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- PDB-6u96: Actin phalloidin at BeFx state -

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Basic information

Entry
Database: PDB / ID: 6u96
TitleActin phalloidin at BeFx state
Components
  • Actin, alpha skeletal muscle
  • PHALLOIDIN Derivative
KeywordsSTRUCTURAL PROTEIN / Actin / phalloidin / beryllium fluoride
Function / homology
Function and homology information


cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament ...cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDas, S. / Ge, P. / Durer, Z.A.O. / Grintsevich, E.E. / Zhou, Z.H. / Reisler, E.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM077190 United States
National Science Foundation (NSF, United States)XSEDE MCB140140 United States
CitationJournal: Structure / Year: 2020
Title: D-loop Dynamics and Near-Atomic-Resolution Cryo-EM Structure of Phalloidin-Bound F-Actin.
Authors: Sanchaita Das / Peng Ge / Zeynep A Oztug Durer / Elena E Grintsevich / Z Hong Zhou / Emil Reisler /
Abstract: Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies ...Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics.
History
DepositionSep 6, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0May 13, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 13, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 20, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Apr 2, 2025Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_value_order / _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 1.1Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: database_2 / em_admin / Data content type: EM metadata / EM metadata / EM metadata
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Actin, alpha skeletal muscle
B: Actin, alpha skeletal muscle
C: Actin, alpha skeletal muscle
D: Actin, alpha skeletal muscle
E: Actin, alpha skeletal muscle
F: PHALLOIDIN Derivative
G: PHALLOIDIN Derivative
H: PHALLOIDIN Derivative
I: PHALLOIDIN Derivative
J: PHALLOIDIN Derivative
hetero molecules


Theoretical massNumber of molelcules
Total (without water)216,73015
Polymers214,59410
Non-polymers2,1365
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 42109.973 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Details: ACTA1, ACTA / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135
#2: Protein/peptide
PHALLOIDIN Derivative


Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Actin phalloidin, BeFx state / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Buffer solutionpH: 7.4
Details: pH7.4, 10mM Tris, 50mM KCl, 1 mM MgC2, 0.2mM CaCl2, 1mM ATP, 1mM DTT, 0.2mM EGTA, 0.2mM BeCl2, 5mM NaF
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES1
2130 mMSaltNaCl1
30.2 mMCalcium ChlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 3000 nm / Calibrated defocus min: 2200 nm / Calibrated defocus max: 4200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 81 K / Temperature (min): 80 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5874
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 32 / Used frames/image: 3-12

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN1.8particle selection
2Leginon3.2image acquisition
4RELION3CTF correction
9PHENIX1.13model refinement
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.967625 ° / Axial rise/subunit: 29.508839 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 214555
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181626 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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