Mutant/mutation
Different mutants are generated and described that contain a modified/mutated trap gene locus. 

Generation of S210C, S210c/Q216C and S210C/F224C parasites 
To generate the parasite lines S210C, S210C/Q216C and S210C/F224C we made use of a synthetic trap gene that had been codon modified for E. coli K12 and used previously to create I domain exchange mutants (Klug et al., 2020). The synthetic trap gene cloned in the pMK-RQ vector (Invitrogen) was targeted by site-directed mutagenesis using the primers P1149/P1150 (S210C), P1153/P1154 (Q216C) and P1151/P1152 (F224C). S210C/Q216C and S210C/F224C mutations were introduced sequentially. Mutated sequences were cloned into the Pb238-TRAP-NdeI/PacI vector (NdeI/PacI), linearized (ScaI-HF) and transfected into P. berghei ANKA using standard protocols.

To generate TRAP mutants with an I domain stabilized in the closed state, we substituted cysteines for Ser-210 and Phe-224 in a codon modified version of trap. Similarly, we mutated Ser-210 and Gln-216 to cysteine to stabilize the I domain in the open state, and used a single S210C mutant as a control. These TRAP constructs were ligated into a previous vector (Klug et al., 2020) in order to replace the endogenous TRAP with the three different constructs. Constructs were either transfected into the fluorescent line cFluo, expressing EGFP constitutively from the ef1α promoter and mCherry in insect stages from the csp promoter, or a non-fluorescent trap(-) line (Bane et al., 2016; Klug et al., 2020).
Generation of clonal lines yielded the parasite lines S210C (control, fluorescent), S210C/F224C (closed, non-fluorescent) and S210C/Q216C (open, fluorescent). 

Protein (function)
TRAP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells.

Phenotype
Sporozoites egressed efficiently from oocysts and could be isolated from the hemolymph. Western blotting of hemolymph-derived sporozoites showed similar expression of TRAP in the wild type, control and conformationally stabilized parasite lines. 
Immunofluorescence analysis of hemolymph derived sporozoites that were fixed but not permeabilized revealed similar signals of TRAP on the surface in all lines (Figure 2C). The numbers of sporozoites were similar in the midgut and hemolymph for all lines but showed a dramatic drop in salivary gland residency of the lines expressing TRAP in the closed and open conformations. 
To investigate the capacity to infect mice, we isolated sporozoites of all three lines as well as the wild type from the hemolymph and injected 10,000 sporozoites of each line into individual naïve C57Bl/6 mice. This showed the expected 100% infection rate in wild-type and S210C control  parasites with a prepatent period of around 5 days. However, hemolymph sporozoites expressing the closed or open TRAP I domains infected only around 50% of mice and those infected showed a prolonged prepatent period of 6-7 days. 
Infection of mice by mosquito bite showed the following: the two control lines (wild type and S210C) infected all mice and showed prepatent periods of 3-4 days. In contrast,   mosquitoes that were infected by the closed mutant (S210C/F224C) could not infect mice while one out of ten mice bitten by mosquitoes harboring the open mutant (S210C/Q216C) was infected. 
The number of salivary gland-derived sporozoites in the open mutant (S210C/Q216C) was higher, but not significantly higher, than in the closed mutant (S210C/F224C); however, we never managed to isolate sufficient numbers of sporozoites from salivary glands of the closed mutant (S210C/F224C) to conduct further experiments.

Additional information
Evidence is presented that:
- Conformationally stabilized open mutants do not glide.

Other mutants