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- PDB-8y6k: Cryo-EM structure of full-length MICAL1 in the autoinhibited state -

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Basic information

Entry
Database: PDB / ID: 8y6k
TitleCryo-EM structure of full-length MICAL1 in the autoinhibited state
Components[F-actin]-monooxygenase MICAL1
KeywordsOXIDOREDUCTASE / MICAL1 / Monooxygenase / F-actin disassembly / autoinhibition
Function / homology
Function and homology information


hippocampal mossy fiber expansion / : / F-actin monooxygenase / F-actin monooxygenase activity / NAD(P)H oxidase (H2O2-forming) / sulfur oxidation / regulation of regulated secretory pathway / NAD(P)H oxidase H2O2-forming activity / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen / actin filament depolymerization ...hippocampal mossy fiber expansion / : / F-actin monooxygenase / F-actin monooxygenase activity / NAD(P)H oxidase (H2O2-forming) / sulfur oxidation / regulation of regulated secretory pathway / NAD(P)H oxidase H2O2-forming activity / oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen / actin filament depolymerization / intermediate filament / actin filament bundle assembly / intercellular bridge / cytoskeleton organization / FAD binding / actin filament / monooxygenase activity / SH3 domain binding / small GTPase binding / actin filament binding / actin cytoskeleton / Factors involved in megakaryocyte development and platelet production / actin binding / midbody / endosome membrane / cilium / ciliary basal body / protein kinase binding / negative regulation of apoptotic process / signal transduction / nucleoplasm / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Mical, Rossman domain / bMERB domain / Bivalent Mical/EHBP Rab binding domain / bMERB domain profile. / DUF3585 / : / LIM zinc-binding domain signature. / LIM domain / Zinc-binding domain present in Lin-11, Isl-1, Mec-3. / Zinc finger, LIM-type ...Mical, Rossman domain / bMERB domain / Bivalent Mical/EHBP Rab binding domain / bMERB domain profile. / DUF3585 / : / LIM zinc-binding domain signature. / LIM domain / Zinc-binding domain present in Lin-11, Isl-1, Mec-3. / Zinc finger, LIM-type / LIM domain profile. / FAD-binding domain / FAD binding domain / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / FAD/NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / [F-actin]-monooxygenase MICAL1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.94 Å
AuthorsNiu, F. / Wei, Z.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971131 China
National Natural Science Foundation of China (NSFC)32170697 China
CitationJournal: Nat Commun / Year: 2024
Title: Autoinhibition and relief mechanisms for MICAL monooxygenases in F-actin disassembly.
Authors: Leishu Lin / Jiayuan Dong / Shun Xu / Jinman Xiao / Cong Yu / Fengfeng Niu / Zhiyi Wei /
Abstract: MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation ...MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.
History
DepositionFeb 2, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release
Revision 2.0Sep 25, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_residues / pdbx_validate_torsion / struct_conf / struct_conn / struct_ref_seq / struct_sheet_range
Item: _atom_site.auth_seq_id / _em_admin.last_update ..._atom_site.auth_seq_id / _em_admin.last_update / _pdbx_nonpoly_scheme.pdb_seq_num / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_validate_torsion.auth_seq_id / _struct_conf.beg_auth_seq_id / _struct_conf.end_auth_seq_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr2_auth_seq_id / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.end_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: [F-actin]-monooxygenase MICAL1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,9314
Polymers118,0151
Non-polymers9163
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein [F-actin]-monooxygenase MICAL1 / Molecule interacting with CasL protein 1 / MICAL-1 / NEDD9-interacting protein with calponin ...Molecule interacting with CasL protein 1 / MICAL-1 / NEDD9-interacting protein with calponin homology and LIM domains


Mass: 118014.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MICAL1, MICAL, NICAL / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
References: UniProt: Q8TDZ2, F-actin monooxygenase, NAD(P)H oxidase (H2O2-forming)
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: full-length MICAL1 protein / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.106 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 7.5
Details: 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM DTT.
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.1particle selection
2Topazparticle selection
3SerialEM3.7image acquisition
5CTFFIND4CTF correction
8Cootwincoot 0.9.4model fitting
10cryoSPARC3.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
13cryoSPARC3.13D reconstruction
14PHENIX1.18.2model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 9063162
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122086 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12BRY

2bry

12BRY1PDBexperimental model
22DK9

2dk9

12DK92PDBexperimental model
32CO8

2co8

12CO83PDBexperimental model
45LE0

5le0

15LE04PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056006
ELECTRON MICROSCOPYf_angle_d1.0468148
ELECTRON MICROSCOPYf_dihedral_angle_d5.742803
ELECTRON MICROSCOPYf_chiral_restr0.054892
ELECTRON MICROSCOPYf_plane_restr0.0081048

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