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- EMDB-42511: Structural and biochemical investigations of a HEAT-repeat protei... -

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Basic information

Entry
Database: EMDB / ID: EMD-42511
TitleStructural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway
Map dataPrimary Map (z-flipped)
Sample
  • Complex: QUATERNARY COMPLEX OF MET18 TETRAMER
    • Protein or peptide: DNA repair/transcription protein MET18/MMS19
KeywordsIRON-SULFUR CLUSTER / METALLOCOFACTOR / ASSEMBLY / METAL TRANSPORT
Function / homology
Function and homology information


cytosolic [4Fe-4S] assembly targeting complex / protein maturation by iron-sulfur cluster transfer / DNA metabolic process / iron-sulfur cluster assembly / response to UV / DNA repair / nucleus / cytosol / cytoplasm
Similarity search - Function
MMS19, C-terminal / MMS19, N-terminal / DNA repair/transcription protein MET18/MMS19 / RNAPII transcription regulator C-terminal / Dos2-interacting transcription regulator of RNA-Pol-II / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
DNA repair/transcription protein MET18/MMS19
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.77 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_42511.png

Downloads & links

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Map

FileDownload / File: emd_42511.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary Map (z-flipped)
Projections & slices

Image control

Size
Brightness
Contrast
Others
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5998 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.36368498 - 1.2943708
Average (Standard dev.)-0.0009510647 (±0.07864057)
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: Half map 1

Fileemd_42511_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42511_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: DNA repair/transcription protein 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 TETRAMER IMAGED AND SOLVED BY CRYO-EM.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288C
Molecular weightTheoretical: 118 kDa/nm

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Macromolecule #1: DNA repair/transcription protein MET18/MMS19

MacromoleculeName: DNA repair/transcription protein MET18/MMS19 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288C
Molecular weightTheoretical: 118.007078 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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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
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.039 kPa
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 30104
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
Output model

PDB-8usq:
Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway

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