Proteins are known to undergo a dynamical transition at around 200 K but the underlying mechanism, physical origin, and relationship to water are controversial. Here we report an observation of a protein dynamical transition as low as 110 K. This unexpected protein dynamical transition precisely correlated with the cryogenic phase transition of water from a high-density amorphous to a low-density amorphous state. The results suggest that the cryogenic protein dynamical transition might be directly related to the two liquid forms of water proposed at cryogenic temperatures.conformation fluctuations | water phase transition | high-pressure cryocooling | X-ray crystallography I t is known that a hydrated protein undergoes a dynamical transition at around 200 K (1). Proteins below the transition temperature are in a glassy state with little conformational flexibility and no appreciable biological function; above the transition temperature flexibility is restored and the protein becomes biologically active (2-4). This protein dynamical transition has been extensively studied by measuring the mean square atomic displacement, hx 2 i, of protein atoms as a function of temperature with various methods, including Mössbauer and terahertz time domain spectroscopies (5-7), X-ray crystallography (4, 8-11), neutron scattering (2,(12)(13)(14)(15)(16)(17)(18)(19), and molecular dynamics simulations (20)(21)(22)(23)(24)(25).It is believed that the protein dynamical transition involves a strong coupling to the hydration water (10,26,27), as a protein dehydrated below a critical level (water/protein mass ratio of ∼0.2) does not show the dynamical transition (13,14,28). Several mechanisms have been proposed to explain the microscopic origin of protein dynamical transition, including α and β fluctuations in the bulk solvent and the hydration shell (29, 30), liquid-glass transition of hydration water (31), a frequency window scenario (32, 33), and a fragile to strong transition of the hydration water (15,20).In this work, we studied a protein dynamical transition inside protein crystals using a high-pressure cryocooling method (34) and temperature-dependent X-ray diffraction (8,35,36). Protein crystals contain large amounts of water in solvent channels between the proteins in the crystal lattice; water fractions of greater than 50% are very common. It has been reported that this intracrystalline water can be cryocooled under high pressure into a high-density amorphous (HDA) state that subsequently transforms upon heating to the low-density amorphous (LDA) (36) water typical of ambient-pressure flash-cooling [in bulk water the respective densities of HDA and LDA are 1.17 and 0.94 g cm −3 at 77 K and 0.1 MPa. (37, 38)]. In this study, the temperature of the HDA-LDA transition was adjusted by controlling solvent conditions inside the protein crystals. X-ray diffraction data from five thaumatin crystals were used for this study, including four high-pressure cryocooled crystals (Thau-0M-1, Thau-0M-2, Thau-0.45M, Thau-0.9M; 0.9M indicates w...