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- PDB-7s9z: Helicobacter Hepaticus CcsBA Closed Conformation -

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Basic information

Entry
Database: PDB / ID: 7s9z
TitleHelicobacter Hepaticus CcsBA Closed Conformation
ComponentsCytochrome c biogenesis protein
KeywordsMEMBRANE PROTEIN / Cytochrome c biogenesis / Heme transporter / Heme lyase
Function / homology
Function and homology information


cytochrome complex assembly / membrane => GO:0016020 / heme binding
Similarity search - Function
ResB-like domain / ResB-like family / Cytochrome c-type biogenesis protein CcsA/CcmC / Cytochrome c assembly protein / Cytochrome C assembly protein
Similarity search - Domain/homology
HEME B/C / PHOSPHATIDYLETHANOLAMINE / Cytochrome c biogenesis protein
Similarity search - Component
Biological speciesHelicobacter hepaticus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.14 Å
AuthorsMendez, D.L. / Lowder, E.P. / Tillman, D.E. / Sutherland, M.C. / Collier, A.L. / Rau, M.J. / Fitzpatrick, J.A. / Kranz, R.G.
Funding support5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM47909
Childrens Discovery Institute of Washington University and St. Louis Childrens Hospital2015-505
Childrens Discovery Institute of Washington University and St. Louis Childrens Hospital2019-813
Foundation for Barnes-Jewish Hospital3770
Chan Zuckerberg Initiative2020-225726
CitationJournal: Nat Chem Biol / Year: 2022
Title: Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport.
Authors: Deanna L Mendez / Ethan P Lowder / Dustin E Tillman / Molly C Sutherland / Andrea L Collier / Michael J Rau / James A J Fitzpatrick / Robert G Kranz /
Abstract: Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We ...Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome.
History
DepositionSep 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 12, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Cytochrome c biogenesis protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,6443
Polymers107,2911
Non-polymers1,3532
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cytochrome c biogenesis protein


Mass: 107291.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter hepaticus (bacteria) / Gene: ccsBA / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q7VHG9
#2: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE / Phosphatidylethanolamine


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#3: Chemical ChemComp-HEB / HEME B/C / HYBRID BETWEEN B AND C TYPE HEMES (PROTOPORPHYRIN IX CONTAINING FE)


Mass: 618.503 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H34FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of CcsBA with one heme present / Type: COMPLEX / Details: Purified from E. coli in DDM / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Helicobacter hepaticus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43 / Plasmid: pGEX
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2100 mMSodium chlorideNaCLSodium chloride1
30.03 %DDMC24H46O111
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Blot for 2 seconds at a blot force of -1 and plunge frozen

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2500 nm / Cs: 0.01 mm / C2 aperture diameter: 150 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 84 K / Temperature (min): 82 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 66 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8676
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Specific energy filter was a Gatan BioQuantum 968. / Energyfilter slit width: 20 eV
Spherical aberration corrector: Microscope is outfitted with a Cs image corrector with two hexapole elements.
Image scansWidth: 3832 / Height: 3704 / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2particle selection
2EPU2.10.0.1941image acquisitionEPU was used to automated the process of single particle acquisition
4cryoSPARC3.2CTF correction
7Coot0.8.9.2model fittingwinCOOT was used to create a poly-alanine model, mutate this model to correct sequence, and further improve fit to density map
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstruction
13PHENIX1.17.1model refinementPhenix was used to refine the model using Phenix Real Space Refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1778157
Details: Initial particle picking was facilitated using the blob picker in cryoSPARC. Picked particles were then subjected to 2D classification. Once converged, a sub-set of 2D classes were manually ...Details: Initial particle picking was facilitated using the blob picker in cryoSPARC. Picked particles were then subjected to 2D classification. Once converged, a sub-set of 2D classes were manually picked and reclassified using a smaller number of classes.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117488 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Model was built de novo into density map using COOT. Both Phenix and ISOLDE were used for refinement

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