+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-30179 | |||||||||
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Title | Phalloidin bound F-actin complex | |||||||||
Map data | phalloidin stabilised F-actin | |||||||||
Sample |
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Function / homology | Function and homology information Striated Muscle Contraction / skeletal muscle thin filament assembly / striated muscle thin filament / stress fiber / skeletal muscle fiber development / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / toxin activity / hydrolase activity / ATP binding Similarity search - Function | |||||||||
Biological species | Gallus gallus (chicken) / Chicken (chicken) / Amanita phalloides (death cap) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | Kumari A / Ragunath VK / Sirajuddin M | |||||||||
Funding support | India, 2 items
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Citation | Journal: EMBO J / Year: 2020 Title: Structural insights into actin filament recognition by commonly used cellular actin markers. Authors: Archana Kumari / Shubham Kesarwani / Manjunath G Javoor / Kutti R Vinothkumar / Minhajuddin Sirajuddin / Abstract: Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely ...Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_30179.map.gz | 60 MB | EMDB map data format | |
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Header (meta data) | emd-30179-v30.xml emd-30179.xml | 15.9 KB 15.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_30179_fsc.xml | 9.1 KB | Display | FSC data file |
Images | emd_30179.png | 41.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-30179 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-30179 | HTTPS FTP |
-Validation report
Summary document | emd_30179_validation.pdf.gz | 521.7 KB | Display | EMDB validaton report |
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Full document | emd_30179_full_validation.pdf.gz | 521.2 KB | Display | |
Data in XML | emd_30179_validation.xml.gz | 10.9 KB | Display | |
Data in CIF | emd_30179_validation.cif.gz | 14.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30179 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30179 | HTTPS FTP |
-Related structure data
Related structure data | 7btiMC 6m5gC 7bt7C 7bteC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_30179.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | phalloidin stabilised F-actin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.38 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Filamentous actin in ADP state
Entire | Name: Filamentous actin in ADP state |
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Components |
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-Supramolecule #1: Filamentous actin in ADP state
Supramolecule | Name: Filamentous actin in ADP state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Gallus gallus (chicken) |
-Macromolecule #1: Actin, alpha skeletal muscle
Macromolecule | Name: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Chicken (chicken) |
Molecular weight | Theoretical: 42.096953 KDa |
Sequence | String: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY ...String: MCDEDETTAL VCDNGSGLVK AGFAGDDAPR AVFPSIVGRP RHQGVMVGMG QKDSYVGDEA QSKRGILTLK YPIEHGIITN WDDMEKIWH HTFYNELRVA PEEHPTLLTE APLNPKANRE KMTQIMFETF NVPAMYVAIQ AVLSLYASGR TTGIVLDSGD G VTHNVPIY EGYALPHAIM RLDLAGRDLT DYLMKILTER GYSFVTTAER EIVRDIKEKL CYVALDFENE MATAASSSSL EK SYELPDG QVITIGNERF RCPETLFQPS FIGMESAGIH ETTYNSIMKC DIDIRKDLYA NNVMSGGTTM YPGIADRMQK EIT ALAPST MKIKIIAPPE RKYSVWIGGS ILASLSTFQQ MWITKQEYDE AGPSIVHRKC F |
-Macromolecule #2: Phalloidin
Macromolecule | Name: Phalloidin / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Amanita phalloides (death cap) |
Molecular weight | Theoretical: 808.899 Da |
Sequence | String: (HYP)AW(G5G)A(ALO)C |
-Macromolecule #3: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 5 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 0.0002 mg/mL |
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Buffer | pH: 7.5 Details: 50mM KCl,1mM MgCl2,0.2mM EGTA, 10mM Imidazole buffer pH 7.5 |
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK I / Details: blot for 3.5 seconds. |
Details | 10 mole excess of lifeact was mixed with F-actin and used for sample preparation. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 100.0 K / Max: 120.0 K |
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 529 / Average exposure time: 2.0 sec. / Average electron dose: 49.2 e/Å2 / Details: 30 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated magnification: 75000 / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |