_scbond_it25.24212.4377464
ELECTRON MICROSCOPYr_scbond_other25.2412.4367465
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other41.44817.2811005
ELECTRON MICROSCOPYr_long_range_B_refined47.41193.74362298
ELECTRON MICROSCOPYr_long_range_B_other47.41193.74362299
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11A43280
12B43280
21A43284
22C43284
31A43280
32D43280
41A43272
42E43272
51A43300
52F43300
61B43290
62C43290
71B43282
72D43282
81B43298
82E43298
91B43284
92F43284
101C43310
102D43310
111C43278
112E43278
121C43282
122F43282
131D43278
132E43278
141D43280
142F43280
151E43276
152F43276
LS refinement shellResolution: 3.65→3.745 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.88 5500 -
Rfree-0 -
obs--100 %

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