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- EMDB-42514: Structural and biochemical investigations of a HEAT-repeat protei... -

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Basic information

Entry
Database: EMDB / ID: EMD-42514
TitleStructural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway
Map data
Sample
  • Complex: QUATERNARY COMPLEX OF MET18 TETRAMER
    • Protein or peptide: Met18/MMS19
KeywordsIRON-SULFUR CLUSTER / METALLOCOFACTOR / ASSEMBLY / METAL TRANSPORT
Biological speciesSaccharomyces (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.43 Å
AuthorsVasquez S / Drennan CL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121673 United States
National Science Foundation (NSF, United States)DGE-1247312 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM007287 United States
CitationJournal: 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
DepositionOct 29, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42514.png

Downloads & links

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Map

FileDownload / File: emd_42514.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.6 Å/pix.
x 270 pix.
= 431.946 Å		1.6 Å/pix.
x 270 pix.
= 431.946 Å		1.6 Å/pix.
x 270 pix.
= 431.946 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5998 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.6
Minimum - Maximum-11.337039000000001 - 13.036395000000001
Average (Standard dev.)0.00009884565 (±0.40186584)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 431.94598 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42514_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_42514_half_map_2.map
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Sample components

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Entire : QUATERNARY COMPLEX OF MET18 TETRAMER

EntireName: QUATERNARY COMPLEX OF MET18 TETRAMER
Components
  • Complex: QUATERNARY COMPLEX OF MET18 TETRAMER
    • Protein or peptide: Met18/MMS19

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Supramolecule #1: QUATERNARY COMPLEX OF MET18 TETRAMER

SupramoleculeName: QUATERNARY COMPLEX OF MET18 TETRAMER / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: MET18 HEXAMER IMAGED AND SOLVED BY CRYO-EM.
Source (natural)Organism: Saccharomyces (fungus)
Molecular weightTheoretical: 118 kDa/nm

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Macromolecule #1: Met18/MMS19

MacromoleculeName: Met18/MMS19 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AVVTFMANLN IDDSKANETA STVTDSIVHR SIKLLEVVVA LKDYFLSENE VERKKALTCL TTILAKTPKD HLSKNECSV IFQFYQSKLD DQALAKEVLE GFAALAPMKY VSINEIAQLL RLLLDNYQQG QHLASTRLWP F KILRKIFD RFFVNGSSTE QVKRINDLFI ...String:
AVVTFMANLN IDDSKANETA STVTDSIVHR SIKLLEVVVA LKDYFLSENE VERKKALTCL TTILAKTPKD HLSKNECSV IFQFYQSKLD DQALAKEVLE GFAALAPMKY VSINEIAQLL RLLLDNYQQG QHLASTRLWP F KILRKIFD RFFVNGSSTE QVKRINDLFI ETFLHVANGE KDPRNLLLSF ALNKSITSSL QNVENAKEDL FD VLFCYFA ALKTALRSAI TATPLFAEDA YSNLLDKLTA SSPVVKNDTL LTLLECVRKF GGSSILENWT LLW NALKFE IMQNYTNYDA CLKIINLMAL QLYNFDKVSF EKFFTHVLDE LKPNFKYEKD LKQTCQILSA IGSG NVEIF NKVISSTFPL FLINTSEVAK LKLLIMNFSF FVDSYIDLFG RTSKESLGTP VPNNKMAEYK DEIIM ILSM ALTRSSKAEV TIRTLSVIQF TKMIKMKGFL TPEEVSLIIQ YFTEEILTDN NKNIYYACLE GLKTIS EIY EDLVFEISLK KLLDLLPDCF EEKIRVNDEE NIHIETILKI ILDFTTSRHI LVKESITFLA TKLNRVA KI SKSREYCFLL ISTIYSLFNN NNQNENVLNE EDALALKNAI EPKLFEIITQ ESAIVSDNYN LTLLSNVL F FTNLKIPQAA HQEELDRYNE LFISEGKIRI LDTPNVLAIS YAKILSALNK NCQFPQKFTV LFGTVQLLK KHAPRMTETE KLGYLELLLV LSNKFVSEKD VIGLFDWKDL SVINLEVMVW LTKGLIMQNS LESSEIAKKF IDLLSNEEI GSLVSKLFEV FVMDISSLKK FKGISWNNNV KILYKQKFFG DIFQTLVSNY KNTVDMTIKC N YLTALSLV LKHTPSQSVG PFINDLFPLL LQALDMPDPE VRVSALETLK DTTDKHHTLI TEHVSTIVPL LL SLSLPHK YNSVSVRLIA LQLLEMITTV VPLNYCLSYQ DDVLSALIPV LSDKKRIIRK QCVDTRQVYY ELG QI

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMTris-HCl
100.0 mMNaCl
1.0 %Glycerol
5.0 mMBetamercaptoethanol
VitrificationCryogen name: ETHANE / Chamber humidity: 77 % / Chamber temperature: 297.15 K / Details: SAMPLE WAS PREPARED ON THE CHAMELEON.

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
SoftwareName: EPU
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 53.47 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 92000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.4 µm / Nominal defocus min: 1.3 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 139569
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 36582
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 30000 / Software - Name: cryoSPARC
Details: Classes were made by asking for 3 ab initio maps during data processing

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Atomic model buiding 1

Initial modelPDB ID:

6tc0


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: PHENIX
RefinementSpace: REAL / Protocol: AB INITIO MODEL

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