Plant metacaspase: A case study of microcrystal structure determination and analysis

Metacaspases are highly conserved in plants and play essential roles in mediating programmed cell death, biotic and abiotic stress responses, and damage-induced innate immunity. Ca²⁺ signaling induced by plant damage leads to activation of metacaspase 4 from Arabidopsis thaliana (AtMC4), which subsequently processes a plant elicitor peptide to trigger downstream immuno-response. To understand the structural basis of AtMC4 activation by Ca²⁺, we previously determined its crystal structure and performed in-crystal Ca²⁺ treatment to probe activation-associated conformational changes. To enable structure determination and in-crystal Ca²⁺ activation analysis, we used microcrystals and related methods which were essential for our successful approach. Here we describe in detail the methods that we used for determination of AtMC4 structure using single-wavelength isomorphous replacement with anomalous signals assembled from 22 microcrystals. We also describe the method for in-crystal Ca²⁺ soaking, microcrystal data collection, data assembly and analysis to obtain the activated structure of AtMC4 from 91 micro-sized crystals. The described methods may be useful to study other plant metacaspases and more broadly other plant enzymes for their structure determination and in-crystal functional characterization.