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- EMDB-21005: Negative stain EM map of CODH/ACS in the closed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-21005
TitleNegative stain EM map of CODH/ACS in the closed conformation
Map dataNegative stain map of CODH/ACS in the closed conformation
Sample
  • Complex: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer
    • Protein or peptide: Carbon monoxide dehdyrogenase
    • Protein or peptide: Acetyl-CoA synthase
Biological speciesMoorella thermoacetica (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 44.8 Å
AuthorsCohen SE / Brignole EJ / Drennan CL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM069857 United States
National Institutes of Health/National Institute of General Medical SciencesGM039451 United States
National Institutes of Health/National Institute of General Medical SciencesGM126982 United States
National Institutes of Health/National Institute of General Medical SciencesGM008334 United States
CitationJournal: Structure / Year: 2021
Title: Negative-Stain Electron Microscopy Reveals Dramatic Structural Rearrangements in Ni-Fe-S-Dependent Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase.
Authors: Steven E Cohen / Edward J Brignole / Elizabeth C Wittenborn / Mehmet Can / Samuel Thompson / Stephen W Ragsdale / Catherine L Drennan /
Abstract: The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH), and CoA. To accomplish this feat, ACS must bind CoA and ...The Ni-Fe-S-containing A-cluster of acetyl-coenzyme A (CoA) synthase (ACS) assembles acetyl-CoA from carbon monoxide (CO), a methyl group (CH), and CoA. To accomplish this feat, ACS must bind CoA and interact with two other proteins that contribute the CO and CH, respectively: CO dehydrogenase (CODH) and corrinoid Fe-S protein (CFeSP). Previous structural data show that, in the model acetogen Moorella thermoacetica, domain 1 of ACS binds to CODH such that a 70-Å-long internal channel is created that allows CO to travel from CODH to the A-cluster. The A-cluster is largely buried and is inaccessible to CFeSP for methylation. Here we use electron microscopy to capture multiple snapshots of ACS that reveal previously uncharacterized domain motion, forming extended and hyperextended structural states. In these structural states, the A-cluster is accessible for methylation by CFeSP.
History
DepositionNov 19, 2019-
Header (metadata) releaseDec 18, 2019-
Map releaseSep 2, 2020-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.89
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.89
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21005.png

Downloads & links

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Map

FileDownload / File: emd_21005.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain map of CODH/ACS in the closed conformation
Voxel sizeX=Y=Z: 7.16 Å
Density
Contour LevelBy AUTHOR: 4.89 / Movie #1: 4.89
Minimum - Maximum-5.11331 - 10.716523
Average (Standard dev.)0.032466616 (±0.6741924)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 458.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.167.167.16
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z458.240458.240458.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-5.11310.7170.032

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Supplemental data

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Sample components

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Entire : carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer

EntireName: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer
Components
  • Complex: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer
    • Protein or peptide: Carbon monoxide dehdyrogenase
    • Protein or peptide: Acetyl-CoA synthase

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Supramolecule #1: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer

SupramoleculeName: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Moorella thermoacetica (bacteria)

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Macromolecule #1: Carbon monoxide dehdyrogenase

MacromoleculeName: Carbon monoxide dehdyrogenase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: anaerobic carbon monoxide dehydrogenase
Source (natural)Organism: Moorella thermoacetica (bacteria)
SequenceString: MPRFRDLSHN CRPSEAPRVM EPKNRDRTVD PAVLEMLVKS KDDKVITAFD RFVAQQPQCK IGYEGICCR FCMAGPCRIK ATDGPGSRGI CGASAWTIVA RNVGLMILTG AAAHCEHGNH I AHALVEMA EGKAPDYSVK DEAKLKEVCR RVGIEVEGKS VLELAQEVGE ...String:
MPRFRDLSHN CRPSEAPRVM EPKNRDRTVD PAVLEMLVKS KDDKVITAFD RFVAQQPQCK IGYEGICCR FCMAGPCRIK ATDGPGSRGI CGASAWTIVA RNVGLMILTG AAAHCEHGNH I AHALVEMA EGKAPDYSVK DEAKLKEVCR RVGIEVEGKS VLELAQEVGE KALEDFRRLK GE GEATWLM TTINEGRKEK FRTHNVVPFG IHASISELVN QAHMGMDNDP VNLVFSAIRV ALA DYTGEH IATDFSDILF GTPQPVVSEA NMGVLDPDQV NFVLHGHNPL LSEIIVQAAR EMEG EAKAA GAKGINLVGI CCTGNEVLMR QGIPLVTSFA SQELAICTGA IDAMCVDVQC IMPSI SAVA ECYHTRIITT ADNAKIPGAY HIDYQTATAI ESAKTAIRMA IEAFKERKES NRPVYI PQI KNRVVAGWSL EALTKLLATQ NAQNPIRVLN QAILDGELAG VALICGCNNL KGFQDNS HL TVMKELLKNN VFVVATGCSA QAAGKLGLLD PANVETYCGD GLKGFLKRLG EGANIEIG L PPVFHMGSCV DNSRAVDLLM AMANDLGVDT PKVPFVASAP EAMSGKAAAI GTWWVSLGV PTHVGTMPPV EGSDLIYSIL TQIASDVYGG YFIFEMDPQV AARKILDALE YRTWKLGVHK EVAERYETK LCQGY

