1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Atomic structure of a mitochondrial complex I intermediate from vascular plants

  1. Maria Maldonado
  2. Abhilash Padavannil
  3. Long Zhou
  4. Fei Guo
  5. James A Letts  Is a corresponding author
  1. Department of Molecular and Cellular Biology, University of California Davis, United States
  2. BIOEM Facility, University of California Davis, United States
Research Article
Cite this article as: eLife 2020;9:e56664 doi: 10.7554/eLife.56664
6 figures, 1 video, 7 tables and 1 additional file

Figures

Figure 1 with 4 supplements
The structure of CI* from Vigna radiata.

(A) An overview of the conserved modular structure of CI using the Thermus thermophilus bacterial core subunits as a simple model (PDB: 4HEA) (Baradaran et al., 2013). (B) CryoEM density map of CI* from V. radiata highlighting its modular architecture. N, NADH-binding module; Q, quinone-binding module; PP, proximal-pump module; PD, distal-pump module; γCA, carbonic anhydrase domain, see also Video 1). (C) Atomic model of V. radiata CI* with all 30 assigned subunits labeled. The additional N-terminal helix of NDUS8 is indicated with an asterisk (*).

Figure 1—figure supplement 1
Schematic CI assembly pathways in metazoans and plants.

(A) CI assembly in metazoans. N-module is added at the last step of assembly. (B) CI assembly in plants. CI* intermediate is assembled before the PD domain is added last. N, NADH-binding module; Q, quinone-binding module; PP, proximal-pumps module; PD, distal-pumps module; γCA, carbonic anhydrase domain. Based on Formosa et al., 2018; Guerrero-Castillo et al., 2017; Garcia et al., 2017; Stroud et al., 2016; Ligas et al., 2019.

Figure 1—figure supplement 2
Purification and characterization of CI*.

(A-F) Representative preparation of CI* sample. (A) Digitonin-extracted, amphipol-stabilized mitochondrial membrane complexes were run on a 3–12% blue-native polyacrylamide gel electrophoresis (BN-PAGE) and subjected to in-gel NADH dehydrogenase activity assay to detect CI activity (purple bands). The band labeled Peak 2 corresponds to CI*. (B) The digitonin-extracted, amphipol-stabilized sample was separated by a 10–45% (w:v) linear sucrose (suc) gradient and fractionated. (C) Samples of the sucrose gradient fractions from (B) were run on BN-PAGE gels and subjected to in-gel NADH-dehydrogenase activity assay. Relevant fractions as indicated by the dashed boxes were separately pooled and concentrated. (D) The pooled fractions from (C) were furthered purified using size-exclusion chromatography. The trace for the absorbance at 280 nm is shown. Relevant peak fractions were pooled and concentrated. (E) The activity of the purified fractions from (D) was re-tested with an NADH-dehydrogenase in-gel activity assay. (F) The activity of the purified samples from (D) was further tested with a spectroscopic NADH-decylubiquinone (DQ) activity assay in the presence or absence of 100 µM DQ. Four independent repeat measurements were done for each sample. The background-corrected average of the repeats is shown, together with the standard deviation (error bars). Significance (**, p<0.01) was tested with a two-tailed t-test. p-values: 0.0005 (peak 2) and 0.0063 (peak 3). (G-H) Preparation of CI* for the cryoEM dataset presented in this paper. (G) The digitonin-extracted, amphipol-stabilized mitochondrial membrane complexes were separated by a 15–45% (w:v) linear sucrose gradient and fractionated. (H) Sucrose gradient fractions from (G) were subjected to in-gel NADH-dehydrogenase activity assay. Fractions 10–11 were pooled, concentrated, buffer-exchanged and used as the sample for the cryoEM grid used in the determination of the structure of CI* presented here.

Figure 1—figure supplement 3
CryoEM processing steps.

(A) A representative micrograph of the 8541 used for further processing (9816 collected). Scale bar, 100 nm. (B) Representative 2D class averages from reference-free classification in Relion. (C) Classification and refinement procedures used. The local refinement map, a local refinement slice and the gold-standard FSC curves are shown next to their respective final reconstructions.

Figure 1—figure supplement 4
CryoEM model-to-map correlation.

