Reconstructing complete native genome of a virus from cryo-EM density: correspondence between the measured resolution and RNA flexibility Dmitry Nerukh 1 and Vladimir Farafonov 2 1 Aston University, UK, 2 Kharkiv National University, Ukraine Cryo-EM measures the density of biomolecular systems at resolution allowing fitting the atomistic structure of systems as large as whole organelles or complete viruses. However, the resolution is not uniform, at some locations being significantly lower than needed for direct fitting at atomistic detail. Here we use the density of a whole bacteriophage MS2 to reconstruct the complete all-atom model of the virus, including its native genome, which is measured at mixed resolution. Using our model, we show quantitatively that the low resolution regions of measurements are most probably caused by the flexibility of the RNA at these regions, as intuitively expected, but not quantitatively demonstrated so far. The available cryo-EM density of MS2 genome contains regions at three levels of detail: high-resolution, moderate resolution, and unresolved. The former regions constituted 16 stem-loops contacting the capsid, their atomistic reconstruction was possible. The regions of moderate resolution allowed recognise the nucleic backbone only without identifying the particular nucleotides. They are adjacent to the capsid mostly. The unresolved region is located deeper in the capsid. Possible reasons for lower resolution can be both the properties of the molecule (flexibility, mobility) and cryo-EM method limitations of various nature. Recently we have reconstructed the complete genome including poorly and non-resolved regions [1]. For this, an algorithm that included models at various resolutions was devised. The genome was then MD simulated together with the capsid at laboratory conditions (physiological saline, 25°С). With the model it is now possible to elucidate the reason for the inhomogeneous character of cryo-EM density. Here we examine the correspondence between the experimental resolution of the nucleotides and their root mean square fluctuation (RMSF) during the simulation. The experimental resolution was represented by a number from the set {0, 1, 2} where 0 means high resolution, 1 – the moderate one, 2 – unresolved. The RMSF was computed for the nucleotide’s phosphorus atoms. The obtained data is presented in the Figure.
Figure. The variation of experimental resolution (red) and RMSF (black) of nucleotides across the genome from MD simulation. Grayed are single-stranded intervals longer than 10 residues. The red curve is scaled down 4 times. There is a pronounced correspondence between the two metrics. The nucleotides having RMSF higher than ~0.5 nm are mostly unresolved. In contrast, the well resolved nucleotides always correspond to local minima: their RMSF is 0.1–0.15 nm. Long single stranded chains also usually correspond to local maxima of RMSF and belong to the poorly resolved regions. Overall, the calculations demonstrate that the likely reason for the deficiency of cryo-EM measurements is, in this case, the flexibility of the macromolecule and higher mobility of some its segments. References 1. Vladimir S. Farafonov, Michael Stich, and Dmitry Nerukh, Reconstruction and validation of entire virus model with complete genome from mixed resolution cryo-EM density, Faraday Discuss. , 240 , 152-167 (2022)
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