Supporting InformationRoh et al. 10.1073/pnas.1704725114

Fig. S1. A typical motion-corrected and radiation damage-compensated K2-summit image created using DE-process-frame.py. (A) Uncorrected sum image of atotal dose of 50 e−/A2. (B) Identical micrograph after drift correction and radiation damage weighting of frozen hydrated GroEL chaperonin. (C) Powerspectrum of an image showing simulated contrast transfer function rings on the top left and experimental results. The rings extend to ∼3 Å. (D) A workflowdiagram for the reconstruction of maps with imposed D7 symmetry. Early frame images with 20 e−/A2 were used for the final map reconstruction (EMD-8750).

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Fig. S2. Gold standard FSC and map vs. model FSC. (A) Gold standard FSC curve showing 3.5 Å resolution at 0.143. The FSC between the 3.5-Å map and therefined model shows a similar resolution at 0.5. (B) Per-residue correlation between the map and the model.

Fig. S3. Exploring the localized flexibility on the E. coli chaperonin GroEL. (A) Color-coded local resolution distribution for the 3.5-Å map created from ex-perimental data. (B) Atomic displacement parameter: color-coded B-factor distribution of the 3.5-Å cryo-EM model (PDB ID code 5W0S) and the 2.8-Å crystalstructure (PDB ID code 1OEL).

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Fig. S4. Detecting conformational variants of the E. coli chaperonin GroEL. (A) Focused classification resulted in three converged classes with differentpopulations: class I, 18%; class II, 30%; class III, 23%; 29% of the total population was discarded because of low-resolution convergence. (B) An identicalprocessing protocol used on simulated single-conformation data resulted in a single dominant population. (C) Identical processing of simulated data with threecryo-EM conformations resulted in three major populations; simulated particles with experimental noise (Top) and without noise (Bottom).

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Fig. S5. Superposition and rmsd of cryo-EM classes and crystal structures. (A) Superposition and (B) rmsd of the C-alpha backbone of subunits in (a) three apo-GroEL crystal structures with imposed symmetry (PDB ID codes 1OEL, 1SS8, and 1GRL), and 14 different conformations in (b) 1XCK and (c) cryo-EM classes.

Fig. S6. Subunit-based structural analysis. (A) Numbers of subunits in GroEL falling into the three major class conformations. (B) Composition of confor-mations in the single GroEL particles.

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Fig. S7. Apical domain flexibility is linked with chemical switches in apo-GroEL. (A) Three superimposed cryo-EM conformations and the locations of saltbridge between K327 (blue) and D83 (red). (B) Distance of each salt bridge in three cryo-M conformations. (C) Superposition of three cryo-EM models, showingthe orientation change of D87 in the ATP-binding pocket according to the change in the salt bridge distance between K327 and D87.

Table S1. Data collection and map processing parameters

Data collection and image processing steps Instrument/software/settings Specifics

Cryo-specimen freezing Leica EM GP One blot; 90% RH; blot time, 2∼3 sElectron microscope JEM3200FSC 300 KeV, in-column energy filter (25 eV)Detector Gatan K2 Summit 4K × 4K, 5 μm pixel sizeSampling interval 0.615 Å/Pixel SuperresolutionExposure rate on specimen 5 e−/Å2/s 5 frames/sExposure time 10 s Total dose, 50 e−/Å2

Movie-mode micrographs 260 used for processing Out of 324 collectedDefocus range 0.7–2.5 μmDefocus determination EMAN2.1Particles picked EMAN2.1 37,367 for the final map reconstructionParticle box size 240 × 240Drift and damage compensation DE_process_frame.pyInitial map generation EMAN2.1Map refinement RELION1.4Resolution 3.5 Å Gold standard FSC at 0.143Modeling Chimera, Coot, and Phenix Initial model 1SS8

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Table S2. Model optimization results

All-atom contacts:Clash score*, all atoms 12.27 Percentile ranking** Expected goal

Protein geometryPoor rotamers 0 0.00% Goal: <0.3%Favored rotamers 5,600 99.01% Goal: >98%Ramachandran outliers 0 0.00% Goal: <0.05%Ramachandran favored 7,168 98.08% Goal: >98%MolProbity score 1.6 100th percentile* (n = 342, 3.25–3.95 Å)Cβ deviations >0.25 Å 0 0.00% Goal: 0Bad bonds 0/54,306 0.00% Goal: 0%Bad angles 0/73,346 0.00% Goal: <0.1%

Peptide omegasCis prolines 0/196 0.00% Expected: ≤1 per chain, or ≤5%

Low-resolution criteriaCa BLAM outliers 56 0.77% Goal: <1.0%Cα geometry outliers 28 0.38% Goal: <0.5%

Fit-to-densityCross-correlation 0.926EmRinger score 1.438

*Clash score is the number of serious steric overlaps (>0.4 Å) per 1,000 atoms.**97th percentile (n = 37, 3 Å–9,999 Å).

Table S3. C-alpha distance between 1XCK and Cryo-EM models

Chain

Cryo-EM map-dervied model

Regions ofcomparison A B C D E F G H I J K L M N Minimum

D7 Overall 0.79 0.97 1.05 0.84 0.96 1.10 0.70 1.05 0.72 0.66 0.86 0.69 0.88 1.31 0.66Apical 1.29 1.84 2.08 1.46 1.83 2.17 1.11 2.07 1.20 1.00 1.57 1.06 1.55 2.77 1.00H6,7 1.29 2.55 2.48 1.44 2.00 2.41 1.11 2.42 1.35 1.03 1.98 1.03 1.67 3.27 1.03

Class I Overall 1.02 0.97 1.71 1.43 1.04 1.75 1.34 1.71 1.36 1.05 0.90 1.23 1.11 1.97 0.90Apical 1.61 1.42 3.59 2.78 1.67 3.68 2.58 3.58 2.70 1.83 1.33 2.27 1.86 4.29 1.33H6,7 1.40 1.33 4.37 3.11 1.44 4.32 2.90 4.31 3.19 2.08 1.16 2.54 1.77 5.19 1.16

Class II Overall 0.96 1.15 1.09 0.90 1.13 1.13 0.77 1.09 0.79 0.83 1.04 0.80 1.02 1.33 0.77Apical 1.46 2.05 1.90 1.34 2.00 1.97 1.02 1.89 1.12 1.16 1.80 1.07 1.66 2.57 1.02H6,7 1.72 2.95 2.11 1.24 2.37 2.02 0.94 2.04 1.17 1.36 2.42 1.05 1.91 2.84 0.94

Class III Overall 1.20 1.43 0.99 0.94 1.38 1.04 0.79 1.00 0.76 1.00 1.31 0.93 1.25 1.21 0.76Apical 2.00 2.70 1.49 1.33 2.56 1.58 0.96 1.50 0.91 1.54 2.42 1.30 2.18 2.10 0.91H6,7 2.47 3.78 1.52 1.28 3.18 1.43 1.03 1.47 1.03 1.88 3.23 1.41 2.65 2.13 1.03

Bold represents minimum C-alpha distance in comparisons.

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Movie S1. The primary difference among the three conformers is confined to the apical domains, which derives from a hinge-like motion around the in-termediate region of the subunit.

Movie S1

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