[논문]EMPAS: Electron Microscopy Screening for Endogenous Protein Architectures

Kim, Gijeong; Jang, Seongmin; Lee, Eunhye; Song, Ji-Joon

doi:10.14348/molcells.2020.0163

[국내논문] EMPAS: Electron Microscopy Screening for Endogenous Protein Architectures 원문보기

Molecules and cells, v.43 no.9, 2020년, pp.804 - 812

Kim, Gijeong (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)) , Jang, Seongmin (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)) , Lee, Eunhye (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST)) , Song, Ji-Joon (Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST))

Abstract ▼ AI-Helper

In cells, proteins form macromolecular complexes to execute their own unique roles in biological processes. Conventional structural biology methods adopt a bottom-up approach starting from defined sets of proteins to investigate the structures and interactions of protein complexes. However, this approach does not reflect the diverse and complex landscape of endogenous molecular architectures. Here, we introduce a top-down approach called Electron Microscopy screening for endogenous Protein ArchitectureS (EMPAS) to investigate the diverse and complex landscape of endogenous macromolecular architectures in an unbiased manner. By applying EMPAS, we discovered a spiral architecture and identified it as AdhE. Furthermore, we performed screening to examine endogenous molecular architectures of human embryonic stem cells (hESCs), mouse brains, cyanobacteria and plant leaves, revealing their diverse repertoires of molecular architectures. This study suggests that EMPAS may serve as a tool to investigate the molecular architectures of endogenous macromolecular proteins.

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표/그림 (5)

$Fig. 1. Schematic workflow of EMPAS: Fractionations, target selection, identification, and structural determination. The lysate containing endogenous protein mixtures was fractionated and examined with negative-stain EM. From the collected micrographs, molecular architectures of interest are selected (indicated with red boxes). After target selection, targeted fractionation followed by mass spectrometry revealed the composition of the target molecular architectures. Further cryo-EM analysis determines the high-resolution structure of the targeted molecular architecture (Scale bar = 50 nm, bottom left panel).$ 그림 Fig. 1. Schematic workflow of EMPAS: Fractionations, target selection, identification, and structural determination. The lysate containing endogenous protein mixtures was fractionated and examined with negative-stain EM. From the collected micrographs, molecular architectures of interest are selected (indicated with red boxes). After target selection, targeted fractionation followed by mass spectrometry revealed the composition of the target molecular architectures. Further cryo-EM analysis determines the high-resolution structure of the targeted molecular architecture (Scale bar = 50 nm, bottom left panel).
$Fig. 2. E. coli sample preparation for EMPAS. (A) An anion exchange chromatogram profile (top panel) and SDS-PAGE gel (bottom panel) of E. coli cell lysate. The fractionated samples are categorized into four groups based on the pattern of proteins analyzed by SDS-PAGE. (B) Gel filtration profile of each group of the samples (left panel). Proteins eluted at near void volume (boxed) were collected and analyzed by SDS-PAGE (right panel).$ 그림 Fig. 2. E. coli sample preparation for EMPAS. (A) An anion exchange chromatogram profile (top panel) and SDS-PAGE gel (bottom panel) of E. coli cell lysate. The fractionated samples are categorized into four groups based on the pattern of proteins analyzed by SDS-PAGE. (B) Gel filtration profile of each group of the samples (left panel). Proteins eluted at near void volume (boxed) were collected and analyzed by SDS-PAGE (right panel).
그림 Fig. 3. Representative negative-EM micrographs and 2D classes of endogenous macromolecular architectures. Representative micrographs of Group 1 (A), Group 2 (B), Group 3 (C), and Group 4 (D). White arrows indicate proteins having prominent architectures, which are enlarged within the boxes. (E) Three representative 2D class averages of the distinct architectures from the micrographs collected using the samples in Groups 1, 2, and 3.
$Fig. 4. Identification and structural determination of aldehyde-alcohol dehydrogenase (AdhE). (A) A negative-stain EM micrograph of the fraction containing the spiral architecture. Scale bar = 50 nm. (B) SDS-PAGE analysis of the fraction containing the spiral architecture (left panel). The top five abundant proteins from mass spectrometry identification are listed (right panel). (C) SDS-PAGE analysis of the purified recombinant E. coli AdhE protein. (D) A negative-stain EM micrograph of the recombinant E. coli AdhE protein showing spiral architectures. Scale bar = 50 nm. (E) Comparison among AdhE spiral architectures from negativestain micrographs from cell lysates (left panel), recombinant protein (middle panel), and the cryo-EM structure (right panel; PDB ID: 6AHC).$ 그림 Fig. 4. Identification and structural determination of aldehyde-alcohol dehydrogenase (AdhE). (A) A negative-stain EM micrograph of the fraction containing the spiral architecture. Scale bar = 50 nm. (B) SDS-PAGE analysis of the fraction containing the spiral architecture (left panel). The top five abundant proteins from mass spectrometry identification are listed (right panel). (C) SDS-PAGE analysis of the purified recombinant E. coli AdhE protein. (D) A negative-stain EM micrograph of the recombinant E. coli AdhE protein showing spiral architectures. Scale bar = 50 nm. (E) Comparison among AdhE spiral architectures from negativestain micrographs from cell lysates (left panel), recombinant protein (middle panel), and the cryo-EM structure (right panel; PDB ID: 6AHC).
$Fig. 5. Representative micrographs of lysates from human stem cells, cyanobacteria, mouse brains and tobacco plant leaves showing the diverse and complex landscape of molecular architectures. Representative negativestain EM micrographs (left panels) from fractionated cell lysates of hESCs (A), mouse brains (B), cyanobacteria (C), and tobacco leaves (D) and SDS-PAGE analysis of the sample. A few interesting forms of molecular architectures are shown in the right panels. Scale bars = 50 nm (B-D) or 100 nm (A).$ 그림 Fig. 5. Representative micrographs of lysates from human stem cells, cyanobacteria, mouse brains and tobacco plant leaves showing the diverse and complex landscape of molecular architectures. Representative negativestain EM micrographs (left panels) from fractionated cell lysates of hESCs (A), mouse brains (B), cyanobacteria (C), and tobacco leaves (D) and SDS-PAGE analysis of the sample. A few interesting forms of molecular architectures are shown in the right panels. Scale bars = 50 nm (B-D) or 100 nm (A).

