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- EMDB-2513: Electron cryo-microscopy of F420-reducing [NiFe] hydrogenase Frh -

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Basic information

Entry
Database: EMDB / ID: EMD-2513
TitleElectron cryo-microscopy of F420-reducing [NiFe] hydrogenase Frh
Map dataFrhABG dodecamer
Sample
  • Sample: F420 reducing hydrogenase
  • Protein or peptide: F420-reducing hydrogenase, subunit alpha
  • Protein or peptide: F420-reducing hydrogenase, subunit beta
  • Protein or peptide: F420-reducing hydrogenase, subunit gamma
Keywordshydrogenase / flavoprotein / [NiFe] hydrogenase / electron transfer / ferredoxin
Function / homology
Function and homology information


coenzyme F420 hydrogenase / coenzyme F420 hydrogenase activity / ferredoxin hydrogenase activity / iron-sulfur cluster binding / nickel cation binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding
Similarity search - Function
Coenzyme F420 hydrogenase, subunit alpha / Coenzyme F420 hydrogenase, subunit alpha / Coenzyme F420 hydrogenase subunit beta, archaea / Coenzyme F420 hydrogenase, subunit gamma / Coenzyme F420 hydrogenase subunit beta, archaea / Coenzyme F420 hydrogenase, subunit gamma / 4Fe-4S dicluster domain / Oxidoreductase FRHB/FDHB/HCAR-like / Coenzyme F420 hydrogenase/dehydrogenase beta subunit, N-terminal / Coenzyme F420 hydrogenase/dehydrogenase beta subunit, C-terminal ...Coenzyme F420 hydrogenase, subunit alpha / Coenzyme F420 hydrogenase, subunit alpha / Coenzyme F420 hydrogenase subunit beta, archaea / Coenzyme F420 hydrogenase, subunit gamma / Coenzyme F420 hydrogenase subunit beta, archaea / Coenzyme F420 hydrogenase, subunit gamma / 4Fe-4S dicluster domain / Oxidoreductase FRHB/FDHB/HCAR-like / Coenzyme F420 hydrogenase/dehydrogenase beta subunit, N-terminal / Coenzyme F420 hydrogenase/dehydrogenase beta subunit, C-terminal / Coenzyme F420 hydrogenase/dehydrogenase, beta subunit N-term / Coenzyme F420 hydrogenase/dehydrogenase, beta subunit C terminus / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / NADH:ubiquinone oxidoreductase-like, 20kDa subunit / NADH ubiquinone oxidoreductase, 20 Kd subunit / [NiFe]-hydrogenase, large subunit / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain
Similarity search - Domain/homology
F420-reducing hydrogenase, subunit beta / F420-reducing hydrogenase, subunit gamma / F420-reducing hydrogenase, subunit alpha
Similarity search - Component
Biological speciesMethanothermobacter marburgensis (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsAllegretti M / Mills DJ / McMullan G / Kuehlbrandt W / Vonck J
CitationJournal: Elife / Year: 2014
Title: Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector.
Authors: Matteo Allegretti / Deryck J Mills / Greg McMullan / Werner Kühlbrandt / Janet Vonck /
Abstract: The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct ...The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes. DOI: http://dx.doi.org/10.7554/eLife.01963.001.
History
DepositionNov 21, 2013-
Header (metadata) releaseDec 18, 2013-
Map releaseFeb 26, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4ci0
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4ci0
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2513.tif

Downloads & links

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Map

FileDownload / File: emd_2513.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFrhABG dodecamer
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum-0.19804479 - 0.62828296
Average (Standard dev.)0.0022398 (±0.02294438)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 295.68002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z295.680295.680295.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.1980.6280.002

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Supplemental data

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Sample components

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Entire : F420 reducing hydrogenase

EntireName: F420 reducing hydrogenase
Components
  • Sample: F420 reducing hydrogenase
  • Protein or peptide: F420-reducing hydrogenase, subunit alpha
  • Protein or peptide: F420-reducing hydrogenase, subunit beta
  • Protein or peptide: F420-reducing hydrogenase, subunit gamma

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Supramolecule #1000: F420 reducing hydrogenase

SupramoleculeName: F420 reducing hydrogenase / type: sample / ID: 1000
Oligomeric state: a dodecamer of the heterotrimer FrhA, FrhB, FrhG
Number unique components: 3
Molecular weightExperimental: 1.2 MDa / Theoretical: 1.2 MDa

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Macromolecule #1: F420-reducing hydrogenase, subunit alpha

MacromoleculeName: F420-reducing hydrogenase, subunit alpha / type: protein_or_peptide / ID: 1 / Name.synonym: FrhA / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: No
Source (natural)Organism: Methanothermobacter marburgensis (archaea) / Strain: DSM 2133 / Location in cell: cytoplasm
Molecular weightExperimental: 440 KDa / Theoretical: 440 KDa
SequenceUniProtKB: F420-reducing hydrogenase, subunit alpha
InterPro: Coenzyme F420 hydrogenase, subunit alpha, Coenzyme F420 hydrogenase subunit beta, archaea, Coenzyme F420 hydrogenase, subunit gamma

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Macromolecule #2: F420-reducing hydrogenase, subunit beta

MacromoleculeName: F420-reducing hydrogenase, subunit beta / type: protein_or_peptide / ID: 2 / Name.synonym: FrhB / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: No
Source (natural)Organism: Methanothermobacter marburgensis (archaea) / Strain: DSM 2133 / Location in cell: cytoplasm
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa
SequenceUniProtKB: F420-reducing hydrogenase, subunit beta
InterPro: Coenzyme F420 hydrogenase, subunit alpha, Coenzyme F420 hydrogenase subunit beta, archaea, Coenzyme F420 hydrogenase, subunit gamma

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Macromolecule #3: F420-reducing hydrogenase, subunit gamma

MacromoleculeName: F420-reducing hydrogenase, subunit gamma / type: protein_or_peptide / ID: 3 / Name.synonym: FrhG / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: No
Source (natural)Organism: Methanothermobacter marburgensis (archaea) / Strain: DSM 2133 / Location in cell: cytoplasm
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa
SequenceUniProtKB: F420-reducing hydrogenase, subunit beta
InterPro: Coenzyme F420 hydrogenase, subunit alpha, Coenzyme F420 hydrogenase subunit beta, archaea, Coenzyme F420 hydrogenase, subunit gamma

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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

Concentration0.7 mg/mL
BufferpH: 7.6 / Details: 50mM Tris-HCl, 0.025mM FAD
GridDetails: freshly glow discharged Quantifoil holey grid (1 micrometer holes)
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 103 K / Instrument: FEI VITROBOT MARK I / Method: blotting 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 106000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: OTHER
TemperatureMin: 77 K / Max: 80 K / Average: 79 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
Detailsdata was collected in movie mode
DateMay 27, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 235 / Average electron dose: 22 e/Å2
Details: Every image is the average of six frames recorded by the direct electron detector aligned to each other
Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each image
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 26000

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Atomic model buiding 1

Initial modelPDB ID:

3zfs


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C
SoftwareName: Coot
DetailsThe structure was built using Coot based on an earlier model built into a lower resolution EM map
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4ci0:
Electron cryo-microscopy of F420-reducing NiFe hydrogenase Frh

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