(A) Representative density from the composite map showing the fit between the model and thee map for elements of secondary structure from the peripheral and membrane arms of CI*. (B–D) Map-Model FSC curves are shown for the peripheral arm focused refinement (B), the membrane arm focused refinement (C) and the composite map (D). (E) Low contour of filtered cryoEM density for CI* colored by module (N, tan; Q, green; PP blue; and γCA pink). The lipid membrane density is shown in grey.

Key differences in CI accessory subunits between V. radiata and opisthokonts.

Accessory subunits NDUS6 and NDUA12, NDUA8 and NDUC2 of V. radiata (this study), Y. lipolytica (PDB:6RFR) and O. aries (PDB: 6QA9) are shown as surface for comparison. (A) NDUS6 (green) and NDUA12 (orange), with an additional label for NDUA9. (B) NDUA8 (maroon), with additional label for the V. radiata’s carbonic anhydrase domain (CA). (C) NDUC2 (blue), with additional label for the V. radiata’s CA.

Figure 3 with 1 supplement
V. radiata γ-carbonic-anhydrase (γCA) domain, Zn2+ coordination and associated lipid cavity.

(A) Top view of the carbonic anhydrase domain with its CA1 (green), CA2 (lime) and CAL2 (lime green). Key residues at subunit interfaces for Zn2+ coordination shown as sticks; Zn2+ shown as grey sphere. Only the CA1-CA2 interface has all three key Zn2+ coordinating histidines in place. (B) Zoom-in of Zn2+ coordination site in (A), with map density for the three histidines and Zn2+ shown as blue meshes. (C) Two phosphatidylcholines (spheres) are placed in the lipid cavity between the γCA and the PP-module. Asterisk indicates the N-terminal amphipathic helices of CA1 and CA2. (D Zoom-in of the lipid cavity in C), with lipid density shown as blue mesh and key interacting residues shown as sticks.

Figure 3—figure supplement 1
Schematic representation of the γ-carbonic-anhydrase (γCA) domain interfaces and potential active sites in V. radiata.

The Zn2+-adjacent amino acids, as per sequence alignment with CamH γCA homologue subunits, are shown. A red cross indicates amino acids that are incapable of coordinating Zn2+. A green tick indicates the potential active sites that are capable of coordinating Zn2+. CA1, carbonic-anhydrase-1 subunit; CA2, carbonic-anhydrase-2 subunit; CAL-2, carbonic-anhydrase-like-2 subunit.

Unassigned density in V. radiata CI* map.

Four stretches of unassigned, continuous densities in the map are shown with their positions on CI* indicated. Insets (A-D) show the density (blue mesh) and the poly-alanine chains (red) (A, C, D) or the putative NDUS6 C-terminal residues (B).

Structure of the redox centers, Q cavity and the hydrophilic axis of V. radiata CI*.

(A) V. radiata’s FMN (stick) and iron-sulfur clusters (spheres) are labeled by nearest-atom center-to-center distances, overlaid with those from T. thermophilus (transparent grey). (B) Key residues (stick) delineating the Q cavity and the nearby N2 iron-sulfur cluster (spheres). Unassigned density in the Q cavity, potentially corresponding to quinone, shown as blue mesh. (C) Key CI* residues constituting the hydrophilic axis within the membrane domain shown as sticks.

Appendix 1—figure 1
The Gibbs energy change of the CI reaction (ΔGCI) as a function of the redox poise of the mitochondrial NADH pool.

The Gibbs energy change was calculated using equation 10 and the values presented in Table A1, for reactions in which CI pumps 4 H+ (blue; representative of the standard, full-length CI pumping with a 4H+:2e- ratio) or 2 H+ (red; representative of a putative CI* pumping with a 2H+/2e- ratio). The horizontal dashed line indicates equilibrium state (ΔGCI = 0) for the different [NAD+]/[NADH] ratios. The vertical dashed line indicates the [NAD+]/[NADH] ratio at which full-length CI (blue) attains equilibrium (ϵ = 1). The highlighted orange region corresponds to conditions in which thermodynamics would favor reverse electron transport (RET) by full-length CI (ϵ > 1).

Videos

Video 1
CryoEM density for the CI* composite map.

