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Yorodumi- EMDB-42510: Structural and biochemical investigations of a HEAT-repeat protei... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-42510 | |||||||||||||||
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Title | Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway | |||||||||||||||
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Sample |
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Keywords | IRON-SULFUR CLUSTER / ASSEMBLY / CIA PATHWAY / MET18 / METAL TRANSPORT | |||||||||||||||
Function / homology | Function and homology information cytosolic [4Fe-4S] assembly targeting complex / protein maturation by iron-sulfur cluster transfer / iron-sulfur cluster assembly / DNA metabolic process / response to UV / : / DNA repair / nucleus / cytoplasm / cytosol Similarity search - Function | |||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||
Authors | Vasquez S / Drennan CL | |||||||||||||||
Funding support | United States, 4 items
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Citation | Journal: Commun Biol / Year: 2023 Title: Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway. Authors: Sheena Vasquez / Melissa D Marquez / Edward J Brignole / Amanda Vo / Sunnie Kong / Christopher Park / Deborah L Perlstein / Catherine L Drennan / Abstract: Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the ...Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed. | |||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_42510.map.gz | 10.3 MB | EMDB map data format | |
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Header (meta data) | emd-42510-v30.xml emd-42510.xml | 15.6 KB 15.6 KB | Display Display | EMDB header |
Images | emd_42510.png | 118.4 KB | ||
Filedesc metadata | emd-42510.cif.gz | 6.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-42510 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-42510 | HTTPS FTP |
-Validation report
Summary document | emd_42510_validation.pdf.gz | 386.4 KB | Display | EMDB validaton report |
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Full document | emd_42510_full_validation.pdf.gz | 386 KB | Display | |
Data in XML | emd_42510_validation.xml.gz | 6.7 KB | Display | |
Data in CIF | emd_42510_validation.cif.gz | 7.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-42510 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-42510 | HTTPS FTP |
-Related structure data
Related structure data | 8uspMC 8usqC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_42510.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.059 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX.
Entire | Name: QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX. |
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Components |
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-Supramolecule #1: QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX.
Supramolecule | Name: QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288C |
Molecular weight | Theoretical: 118 kDa/nm |
-Macromolecule #1: DNA repair/transcription protein MET18/MMS19
Macromolecule | Name: DNA repair/transcription protein MET18/MMS19 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288C |
Molecular weight | Theoretical: 118.007078 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTPDELNSAV VTFMANLNID DSKANETAST VTDSIVHRSI KLLEVVVALK DYFLSENEVE RKKALTCLTT ILAKTPKDHL SKNECSVIF QFYQSKLDDQ ALAKEVLEGF AALAPMKYVS INEIAQLLRL LLDNYQQGQH LASTRLWPFK ILRKIFDRFF V NGSSTEQV ...String: MTPDELNSAV VTFMANLNID DSKANETAST VTDSIVHRSI KLLEVVVALK DYFLSENEVE RKKALTCLTT ILAKTPKDHL SKNECSVIF QFYQSKLDDQ ALAKEVLEGF AALAPMKYVS INEIAQLLRL LLDNYQQGQH LASTRLWPFK ILRKIFDRFF V NGSSTEQV KRINDLFIET FLHVANGEKD PRNLLLSFAL NKSITSSLQN VENFKEDLFD VLFCYFPITF KPPKHDPYKI SN QDLKTAL RSAITATPLF AEDAYSNLLD KLTASSPVVK NDTLLTLLEC VRKFGGSSIL ENWTLLWNAL KFEIMQNSEG NEN TLLNPY NKDQQSDDVG QYTNYDACLK IINLMALQLY NFDKVSFEKF FTHVLDELKP NFKYEKDLKQ TCQILSAIGS GNVE IFNKV ISSTFPLFLI NTSEVAKLKL LIMNFSFFVD SYIDLFGRTS KESLGTPVPN NKMAEYKDEI IMILSMALTR SSKAE VTIR TLSVIQFTKM IKMKGFLTPE EVSLIIQYFT EEILTDNNKN IYYACLEGLK TISEIYEDLV FEISLKKLLD LLPDCF EEK IRVNDEENIH IETILKIILD FTTSRHILVK ESITFLATKL NRVAKISKSR EYCFLLISTI YSLFNNNNQN ENVLNEE DA LALKNAIEPK LFEIITQESA IVSDNYNLTL LSNVLFFTNL KIPQAAHQEE LDRYNELFIS EGKIRILDTP NVLAISYA K ILSALNKNCQ FPQKFTVLFG TVQLLKKHAP RMTETEKLGY LELLLVLSNK FVSEKDVIGL FDWKDLSVIN LEVMVWLTK GLIMQNSLES SEIAKKFIDL LSNEEIGSLV SKLFEVFVMD ISSLKKFKGI SWNNNVKILY KQKFFGDIFQ TLVSNYKNTV DMTIKCNYL TALSLVLKHT PSQSVGPFIN DLFPLLLQAL DMPDPEVRVS ALETLKDTTD KHHTLITEHV STIVPLLLSL S LPHKYNSV SVRLIALQLL EMITTVVPLN YCLSYQDDVL SALIPVLSDK KRIIRKQCVD TRQVYYELGQ IPFE UniProtKB: DNA repair/transcription protein MET18/MMS19 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.20 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Pressure: 0.039 kPa Details: -15 mA Electron Microscopy Science Quantifoil 1.2/1.3 Cu 300 mesh | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK I |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.59 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Software | Name: PHENIX |
Refinement | Protocol: RIGID BODY FIT |
Output model | PDB-8usp: |