Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu

Abstract

X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

Figure 1 ∣. Endogenous structural proteomics workflow.

a, Protein complexes are enriched by sucrose gradient fractionation. b-c, Fractions are evaluated by SDS-PAGE (b) and negative stain electron microscopy (c). d, Mass spectrometry identifies a list of all proteins in each fraction. e, cryoEM analysis yields near-atomic resolution cryoEM maps. Scale bars, 30 nm (micrograph, top); 10 nm (2D class averages, bottom). f, The proteins in the cryoEM maps are identified using cryoID.

Figure 2 ∣. Simplified 6-Letter Code.

a,The 20 amino acid residues are clustered into 6 simplified groups, based on the similarity of their side chain densities in typical cryoEM density maps. One residue from each group is chosen as the representative of the entire group, denoted by the large colored single letter label in each group shown here. (i.e., G group = small-size side chain density, L group = medium-size side chain density, K group = long and thin side chain density, P group = typical proline side chain density, Y group = long and bulky side chain density) b, cryoID predicts the identity of each residue in the density on the left, and then simplifies the resulting sequence of the entire segment into the degenerate 6-letter code shown on the right.

Figure 3 ∣. Searching in cryoID.

a, cryoID runs alignments of the simplified query sequences obtained from the cryoEM maps against each protein in a pool of candidate proteins (also simplified). b, We created a customized alignment scoring matrix for cryoID by adapting the BLASTP PAM30 scoring matrix to work with the simplified 6-letter code used by cryoID. c, d, cryoID calculates a composite E-value for the alignment between each protein candidate against the query set, and then ranks the candidates by E-value to determine the most likely match to the query set.

Figure 4 ∣. CryoEM structures of proteins enriched directly from P. falciparum parasite lysates.

a,e, 3.2Å cryoEM density map and atomic model of P. falciparum M18 aspartyl aminipeptidase (a) and glutamine synthetase (e). b,f, Enlarged view of the P. faciparum M18 aspartyl aminopeptidase (b) and glutamine synthetase (f) monomer. Segments from which the queries for cryoID were generated are highlighted in pink. c,g, Local resolution (in Å) calculated using Resmap and two unfiltered halves of the reconstruction for P faciparum M18 aspartyl aminopeptidase (c) and glutamine synthetase (g). d,h, Detailed view of regions boxed in (b & f), displayed with corresponding cryoEM density.

Figure 5 ∣. Details of the M18 aspartyl aminopeptidase and glutamine synthetase monomers.

a, A single monomer from our atomic model of the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP), solved by cryoEM using our endogenous structural proteomics workflow, colored to indicate the regulatory (pink) and catalytic (sea green) domains. b, Our atomic model of PfM18AAP (sea green), solved by cryoEM using our endogenous structural proteomics workflow, is shown superimposed with the previously published structure of PfM18AAP (gold), solved using X-ray crystallography. The structures align with an RMSD of 0.548Å. c, The previously published structure of the S. enterica glutamine synthetase, solved using X-ray crystallography, colored dark grey. d, Our cryoEM structure of the P. falciparum glutamine synthetase, colored cornflower blue, is shown superimposed with the S. enterica glutamine synthetase crystallographic structure. The two structures align with an RMSD of 1.5Å. e, A single monomer from our atomic model of the P. falciparum glutamine synthetase, determined by cryoEM using our endogenous structural proteomics workflow, colored in cornflower blue. We observed an extra 50-residue insertion in the P. falciparum structure (colored red) that is absent in the S. enterica structure. This long insertion forms a large flap that curls away from the active site, unlike the shorter flap formed by the corresponding region in the S. enterica glutamine synthetase (colored green), which curls toward the active site.