Tables

Table 1
Cryo-EM data collection, refinement and validation statistics.
Data Collection and processing
MicroscopeTitan krios, (UCSF)
CameraK3
Magnification60,010
Voltage (kV)300 kV
Electron exposure (e-2)86.4
Defocus range (µm)−0.5 to −2.0
Pixel size (Å)0.8332
SoftwareSerialEM
ReconstructionCI* Peripheral ArmCI* Membrane ArmCI* Composite Map
Number of particles34,40734,407The CI* Peripheral Arm and Membrane Arm Maps were combined in Phenix to generate this composite map
Accuracy of rotations (°)0.681.489
Accuracy of translations (pixels)0.6550.881
Box size (pixels)512512
Final resolution (Å)3.83.9
Map sharpening B factor (Å2)−90−96
EMDB ID220932209222090
Refinement
SoftwarePhenix
Initial model (PDB code)6Q9D6Q9B and 1QRG6Q9D, 6Q9B and 1QRG
Map/model correlation
Model resolution (Å)3.94.03.9
d99 (Å)3.94.04.0
FSC model 0.5 (Å)3.93.93.9
Map CC (around atoms)0.820.860.87
Model composition
Non-hydrogen atoms26,00119,05245047
Protein residues328424535736
Number of chains171834
Number of ligands and cofactors11112
Number of lipids066
Atomic Displacement Parameters (ADP)
Protein average (Å2)68.7858.4064.39
Ligand average (Å2)48.5948.5948.59
R.m.s. deviations
Bond lengths (Å)0.0070.0070.007
Bond angles (°)1.1871.1220.845
Ramachandran Plot
Favored (%)82.9088.0384.98
Allowed (%)16.7611.8814.79
Disallowed (%)0.340.080.23
Validation
MolProbity score2.412.312.38
Clash score16.7916.2116.42
Rotamer outliers (%)0.250.200.23
EMRinger score1.472.092.17
PDB ID------6X89
Table 2
Model building statistics by subunit.
Subunit nameUniprot IDChain IDTotal residuesAtomic residuesPoly-AlaUn-modeled residues% atomicTMHIdentified RNA editing sites*Ligands, lipids
Peripheral arm core subunits
NDUS1A0A1S3TQ85S174657–744571–56, 745–74692.1%4Fe4S×2, 2Fe2S
NDUS2E9KZN6S23949–17,21-3941–8, 18–2098.0%S26L, 246L, S67F, H82Y, S84L, R106C, S112L, S193L, S233L, H242Y, S245L, P247F, R257C, R353C, S360F, S363L, S368F, P375L
NDUS3E9KZM7S31901–184185–19096.8%S31F, S56L, P100S, R110W, S133L, L147F
NDS7A0A1S3U8J5S721356–2131–5574.2%4Fe4S, PC
NDS8A0A1S3VGS8S822242–2221–4181.5%4Fe4S×2
NDUV1A0A1S3V7V2V149159–4911–5888.2%4Fe4S, FMN
NDUV2A0A1S3U769V225128–2431–27, 244–25186.1%2Fe2S
Peripheral arm accessory subunits
NDUA2A0A1S3TVC7A2982–931, 94–9893.9%
NDUA5A0A1S3U023A516912–1371–11, 138–16974.6%
NDUA6A0A1S3W1K8A6132118–1311–117, 13211.4%
NDUA7A0A1S3UVC7A712719–1271–1885.8%
NDUA9A0A1S3V8W7A939647–3811–46, 382–39684.6%NADPH
NDUA12A0A1S3VNK7AL15621–1551–20, 15686.5%
NDUS4A0A1S3UIW7S414642–1421421–41, 143–14669.2%
NDUS6A0A1S3VYF3S610331–1021–30, 10369.9%Zn2+
Membrane arm core subunits
NU1MA0A1S4ETV6/E9KZL01M3252–213, 220–3251, 214–21997.8%8R89W, P164S, R165C, S167L, S179F, R225C, P242L, P248L, P252L, R300W, R310W
NU2ME9KZK92M4881–48748899.8%14S19F, S103F, S104F, P119L, P121S, R123C, H132Y, P143L, S166LL, S221F, P307L, H310Y, R320C, S376L, S467L, S468F, S486LPC×2
NU3MQ9XPB43M1181–28, 56–11829–5577.1%3P70F, P83S, P84L, S115L, R117W
NU4LMA0A1S4ETY3/E9KZN84L1001–8687–10086.0%3S14F, P29L, S32L, P34S, S37L, S53L, S63L, S66L
NU6ME9KZM56M2051–72, 111–17273–110173–20565.4%5P9L, A18V, P30F, P32L, R35C, P54L, H57Y
Membrane arm accessory subunits
CA1A0A1S3VT00G12703–222223–2331–2, 234–27081.5%
CA2A0A1S3U544G22732–2371, 238–27386.4%
CAL2A0A1S3UI49L225649–129, 134–2541–48, 130–133, 255–25680.5%
NDUX1A0A1S3VI15X11011–10010199.0%2
NDUC2A0A1S3UPL8C2815–681–4, 69–8179.0%2
NDUA8A0A1S3VVN6A81062–106199.1%
NDUA13A0A1S3UYW0AM1432–143199.3%1
NDUA1A0A1S3TU57A1652–631, 64–6595.4%1PC
NDS5A0A1S3TQ33S53992–701, 71–39917.3%
NDUA3A0A1S3TCK0A3632–451, 46–6369.8%1
P2A0A1S3TGE7P211583–10677–821–76, 107–11520.9%
Unassigned density
A1–18
B1–24
C1–431
  1. *RNA editing of mitochondrially encoded subunits: amino acids were changed at the listed positions as detailed. The changes were based on the reported equivalent A. thaliana RNA edits (Giegé and Brennicke, 1999; Bentolila et al., 2008) and were only made when density was unambiguously correct for the edited V. radiata amino acid in the cryoEM map.

