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- EMDB-49495: Thermothelomyces thermophilus SAM complex open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-49495
TitleThermothelomyces thermophilus SAM complex open conformation
Map datanon-sharpened map SAM complex open conformation
Sample
  • Complex: Thermothelomyces thermophilus SAM complex open conformation
    • Protein or peptide: Sam35
    • Protein or peptide: Sam50
    • Protein or peptide: Sam37
  • Ligand: ERGOSTEROL
KeywordsMembrane protein / beta-barrel / sorting and assembly machinery
Function / homology
Function and homology information


SAM complex / protein insertion into mitochondrial outer membrane / mitochondrion organization / protein transport / mitochondrial outer membrane
Similarity search - Function
Metaxin, glutathione S-transferase domain / Glutathione S-transferase, C-terminal domain / Glutathione S-transferase N-terminal domain / Mitochondrial outer membrane transport complex Sam37/metaxin, N-terminal domain / : / Outer mitochondrial membrane transport complex protein / Thioredoxin-like fold / Surface antigen D15-like / Bacterial surface antigen (D15) / Omp85 superfamily domain
Similarity search - Domain/homology
Mitochondrial outer membrane transport complex Sam37/metaxin N-terminal domain-containing protein / Thioredoxin-like fold domain-containing protein / Bacterial surface antigen (D15) domain-containing protein
Similarity search - Component
Biological speciesThermothelomyces thermophilus (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsDiederichs K / Botos I / Buchanan SK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)ZIA DK036139 United States
CitationJournal: Nat Commun / Year: 2025
Title: The dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A.
Authors: Kathryn A Diederichs / Istvan Botos / Scout Hayashi / Gvantsa Gutishvili / Vadim Kotov / Katie Kuo / Akira Iinishi / Gwendolyn Cooper / Benjamin Schwarz / Herve Celia / Thomas C Marlovits / ...Authors: Kathryn A Diederichs / Istvan Botos / Scout Hayashi / Gvantsa Gutishvili / Vadim Kotov / Katie Kuo / Akira Iinishi / Gwendolyn Cooper / Benjamin Schwarz / Herve Celia / Thomas C Marlovits / Kim Lewis / James C Gumbart / Joseph A Mindell / Susan K Buchanan /
Abstract: The folding and insertion of β-barrel proteins into the mitochondrial outer membrane is facilitated by the sorting and assembly machinery (SAM) complex. Here we report two 2.8 Å cryo-EM ...The folding and insertion of β-barrel proteins into the mitochondrial outer membrane is facilitated by the sorting and assembly machinery (SAM) complex. Here we report two 2.8 Å cryo-EM structures of the Thermothelomyces thermophilus SAM complex in the absence of substrate in which the Sam50 lateral gate adopts two different conformations: the first is a closed lateral gate as observed in previously published structures, while the second contains a Sam50 with the first four β-strands rotated outwards by approximately 45°, resulting in an open lateral gate. The observed monomeric open conformation contrasts our previous work where the open conformation was adopted by non-physiological up-down dimers. To understand how these lateral gate dynamics are influenced by substrate, we studied the interaction of the SAM complex with a β-signal peptide mimic, darobactin A. Darobactin A binds to the SAM complex with nanomolar affinity and inhibits the import and assembly of mitochondrial β-barrel proteins in vitro. Lastly, we solved a 3.0 Å cryo-EM structure of the Thermothelomyces thermophilus SAM complex bound to darobactin A, which reveals that darobactin A stabilizes the Sam50 lateral gate similar to the open conformation by binding to strand β1, therefore blocking β-barrel biogenesis.
History
DepositionFeb 28, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 3, 2025-
Current statusDec 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49495.png

Downloads & links

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Map

FileDownload / File: emd_49495.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnon-sharpened map SAM complex open conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 296.64 Å		0.82 Å/pix.
x 360 pix.
= 296.64 Å		0.82 Å/pix.
x 360 pix.
= 296.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.824 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.28274375 - 0.5543433
Average (Standard dev.)0.0004475086 (±0.012734168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 296.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half-map A SAM complex open conformation

Fileemd_49495_half_map_1.map
Annotationhalf-map A SAM complex open conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map B SAM complex open conformation

Fileemd_49495_half_map_2.map
Annotationhalf-map B SAM complex open conformation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermothelomyces thermophilus SAM complex open conformation

EntireName: Thermothelomyces thermophilus SAM complex open conformation
Components
  • Complex: Thermothelomyces thermophilus SAM complex open conformation
    • Protein or peptide: Sam35
    • Protein or peptide: Sam50
    • Protein or peptide: Sam37
  • Ligand: ERGOSTEROL

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Supramolecule #1: Thermothelomyces thermophilus SAM complex open conformation

SupramoleculeName: Thermothelomyces thermophilus SAM complex open conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Thermothelomyces thermophilus (fungus)

