BioelectricaAlterspan Labs
Bioenergetics  ·  Article

Conformational variability and focused refinement in supercomplex cryo-EM

Respiratory supercomplexes are not uniform in cryo-EM samples. Particles differ in composition and in the relative positions of their parts. A single averaged map therefore blurs the structure. Sousa and colleagues used 3D classification to sort particles12. Letts and colleagues added focused refinement of separate regions56. The same approach was applied to dimeric ATP synthase9.

Earliest held
2016, Sousa JS, Mills DJ, Vonck J…
Most discussed in
Structures of Respiratory Supercomplex…, 2019
In the library
14 passages in 3 works
Rewritten
2026-11-01
18501875190019251950197520002025Sousa JS, Mills DJ, Vonck J, Kühlbrandt…, 20161Sousa JS, Mills DJ, Vonck J, Kühlbrandt…, 20162Sousa JS, Mills DJ, Vonck J, Kühlbrandt…, 20163Sousa JS, Mills DJ, Vonck J, Kühlbrandt…, 20164Letts JA, Fiedorczuk K, Degliesposti G…, 20195Letts JA, Fiedorczuk K, Degliesposti G…, 20196Letts JA, Fiedorczuk K, Degliesposti G…, 20197Letts JA, Fiedorczuk K, Degliesposti G…, 20198Spikes TE, Montgomery MG, Walker JE, 20209
01

Blurred consensus maps

Sousa, Mills, Vonck and Kühlbrandt (2016) prepared the bovine supercomplex I1III2IV1 in amphipol A8-35 for cryo-EM. They picked 156,519 particles by hand and kept 137,606 after class selection for a first consensus refinement4. The overall map reached 8.1 Å. Its definition varied across the structure, which suggested an average of several assemblies or conformations1. Repeated 3D classification confirmed a high degree of compositional and conformational heterogeneity2.

02

Classification as sorting

In the same study, the most homogeneous class held 17,094 particles, 11% of the total. It gave a respirasome structure at 9.1 Å3. That figure is a slightly lower nominal resolution than the consensus map, and it came from fewer particles. The authors therefore traded particle number for homogeneity. Their report supports the view that the average map hid distinct states rather than one rigid structure.

03

Focused refinement of regions

Letts, Fiedorczuk, Degliesposti, Skehel and Sazanov (2019) studied supercomplex I+III2. They found that the relative orientation between the two arms of complex I, and between complex I and CIII2, was variable. This lowered resolution at the edges of the peripheral arm and CIII25. They refined three regions independently: the CI peripheral arm, the CI membrane arm and CIII26. The best peripheral arm class of 178,121 particles gave a 3.8 Å map suitable for atomic modeling7.

04

Further uses

Classification also exposed small populations. Letts and colleagues found a minor class of particles lacking the NADH-binding N-module of the peripheral arm8. Focused refinement was not limited to respiratory supercomplexes. Spikes, Montgomery and Walker (2020) improved the map quality and regional resolution of dimeric bovine ATP synthase by focused refinement of the stator, PS and membrane domain9.

SourcesEach quotation was checked word for word against the passage it opens.
  1. The map definition varied considerably across the structure, suggesting that it might be an average of several different assemblies or conformations.Sousa JS, Mills DJ, Vonck J, Kühlbrandt W, 2016 · Functional asymmetry and electron flow in the bovine respirasome · open at passage 11
  2. Multiple rounds of 3D classification indeed revealed a high degree of compositional and conformational heterogeneity.Sousa JS, Mills DJ, Vonck J, Kühlbrandt W, 2016 · Functional asymmetry and electron flow in the bovine respirasome · open at passage 11
  3. The most homogeneous class of 17,094 particles (class 1; 11% of the total) yielded a structure of the respirasome at a final resolution of 9.1 Å.Sousa JS, Mills DJ, Vonck J, Kühlbrandt W, 2016 · Functional asymmetry and electron flow in the bovine respirasome · open at passage 11
  4. Visual selection of particle classes with interpretable features resulted in a dataset of 137,606 particle images for the first 3D consensus refinement.Sousa JS, Mills DJ, Vonck J, Kühlbrandt W, 2016 · Functional asymmetry and electron flow in the bovine respirasome · open at passage 35
  5. the relative orientation between the two arms of CI and between CI and CIII2 in SC I+III2 was variable, which resulted in lower resolutionLetts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA, 2019 · Structures of Respiratory Supercomplex I+III<sub>2</sub> Reveal… · open at passage 61
  6. To improve the maps, focused refinements were independently performed around three regions of the SC: the CI peripheral arm, the CI membrane arm and CIII2 (Figure S6).Letts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA, 2019 · Structures of Respiratory Supercomplex I+III<sub>2</sub> Reveal… · open at passage 61
  7. Interestingly, a small class of particles was identified that lacked the NADH-binding N-module of the peripheral arm (Figure S5A).Letts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA, 2019 · Structures of Respiratory Supercomplex I+III<sub>2</sub> Reveal… · open at passage 61
  8. When we used the best class of 178,121 particles for 3D-refinement around the CI peripheral arm, we achieved a reconstruction at 3.8 ÅLetts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA, 2019 · Structures of Respiratory Supercomplex I+III<sub>2</sub> Reveal… · open at passage 62
  9. The quality of the map and the resolution of specific regions were improved by focused refinement of the stator, PS, and membrane domain.Spikes TE, Montgomery MG, Walker JE, 2020 · Structure of the dimeric ATP synthase from bovine mitochondria · open at passage 21
Linked ideas
The respirasome structure is the main object whose heterogeneity and classification are described here.
Conformational states of complex I are the kind of variability that classification sorts into separate classes.
Gives the functional context for why the arrangement of complexes within supercomplexes is worth resolving.
The particles lacking the N-module connect to the assembly account of complex I.
Dimeric ATP synthase maps were also improved by focused refinement of separate domains.
The detergent, amphipol and lipid environment of purified complexes bears on how supercomplexes are prepared for imaging.
Where it is discussedPassages matching focused refinement, 3D classification, relative orientation of CI and CIII2, amphipol-stabilized supercomplex
2019Structures of Respiratory Supercomplex I+III<sub>2</sub> Reveal… · Letts JA, Fiedorczuk K, Degliesposti G…11
2016Functional asymmetry and electron flow in the bovine respirasome · Sousa JS, Mills DJ, Vonck J, Kühlbrandt…2
2020Structure of the dimeric ATP synthase from bovine mitochondria · Spikes TE, Montgomery MG, Walker JE1