Table 3
Complex I subunit homologues in plants, mammals, yeast and bacteria.

V. radiata homologues were obtained by performing BLASTp searches of the Arabidopsis thaliana genes (Meyer et al., 2019; Braun et al., 2014). Mammalian, yeast and bacterial homologues were obtained from Letts and Sazanov, 2015. Additional BLASTp searches were performed wherever necessary. Given the high sequence similarity between the carbonic anhydrase (CA) paralogues, the names of the V. radiata CA proteins appear to have been mis-assigned in the genetic databases relative to their A. thaliana homologues. The CA1, CA2, CA2-like nomenclature used in the table is the one that, based on our sequence alignments, best represents homology to the A. thaliana CA proteins. N, NADH-binding module; Q, quinone-binding module; PP, proximal-pumps module; PD, distal-pumps module; CA, carbonic anhydrase domain.

ModuleVigna radiata protein nameVigna radiata geneVigna radiata uniprot identifierArabidopsis thaliana protein nameArabidopsis thaliana geneHomo sapiens nameOvis aries nameMus musculus nameYarrowia lipolytica nameThermus thermophilus name
CORE peripheral arm
NNDUS1LOC106757688A0A1S3TQ8575 kDaAt5g37510NDUFS1NDUFS1NDUFS1NUAMNqo3
NNDUV1LOC106772405A0A1S3V7V251 kDaAt5g08530NDUFV1NDUFV1NDUFV1NUBMNqo1
NNDUV2LOC106762461A0A1S3U76924 kDaAt4g02580NDUFV2NDUFV2NDUFV2NUHMNqo2
QNDUS2nad7E9KZN6Nad7AtMg00510NDUFS2NDUFS2NDUFS2NUCMNqo4
QNDUS3nad9E9KZM7Nad9AtMg00070NDUFS3NDUFS3NDUFS3NUGMNqo5
QNDS7LOC106762764A0A1S3U8J5PSSTAt5g11770NDUFS7NDUFS7NDUFS7NUKMNqo6
QNDS8LOC106775047A0A1S3VGS8TYKYAt1g79010, At1g16700NDUFS8NDUFS8NDUFS8NUIMNqo9
CORE membrane arm
PPNU1Mnad1A0A1S4ETV6Nad1AtMg00516, AtMg01120, AtMg01275MT-ND1MT-ND1MT-ND1NU1MNqo8
PPNU2Mnad2E9KZK9Nad2AtMg00285, AtMg01320MT-ND2MT-ND2MT-ND2NU2MNqo14
PPNU3Mnad3Q9XPB4Nad3AtMg00990MT-ND3MT-ND3MT-ND3NU3MNqo7
PPNU4LMnad4LA0A1S4ETY3Nad4LAtMg00650MT-ND4LMT-ND4LMT-ND4LNULMNqo11
PPNU6Mnad6E9KZM5Nad6AtMg00270MT-ND6MT-ND6MT-ND6NU6MNqo10
PDNU4M*nad4E9KZL8Nad4AtMg00580MT-ND4MT-ND4MT-ND4NU4MNqo13
PDNU5M*nad5E9KZL1Nad5AtMg00060, AtMg00513, AtMg00665MT-ND5MT-ND5MT-ND5NU5MNqo12
ACCESSORY membrane arm
PPNDUA1LOC106758834A0A1S3TU57MWFEAt3g08610NDUFA1NDUFA1NDUFA1NIMM-
PPNDUA3LOC106754061A0A1S3TCK0B9At2g46540NDUFA3NDUFA3NDUFA3NI9M-
PPNDUA8-BLOC106778955A0A1S3VVN6PGIVAt3g06310, At5g18800NDUFA8NDUFA8NDUFA8NUPM-
PPNDUA13-ALOC106769964A0A1S3UYW0B16.6At2g33220, At1g04630NDUFA13NDUFA13NDUFA13NB6M-
PPNDUC1LOC106771273A0A1S3V2Z3--NDUFC1NDUFC1NDUFC1--
PPNDUS5LOC106757655A0A1S3TQ3315 kDaAt3g62790, At2g47690NDUFS5NDUFS5NDUFS5NIPM-
PPNDUB8LOC106765859A0A1S3UJ95ASHIAt5g47570NDUFB8NDUFB8NDUFB8NIAM-
PPNDUB10-BLOC106774903A0A1S3VGT1PDSWAt1g49140, At3g18410NDUFB10NDUFB10NDUFB10NIDM-