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Macromolecule #2: Acetyl-CoA synthase

MacromoleculeName: Acetyl-CoA synthase / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: CO-methylating acetyl-CoA synthase
Source (natural)Organism: Moorella thermoacetica (bacteria)
SequenceString: MTDFDKIFEG AIPEGKEPVA LFREVYHGAI TATSYAEILL NQAIRTYGPD HPVGYPDTAY YLPVIRCFS GEEVKKLGDL PPILNRKRAQ VSPVLNFENA RLAGEATWYA AEIIEALRYL K YKPDEPLL PPPWTGFIGD PVVRRFGIKM VDWTIPGEAI ILGRAKDSKA ...String:
MTDFDKIFEG AIPEGKEPVA LFREVYHGAI TATSYAEILL NQAIRTYGPD HPVGYPDTAY YLPVIRCFS GEEVKKLGDL PPILNRKRAQ VSPVLNFENA RLAGEATWYA AEIIEALRYL K YKPDEPLL PPPWTGFIGD PVVRRFGIKM VDWTIPGEAI ILGRAKDSKA LAKIVKELMG MG FMLFICD EAVEQLLEEN VKLGIDYIAY PLGNFTQIVH AANYALRAGM MFGGVTPGAR EEQ RDYQRR RIRAFVLYLG EHDMVKTAAA FGAIFTGFPV ITDQPLPEDK QIPDWFFSVE DYDK IVQIA METRGIKLTK IKLDLPINFG PAFEGESIRK GDMYVEMGGN RTPAFELVRT VSESE ITDG KIEVIGPDID QIPEGSKLPL GILVDIYGRK MQADFEGVLE RRIHDFINYG EGLWHT GQR NINWLRVSKD AVAKGFRFKN YGEILVAKMK EEFPAIVDRV QVTIFTDEAK VKEYMEV AR EKYKERDDRM RGLTDETVDT FYSCVLCQSF APNHVCIVTP ERVGLCGAVS WLDAKASY E INHAGPNQPI PKEGEIDPIK GIWKSVNDYL YTASNRNLEQ VCLYTLMENP MTSCGCFEA IMAILPECNG IMITTRDHAG MTPSGMTFST LAGMIGGGTQ TPGFMGIGRT YIVSKKFISA DGGIARIVW MPKSLKDFLH DEFVRRSVEE GLGEDFIDKI ADETIGTTVD EILPYLEEKG H PALTMDPI M

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration.017 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
0.1 MNaClsodium chloride
0.05 MC4H11NO3tris(hydroxymethyl)aminomethane
StainingType: NEGATIVE / Material: uranyl acetate
Details: Samples were prepared by blotting protein from EM grid using filter paper, followed by multiple rounds of staining and blotting.
GridMaterial: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa / Details: -15 mA for 60 seconds
DetailsThe sample was predominantly monodisperse, as seen by negative-stain TEM.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 23.0 e/Å2
Details: Images were collected as tilt pairs at 0 and 55 degrees.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 62000
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 28685
CTF correctionSoftware - Name: SPHIRE
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 118 / Random conical tilt - Tilt angle: 55 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 44.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX
Details: Reconstructions were refined in sxalid3d and sxlocal_ali3d before half-map reconstructions were standard.
Number images used: 316
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1mjg

chain_id: A

1mjg

chain_id: B

1mjg

chain_id: M

1mjg

chain_id: N
DetailsInitial fitting was done using chimera and then MDFF was used for flexible fitting. Fitting was performed with rigid domains connected by flexible linkers.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT

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