X-ray crystallography provides the vast majority of macromolecular structures, 
but the success of the method relies on growing crystals of sufficient size. In 
conventional measurements, the necessary increase in X-ray dose to record data 
from crystals that are too small leads to extensive damage before a diffraction 
signal can be recorded. It is particularly challenging to obtain large, 
well-diffracting crystals of membrane proteins, for which fewer than 300 unique 
structures have been determined despite their importance in all living cells. 
Here we present a method for structure determination where single-crystal X-ray 
diffraction 'snapshots' are collected from a fully hydrated stream of 
nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the 
Linac Coherent Light Source. We prove this concept with nanocrystals of 
photosystem I, one of the largest membrane protein complexes. More than 
3,000,000 diffraction patterns were collected in this study, and a 
three-dimensional data set was assembled from individual photosystem I 
nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation 
damage in crystallography by using pulses briefer than the timescale of most 
damage processes. This offers a new approach to structure determination of 
macromolecules that do not yield crystals of sufficient size for studies using 
conventional radiation sources or are particularly sensitive to radiation 
damage.