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Macromolecule #1: Sam35

MacromoleculeName: Sam35 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus (fungus)
Molecular weightTheoretical: 36.88268 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSASIPSAAP SWRKMQIPRP LQRLFDYFPL RIYEPNELPE RSQQLTSGDL PTLYVFSTDS DARLGLPSFN PGCLKWQTLL RLANLDFRI LPSTNHSSPT GSLPFLLPPR TSPTASPAPI PASGLLSFAR KNPWRPGKAA DLDLGHLDAD LPPRAQAYLA L ITHSLRNA ...String:
MSASIPSAAP SWRKMQIPRP LQRLFDYFPL RIYEPNELPE RSQQLTSGDL PTLYVFSTDS DARLGLPSFN PGCLKWQTLL RLANLDFRI LPSTNHSSPT GSLPFLLPPR TSPTASPAPI PASGLLSFAR KNPWRPGKAA DLDLGHLDAD LPPRAQAYLA L ITHSLRNA WLCALYLDPT HDALLRRLYV DPASSSRAVR AALLHQLRRA AAEQVATASS GGGKIVSLAP VDSADGIDEE AV YRSARDA LDALASLLRE SETAWFFGTE RPGSFDAALF SYTHLMVEYM SEEEDTESAK GRVSLGRMVK EAGNGELAEH RER MLGVAW PEWDGYRR

UniProtKB: Thioredoxin-like fold domain-containing protein

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Macromolecule #2: Sam50

MacromoleculeName: Sam50 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus (fungus)
Molecular weightTheoretical: 55.875574 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSHHHHHHHH HHGSENLYFQ SASSLGFGGS NAVDKVNATT TPGTVATPNS GPTKMLDEHI LTPASISTLE VHGATNTRRS LLDQIFKPV LEDTAAAGTT LGQVLDRVGA ATKKLARFDI FKEEGFGVFL SEAAPPQSAP PTDRTDLDIS IRVKEKSRLV F SAGTDFGN ...String:
MSHHHHHHHH HHGSENLYFQ SASSLGFGGS NAVDKVNATT TPGTVATPNS GPTKMLDEHI LTPASISTLE VHGATNTRRS LLDQIFKPV LEDTAAAGTT LGQVLDRVGA ATKKLARFDI FKEEGFGVFL SEAAPPQSAP PTDRTDLDIS IRVKEKSRLV F SAGTDFGN AEGSAYTNAV VRNIFGGAET LTVNASTGTR TRSAYNATFS TPINGNPDLR LSVEALRSAT QKPWASHEEH LT GANLRLA WLTEKGDTHA LAYSSVWRQL TGLAPTASPT VRADAGDSLK SSLTHTFTRD RRDNPMLPQS GYLFRSVSEL AGW GPLNGD VSFAKTEVEA SGALPVAIPG LAGKSGVSVG GGLRLGVLYP LPLGYSLTGA AQPSRINDRF QLGGPNDVRG FKIG GLGPH DGVDAVGGDV FAAGSVNALL PLPRTGPDSP LRLQLYANAG RLVALNSKGT DKEGKEGLAM DSAAVFKGVK SAVGK LTNG IPSLAAGVGL VYAHPVARFE LNFSLPLVLR RGEEGRKGLQ VGVGISFL

UniProtKB: Bacterial surface antigen (D15) domain-containing protein

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Macromolecule #3: Sam37

MacromoleculeName: Sam37 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothelomyces thermophilus (fungus)
Molecular weightTheoretical: 50.122418 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MSSAWSHPQF EKGGGSGGGS GGSAWSHPQF EKENLYFQGG AVQLHVWGPA FGLPSIDAEC LAAIAYLAQT LGSADYQLIQ SSPSAVPTQ HLPTLYDSRT STWIGGFTSI TAHLHTHPPP TFQSAPQPTD GSSSTTTTTT TTTTAASATA DGTAYTAFLS A HAAPLLAL ...String:
MSSAWSHPQF EKGGGSGGGS GGSAWSHPQF EKENLYFQGG AVQLHVWGPA FGLPSIDAEC LAAIAYLAQT LGSADYQLIQ SSPSAVPTQ HLPTLYDSRT STWIGGFTSI TAHLHTHPPP TFQSAPQPTD GSSSTTTTTT TTTTAASATA DGTAYTAFLS A HAAPLLAL SLYVSSANYG AATRPAYSAV LPLPLPWTEP PAVRAAMARR AAHLGLSSLD ADAAAERARA EERRAAADGW VA VPPHATA GRAAGGGGGG GGGGGKGGGV AAVLTPEQKS RIRLEEAARE VLDVLAEVDW AAGGGGRQVA AEVRCLAFGY LAL MLLPDV PRPWLREIME GRYPALCTFV RDFRARVFPQ GGKLLPWADG GAQASASASA SASAVALRFV RAVMAEVPLV GEWW SRWWT ARKKREVLAS KGAKPAPSND LLLLLGAGLG LTVVGAGVFF YRGLPPFGEA VQVWRKPVVG LSSFGAAGAM FSGAL YGLD

UniProtKB: Mitochondrial outer membrane transport complex Sam37/metaxin N-terminal domain-containing protein

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Macromolecule #4: ERGOSTEROL

MacromoleculeName: ERGOSTEROL / type: ligand / ID: 4 / Number of copies: 12 / Formula: ERG
Molecular weightTheoretical: 396.648 Da
Chemical component information

ChemComp-ERG:
ERGOSTEROL

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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

BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 229346
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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