PPNDUA11*LOC106756741A0A1S3TLY8B14.7At2g42210NDUFA11NDUFA11NDUFA11NUJM-
ModuleVigna radiata protein nameVigna radiata geneVigna radiata Uniprot identifierArabidopsis thaliana protein nameArabidopsis thaliana geneHomo sapiens nameOvis aries nameMus musculus nameYarrowia lipolytica nameThermus thermophilus name
ACCESSORY membrane arm
PDNDUB1*LOC106775330A0A1S3VI15MNLLAt4g16450NDUFB1NDUFB1NDUFB1--
PDNDUC2*LOC106767534A0A1S3UPL8B14.5bAt4g20150NDUFC2NDUFC2NDUFC2NEBM-
PDNDUB2*LOC106754955A0A1S3TFG6AGGGAt1g76200NDUFB2NDUFB2NDUFB2--
PDNDUB3*LOC106769121A0A1S3UVV0B12At2g02510, At1g14450NDUFB3NDUFB3NDUFB3NB2M-
PDNDUB4*LOC106766640A0A1S3ULL3B15At2g31490NDUFB4NDUFB4NDUFB4NB5M-
PDNDUB5*LOC106767179A0A1S3UND4--NDUFB5NDUFB5NDUFB5NUNM-
PDNDUB7*LOC106770979A0A1S3V2B8B18At2g02050NDUFB7NDUFB7NDUFB7NB8M-
PDNDUB9*LOC106760947A0A1S3U1J6B22At4g34700NDUFB9NDUFB9NDUFB9NI2M-
PDNDUB11*LOC106771273A0A1S3V2Z3ESSSAt2g42310, At3g57785NDUFB11NDUFB11NDUFB11NESM-
ACCESSORY peripheral arm
NNDUA2LOC106759195A0A1S3TVC7B8At5g47890NDUFA2NDUFA2NDUFA2NI8M-
NNDUA12LOC106776991A0A1S3VNK7B17.2At3g03100NDUFA12NDUFA12NDUFA12N7BM-
NNDUS4LOC106765762A0A1S3UIW718 kDaAt5g67590NDUFS4NDUFS4NDUFS4NUYM-
NNDUS6LOC106779709A0A1S3VYF313 kDaAt3g03070NDUFS6NDUFS6NDUFS6NUMM-
QNDUA5LOC106760411A0A1S3U023B13At5g52840NDUFA5NDUFA5NDUFA5NUFM-
QNDUA6LOC106780789A0A1S3W1K8B14At3g12260NDUFA6NDUFA6NDUFA6NB4N-
QNDUA7LOC106768957A0A1S3UVC7B14.5aAt5g08060NDUFA7NDUFA7NDUFA7NUZM-
QNDUA9LOC106772694A0A1S3V8W739 kDaAt2g20360NDUFA9NDUFA9NDUFA9NUEM-
Plant-specific accessory
CACA1LOC106778103A0A1S3VT00Gamma-CA 1At1g19580-----
CACA2§LOC106761992, LOC106761993A0A1S3U566, A0A1S3U544Gamma-CA 2At1g47260-----
CACA2-LLOC106765552A0A1S3UI49Gamma CA-like 2At3g48680-----
CACA3*n.a.**n.a.**Gamma-CA 3At5g66510-----
CACA1-L*n.a.**n.a.**Gamma-CA-like 1At5g63510-----
PPNDUX1††LOC106775330A0A1S3VI1520.9 kDaAt4g16450---NUXM-
PPP2/16 kDALOC106755236A0A1S3TGE7P2At2g27730-----
Plant-specific accessory
Unconfirmed plant CI subunits (not seen in CI*)MICOS (DUF543)LOC106779628A0A1S3VY06MICOS subunit Mic10At1g72165-----
Uncharacterized protein LOC106758628LOC106758628A0A1S3TTD7NDU10At4g00585----
P1/11 kDALOC106761134A0A1S3U2B9P1At1g67350-----
P3LOC106755586A0A1S3THM0P3At5g14105-----
P4LOC106767179A0A1S3UND4P4At1g67785-----
TIM22−4 × 1LOC106779665A0A3Q0EN44TIM22-4At1g18320-----
TIM22−4 × 2LOC106779665A0A1S3VZ08TIM22-1At3g10110-----
TIM23-2LOC106761237A0A1S3U2K1TIM23-2At1g72750-----
Uncharacterized protein LOC106768488 isoform X4LOC106768488A0A1S3UST2SH3/FCH domain proteinAt1g68680-----
UDP-galactose transporter 1LOC106762681A0A1S3U838TPT domain-containing proteinAt1g72180-----
Gravitropic in the light 1LOC106779790A0A1S3VYR1DUF641 domain-containing proteinAt2g28430-----
  1. *Not seen in CI*.

