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- EMDB-21006: Negative stain EM map of CODH/ACS in the hyperextended conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-21006
TitleNegative stain EM map of CODH/ACS in the hyperextended conformation
Map dataNegative stain electron microscopy map of CODH/ACS in the hyperextended state
Sample
  • Complex: carbon monoxide dheydrogenase/acetyl-CoA synthase tetramer
    • Protein or peptide: Carbon monoxide dehydrogenase
    • Protein or peptide: Acetyl-CoA synthase
Biological speciesMoorella thermoacetica (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 46.7 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: 5.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21006.png

Downloads & links

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Map

FileDownload / File: emd_21006.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain electron microscopy map of CODH/ACS in the hyperextended state
Voxel sizeX=Y=Z: 7.16 Å
Density
Contour LevelBy AUTHOR: 5.11 / Movie #1: 5.11
Minimum - Maximum-6.7376604 - 10.137042
Average (Standard dev.)0.029528992 (±0.7040544)
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-6.73810.1370.030

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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 dehydrogenase
    • 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 dehydrogenase

MacromoleculeName: Carbon monoxide dehydrogenase / 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 IGYEGICCRF CMAGPCRIKA TDGPGSRGIC GASAWTIVAR NVGLMILTGA AAHCEHGNHI AHALVEMAEG KAPDYSVKDE AKLKEVCRRV GIEVEGKSVL ELAQEVGEKA ...String:
MPRFRDLSHN CRPSEAPRVM EPKNRDRTVD PAVLEMLVKS KDDKVITAFD RFVAQQPQCK IGYEGICCRF CMAGPCRIKA TDGPGSRGIC GASAWTIVAR NVGLMILTGA AAHCEHGNHI AHALVEMAEG KAPDYSVKDE AKLKEVCRRV GIEVEGKSVL ELAQEVGEKA LEDFRRLKGE GEATWLMTTI NEGRKEKFRT HNVVPFGIHA SISELVNQAH MGMDNDPVNL VFSAIRVALA DYTGEHIATD FSDILFGTPQ PVVSEANMGV LDPDQVNFVL HGHNPLLSEI IVQAAREMEG EAKAAGAKGI NLVGICCTGN EVLMRQGIPL VTSFASQELA ICTGAIDAMC VDVQCIMPSI SAVAECYHTR IITTADNAKI PGAYHIDYQT ATAIESAKTA IRMAIEAFKE RKESNRPVYI PQIKNRVVAG WSLEALTKLL ATQNAQNPIR VLNQAILDGE LAGVALICGC NNLKGFQDNS HLTVMKELLK NNVFVVATGC SAQAAGKLGL LDPANVETYC GDGLKGFLKR LGEGANIEIG LPPVFHMGSC VDNSRAVDLL MAMANDLGVD TPKVPFVASA PEAMSGKAAA IGTWWVSLGV PTHVGTMPPV EGSDLIYSIL TQIASDVYGG YFIFEMDPQV AARKILDALE YRTWKLGVHK EVAERYETKL CQGY

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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 YLPVIRCFSG EEVKKLGDLP PILNRKRAQV SPVLNFENAR LAGEATWYAA EIIEALRYLK YKPDEPLLPP PWTGFIGDPV VRRFGIKMVD WTIPGEAIIL GRAKDSKALA ...String:
MTDFDKIFEG AIPEGKEPVA LFREVYHGAI TATSYAEILL NQAIRTYGPD HPVGYPDTAY YLPVIRCFSG EEVKKLGDLP PILNRKRAQV SPVLNFENAR LAGEATWYAA EIIEALRYLK YKPDEPLLPP PWTGFIGDPV VRRFGIKMVD WTIPGEAIIL GRAKDSKALA KIVKELMGMG FMLFICDEAV EQLLEENVKL GIDYIAYPLG NFTQIVHAAN YALRAGMMFG GVTPGAREEQ RDYQRRRIRA FVLYLGEHDM VKTAAAFGAI FTGFPVITDQ PLPEDKQIPD WFFSVEDYDK IVQIAMETRG IKLTKIKLDL PINFGPAFEG ESIRKGDMYV EMGGNRTPAF ELVRTVSESE ITDGKIEVIG PDIDQIPEGS KLPLGILVDI YGRKMQADFE GVLERRIHDF INYGEGLWHT GQRNINWLRV SKDAVAKGFR FKNYGEILVA KMKEEFPAIV DRVQVTIFTD EAKVKEYMEV AREKYKERDD RMRGLTDETV DTFYSCVLCQ SFAPNHVCIV TPERVGLCGA VSWLDAKASY EINHAGPNQP IPKEGEIDPI KGIWKSVNDY LYTASNRNLE QVCLYTLMEN PMTSCGCFEA IMAILPECNG IMITTRDHAG MTPSGMTFST LAGMIGGGTQ TPGFMGIGRT YIVSKKFISA DGGIARIVWM PKSLKDFLHD EFVRRSVEEG LGEDFIDKIA DETIGTTVDE ILPYLEEKGH PALTMDPIM

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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 chloridesodium 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.
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified
DetailsThe sample was predominantly monodisperse, as seen by negative-stain TEM.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 62000
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.
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
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 46.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX
Details: Reconstructions were refined in sxali3d and sxlocal_ali3d before half-map reconstructions were standard.
Number images used: 399
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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