Protein interfaces play an essential role in both natural and man-made materials as stabilizers, sensors, catalysts, and selective channels for ions and small molecules. Probing the molecular arrangement within such interfaces is of prime importance to understand the relation between structure and functionality. Here we report on the preparation and characterization of large area suspended crystalline films of class II hydrophobin HFBI. This small, amphiphilic globular protein readily self-assembles at the air water interface into a 2D hexagonal lattice which can be transferred onto a holey carbon electron microscopy grid yielding large areas of hundreds of square micrometers intact hydrophobin film spun across micronsized holes. Fourier transform analysis of low-dose electron microscopy images and selected area electron diffraction profiles reveal a unit cell dimension a = 5.6 +/- 0.1 nm, in agreement with reported atomic force microscopy studies on solid substrates and grazing incidence X-ray scattering experiments at the air water interface. These findings constitute the first step towards the utilization of large-area suspended crystalline hydrophobin films as membranes for ultrapurification and chiral separation or as biological substrates for ultrafast electron diffraction. (C) 2015 Elsevier Inc. All rights reserved.