    Only the C-terminus seen in CI* (see Main body and Discussion).

  2. Called gamma carbonic anhydrase one in Uniprot.

    §Called gamma carbonic anhydrase 1, mitochondrial in Uniprot (mis-assigned in the database).

  3. Called gamma carbonic anhydrase-like 2, mitochondrial in Uniprot (mis-assigned in the database).

    **Homologue not found using BLASTp.

  4. ††New identified subunit.

Table 4
PP- and PD-module bridging subunits in mammalian, Y. lipolytica and V. radiata CI.

Subunits discussed in the manuscript are marked with two asterisks (**). Bridging interactions are shaded in green. Lack of interactions by existing subunits or lack of homologues are shaded in orange. Lack of the PD subunits in V. radiata CI* is shaded in yellow. PP, proximal pumping domain; PD, distal pumping domain.

LocationSubunitMammalsY. lipolyticaV. radiata
Inter-membrane space (IMS)NDUA8**Extends along membrane arm, bridges NU2M (PP) and NU4M (PD)Does not extend to the PP/PD-module interface but has an additional helix interacts with NU1MC-terminally truncated (does not bridge)
NDUC2**C-terminus bridges NDUB10 (PP) and NDUB11 (PD)C-terminally truncated, but bridging interaction replaced by extended loop on NU4MC-terminally truncated (does not bridge)
NDUB5Bridging interactionsBridging interactionsN- and C-terminally truncated (subunit not present in CI*)
NDUA11Does not bridge in the IMSC-terminal extension binding to NU4MSubunit not present in CI*
MembraneNDUA11Binds to the lateral helix of NU5M, connecting NU5M and NU2MBinds to the lateral helix of NU5M, connecting NU5M and NU2MSubunit not present in CI*
MatrixNDUS2**Bridging interactionsDoes not bridgeN-terminally truncated (does not bridge)
NU5MLateral helix extends into PPLateral helix extends into PPSubunit not present in CI*
NDUA10Bridging interactionsNo homologue presentNo homologue present
NDUB11Bridging interactionsDoes not bridgeSubunit not present in CI*
NDUB4Does not bridgeN-terminus extends along matrix arm and binds to NU2MN-terminally truncated (subunit not present in CI*)
Table 5
Quantification of interfaces within the γ-carbonic-anhydrase (γCA) domain and between γCA and the proximal pumping domain (PP) of CI*.

