ИСТИНА

The GroEL-GroES complex is a bacterial protein foldingmachine working in an ATP-dependent manner. Despite being studied for decades, details of its functional cycle remain underdebate. One of the discussed topics is whether the two rings of GroEL function simultaneously (implying the formation of asymmetric “football-shaped” complex) or alternately (through anasymmetric “bullet-shaped” form). Both football- and bullet-shaped complexes were resolved experimentally using X-ray crys-tallography or cryo-electron microscopy, and the corresponding atomic models are available in the Protein Data Bank. However,the deposited cryo-EM structures of the GroEL-GroES complexare of 7A or lower resolution and often with an imposed C7 symmetry, which limits their interpretation. In this work, we have obtained a cryo-EM structure of the bullet-shaped GroEL-GroES complex at 4.3A with no symmetry applied. A samplefor cryo-EM was prepared as follows: GroEL (1 mkM) was incubated with GroES (3 mkM) in 50 mM Tris-HCl buffer, pH 7.5,containing 10 mM KCl, 10 mM MgCl2, 3 mM ATP for 20 min. Then, the sample was concentrated 10 times using Vivaspin 500with 100,000 MWCO PES membrane centrifugal filters. 3 mkl of the sample was applied to a glow discharged grid (QuatifoilR1.2/1.3) and vitrified in Vitrobot Mark IV at 4.5°C. Cryo-EM movie stacks were recorded on a Titan Krios electron microscope (Thermo-Fisher) equipped with the direct electron detector Fal-con II. 6100 image stacks were collected and further processed inWarp and CryoSPARC. 95208 particles were selected for the final symmetry-free 3D reconstruction. The local resolution ofthe equatorial domains reached 3.5A which allowed us to distinguish extra densities in the ATP-binding pockets of both rings. The resolution variations also indicate that, compared to the GroES-bound ring, the second ring was more flexible at the apical domains. The authors acknowledge funding from the Russian Science Foundation (grant № 19-74-20055).