Bacteria resistant to antibiotics are being selected in a relatively short time, and cases of infections resistant to treatment by all known antibiotics are being identified at alarming rates. The primary mechanism for resistance to beta-lactam antibiotics is the catalytic function of beta-lactamases. However, imipenem (a beta-lactam) resists the action of most beta-lactamases and is virtually the last effective agent against the vancomycin-resistant Gram-positive bacteria, as well as against multiple antibiotic-resistant Gram-negative organisms. Here, we report the crystal structure, to 1.8 Angstrom resolution, of an acyl-enzyme intermediate for imipenem bound to the TEM-1 beta-lactamase from Escherichia coil, the parent enzyme of 67 clinical variants. The structure indicates an unprecedented conformational change for the complex which accounts for the ability of this antibiotic to resist hydrolytic deactivation by beta-lactamases. Computational molecular dynamics underscored the importance of the motion of the acyl-enzyme intermediate, which may be a general feature for catalysis by these enzymes.