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제안 방법

As we aimed to examine large molecular architectures, we subjected the sample of each group to Superose 6 size-exclusion chromatography and collected fractions eluted in the high molecular weight range(Fig. 2B).
After preparing the fractionated samples containing endogenous macromolecular complexes, we moved to the next step of EMPAS, target searching and identification. We examined molecular architectures in each group by using negative-stain EM (Fig.
3C and 3D). To further characterize the structures of the prominent molecular architecture, we collected a number of micrographs from each group and performed 2D class averaging using EMAN2 (Tang et al., 2007) with random particle selection. The 2D class average analysis of the micrographs collected from Groups 1 and 3 shows a tetrameric structure and a long fibric structure, respectively (Fig.
EMPAS is composed of several steps. First, as direct visualization by EM is not possible due to the complexity of endogenous molecular complexes in cell lysates, the complexity is reduced by multiple fractionations followed by grouping the samples based on the compositions of proteins in fractions assessed by SDS-PAGE analysis. Among the molecular architectures visualized by negative-stain EM, a specific architecture of interest, a spiral structure in this case, is targeted.

대상 데이터

The cut gel was washed with 1 ml of distilled water three times and stored at4°C before mass spectrometry analysis. The cut gel was sent to the Taplin Mass Spectrometry Facility at Harvard MedicalSchool.

이론/모형

The stained grid was blotted and air-dried before observation. The prepared grid was analyzed with a Tecnai F20electron microscope (FEI) with a Gatan CCD camera at the KAIST Analysis Center for Research Advancement (KARA).A total of 46, 43, 11, and 16 micrographs were collected for Group 1, 2, 3, and 4 from the E.

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