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- EMDB-11200: S-shaped FtsH dodecamer of A. aeolicus with lamellar-like N-termi... -

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Basic information

Entry
Database: EMDB / ID: EMD-11200
TitleS-shaped FtsH dodecamer of A. aeolicus with lamellar-like N-terminal domains in LMNG micelle
Map dataS-shaped FtsH dodecamer of A. aeolicus with lamellar-like N-terminal domains in LMNG micelle
Sample
  • Complex: FtsH protease of A. aeolicus
    • Other: A. aeolicus FtsH protease
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.5 Å
AuthorsCarvalho V / Prabudiansyah I / Kovacik L / Chami M / Kieffer R / van der Valk R / de Lange N / Engel A / Aubin-Tam M-E
Funding support Netherlands, Switzerland, 2 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)723-016-007 Netherlands
Swiss National Science Foundation18272.1 Switzerland
CitationJournal: J Biol Chem / Year: 2021
Title: The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry.
Authors: Vanessa Carvalho / Irfan Prabudiansyah / Lubomir Kovacik / Mohamed Chami / Roland Kieffer / Ramon van der Valk / Nick de Lange / Andreas Engel / Marie-Eve Aubin-Tam /
Abstract: AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane- ...AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.
History
DepositionJun 18, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateJan 26, 2022-
Current statusJan 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0085
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11200.png

Downloads & links

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Map

FileDownload / File: emd_11200.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationS-shaped FtsH dodecamer of A. aeolicus with lamellar-like N-terminal domains in LMNG micelle
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 320 pix.
= 409.6 Å		1.28 Å/pix.
x 320 pix.
= 409.6 Å		1.28 Å/pix.
x 320 pix.
= 409.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0085 / Movie #1: 0.0085
Minimum - Maximum-0.006271396 - 0.020779662
Average (Standard dev.)0.00027056245 (±0.0015851178)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 409.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z409.600409.600409.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0060.0210.000

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Supplemental data

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Sample components

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Entire : FtsH protease of A. aeolicus

EntireName: FtsH protease of A. aeolicus
Components
  • Complex: FtsH protease of A. aeolicus
    • Other: A. aeolicus FtsH protease

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Supramolecule #1: FtsH protease of A. aeolicus

SupramoleculeName: FtsH protease of A. aeolicus / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET22a
Molecular weightExperimental: 427 KDa

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Macromolecule #1: A. aeolicus FtsH protease

MacromoleculeName: A. aeolicus FtsH protease / type: other / ID: 1 / Classification: other
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString: MNALKNFFIW AIIIGAAIVA FNLFEGKREF TTKVSLNEVV KLVEEGKVSY AEVRGNTAII QTKDGQKLE VTLPPNTNLV DKMVEKGVRV EVANPEPPGG WLVNVFLSWL PILFFIGIWI F LLRQMSGG GNVNRAFNFG KSRAKVYIEE KPKVTFKDVA GIEEVKEEVK ...String:
MNALKNFFIW AIIIGAAIVA FNLFEGKREF TTKVSLNEVV KLVEEGKVSY AEVRGNTAII QTKDGQKLE VTLPPNTNLV DKMVEKGVRV EVANPEPPGG WLVNVFLSWL PILFFIGIWI F LLRQMSGG GNVNRAFNFG KSRAKVYIEE KPKVTFKDVA GIEEVKEEVK EIIEYLKDPV KF QKLGGRP PKGVLLYGEP GVGKTLLAKA IAGEAHVPFI SVSGSDFVEM FVGVGAARVR DLF ETAKKH APCIIFIDEI DAVGRARGAI PVGGGHDERE QTLNQLLVEM DGFDTSDGII VIAA TNRPD ILDPALLRPG RFDRQIFIPK PDVRGRYEIL KVHARNKKLA KDVDLEFVAR ATPGF TGAD LENLLNEAAL LAARKGKEEI TMEEIEEALD RITMGLERKG MTISPKEKEK IAIHEA GHA LMGLVSDDDD KVHKISIIPR GMALGVTQQL PIEDKHIYDK KDLYNKILVL LGGRAAE EV FFGKDGITTG AENDLQRATD LAYRMVSMWG MSDKVGPIAI RRVANPFLGG MTTAVDTS P DLLREIDEEV KRIITEQYEK AKAIVEEYKE PLKAVVKKLL EKETITCEEF VEVFKLYGI ELKDKCKKEE LFDKDRKSEE NKELKSEEVK EEVV
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 8
Component:
ConcentrationNameFormula
10.0 mMTris-HCl
150.0 mMNaCl
0.01 w/vLMNG
5.0 %glycerol
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.005 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 90.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3760 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-35 / Number grids imaged: 1 / Number real images: 3993 / Average exposure time: 16.0 sec. / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.7000000000000001 µm / Calibrated magnification: 78247 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 215000
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

Particle selectionNumber selected: 101726
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: INSILICO MODEL / In silico model: spherical blob
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 2651
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationNumber classes: 7 / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ww0


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient

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