Interface residues, surface areas, solvation free energies and P-values were determined by uploading the molecular model of CI* into the the PDBePISA tool for the exploration of macromolecular interfaces (Krissinel and Henrick, 2007). The table with the full list of interaction surfaces for CI* was filtered for the interfaces involving CA1, CA2 or CAL2. Total values were obtained by adding the relevant two-way interactions, as per PDBePISA guidelines.

Subunit 1Subunit 2Inter-subunit interface
Subunit# Interfacing residuesInterfacing surface area (Å2)Subunit# Interfacing residuesInterfacing surface area (Å2)Interface surface area (Å2)Interface solvation free energy (kcal/mol)Solvation free energy gain P-value
Within γCA domain
CA18820,989CA28022,0882581.4−45.60.000
CA17920,989CAL27220,0732637.8−38.20.001
CA27322,088CAL26620,0732148.1−33.20.000
Total7367.3−117
Between γCA domain and membrane arm (PP)
NU2M167610CA11620,989259.2−17.10.001
NU2M167610CA21622,088261.1−17.10.001
NU2M167610CAL21620,073263.6−18.00.003
NDUC22113,708CA12120,989427.8−20.20.000
NDUC21613,708CA21622,088242.3−15.80.000
NDUC22113,708CAL22220,073416.2−18.70.000
P22110,433CA12120,989362.6−16.50.000
P22610,433CA22222,088512.7−21.50.001
P21710,433CAL21720,073244.6−16.80.001
NDUX11613,955CA11620,989236.5−15.90.000
NDUX11713,955CA21722,088268.1−15.00.000
NDUX11613,955CAL21620,073240.9−16.70.000
Total3735.6−209.3
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IdentifiersAdditional
information
Biological sample (Vigna radiata) V. radiata seedsTodd’s Tactical GroupTS-229Lot SMU2-8HR; DOB 2/25/2019
Commercial assay or kitPierce BCA assay kitThermo Fisher23225
Commercial assay or kit3–12% NativePAGE gels and buffersInvitrogenBN1001BOX; BN2001; BN2002
Chemical compound, drugDigitonin, high purityEMD Millipore300410
Chemical compound, drugA8-35AnatraceA835
Chemical compound, drugGamma-cyclodextrinEMD MilliporeC4892
Chemical compound, drugNADHVWR Life Sciences97061–536
Chemical compound, drugNitrotetrazoleumEMD Millipore74032
Software, algorithmSerialEMUniversity of Colorado, Schorb et al., 2019RRID:SCR_017293
Software, algorithmRELION 3.0Zivanov et al., 2018RRID:SCR_016274
Software, algorithmMotioncor2Zheng et al., 2017
Software, algorithmCtffind4Rohou and Grigorieff, 2015RRID:SCR_016732
Software, algorithmcrYOLOWagner et al., 2019Wagner and Raunser, 2020RRID:SCR_016732
Software, algorithmPhyre2Kelley et al., 2015
Software, algorithmCootEmsley and Cowtan, 2004RRID:SCR_014222
Software, algorithmPHENIXLiebschner et al., 2019Goddard et al., 2018; Pettersen et al., 2004RRID:SCR_014224
Software, algorithmUCSF ChimeraResource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, Pettersen et al., 2004RRID:SCR_004097
Software, algorithmPyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.Schrödinger, LLCRRID:SCR_000305Version 2.0
OtherHoley carbon gridsQuantifoilQ310CR1.31.2/1.3 300 mesh
Appendix 1—table 1
Values used in the calculations.
VariableValueSource
Δp160 mVValues of 140–190 mV have been reported from respiring cells (Ripple et al., 2013); a value of 200 mV was reported for isolated etiolated V. radiata mitochondria after addition of 1 mM NADH, which defines an upper limit for steady-state respiration (Moore and Bonner, 1981)
R8.314 kJ K−1 mol−1Physical Constant
T300 KApproximately 27 °C
F96,485 C mol−1Physical Constant
Em,7CoQ4 mVThis value varies as a function of pH so should only be considered an estimate (Nicholls, 2013)
CoQIMM/ [CoQH2]IMM10Kim et al., 2012
Eh,7CoQ34 mVCalculated from Em,7CoQ and CoQIMM/[CoQH2]IMM using Equation 1
Em,7NADH−320 mVThis value varies as a function of pH, so should only be considered an estimate (Nicholls, 2013)

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