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| Item Type: | Dataset |
|---|---|
| Title: | PXD077526: In vitro PhoX cross-linking of purified hsMic60 |
| Creators: |
Ruwolt, Max and Daumke, Oliver  ORCID: https://orcid.org/0000-0002-6190-1414
|
| Abstract: | DESCRIPTION: Mitochondrial crista junctions (CJs) operate as regulated gateways into the cristae microenvironment, whose protein, metabolite, and ion compositions are finely tuned for mitochondrial function. The Mic60-Mic19 complex of the mitochondrial contact site and cristae organizing system (MICOS) complex was suggested to span across CJs and act as a diffusion barrier, but little is known of how its dynamic architecture facilitates this task. To address this open question, we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic60. These and previous structural and biochemical data were harnessed in molecular dynamic (MD) simulations to develop a dynamic model of the human tetrameric Mic60-Mic19 subcomplex in the CJ environment, to validate its architecture using in organello cross-linking data and to computationally characterize its function as a diffusion barrier. Our integrative structural biology approach enables the functional investigation of flexible, multidomain protein complexes which escape conventional structural biology methods. This in vitro PhoX cross-linking mass spectrometry data of purified hsMic60 supports the in organello studies by showing a similar cross-linking pattern. SAMPLE Processing Protocol: Mic60 HB-CC was diluted to 250 μl of 1 mg/ml using SEC buffer (20 mM HEPES/NaOH pH 7.5, 150 mM NaCl). PhoX (Thermo-Fisher-Scientific) was freshly dissolved in anhydrous DMSO to a concentration of 50 mM and added to the sample in two steps of 20 minutes incubation on ice, reaching a final concentration of 1.2 mM. The reaction was quenched with 20 mM Tris/HCl pH 8.0 for 30 min at room temperature. After quenching, proteins were directly subjected to tryptic digestion in-solution. 8 M urea, 5 mM TCEP, and 40 mM chloroacetamide were added and the mixture was incubated at 37 °C for 1 – 1.5 hours in the dark. Proteolytic digestion was performed using LysC (1:200 ratio (w/w)) and trypsin (1:100 ratio (w/w)). Prior to trypsin addition, samples were pre-digested with LysC at 37 °C for 2 – 4 hours. The solution was then diluted to a final concentration of 2 M urea using 50 mM triethylammonium bicarbonate buffer (pH 8.5). The digestion was stopped with 1% formaldehyde, and peptides were desalted using C8 StageTips. Peptides were dried and stored at -20 °C until measurement. Peptides were resuspended in 1% acetonitrile with 0.05% trifluoroacetic acid and 1 µg was injected into the LC-MS system. Separation was performed via reverse-phase (RP)-HPLC on a Thermo Scientific Vanquish neo system using a PepMap C-18 trap-column (0.075 mm x 50 mm, 3 μm particle size, 100 Å pore size, Thermo Fisher Scientific) and an in-house packed C18 column (Poroshell 120 EC-C18, 2.7 μm, Agilent Technologies) at a flow rate of 250 nL/min. RP separation was achieved using a 180-min method. Samples were analysed on an Orbitrap Exploris 480 mass spectrometer (equipped with a FAIMS Pro Duo interface (Thermo Scientific)). MS1 scans were recorded in the Orbitrap at a resolution of 120,000, 375 – 1,400 m/z scan range, 50 ms maximum injection time, and 300% AGC target. Precursors with charge states 3 – 8 were isolated in 2 s per FAIMS CV (-50/-60/-70) cycles with a 2 m/z window and subjected to MS2 before being dynamically excluded from fragmentation for 60 s. Fragment ions were generated with NCE 30% and detected in the Orbitrap at a resolution of 30,000, automatic scan range, 100 ms maximum injection time, and 200% AGC target. DATA PROCESSING PROTOCOL: RAW files were analysed using XlinkX implemented in Proteome Discoverer version 3.2, operating in non-cleavable mode. After recalibration of RAW files, the search parameters were set as follows: a mass tolerance of 10 ppm for MS1 and 20 ppm for MS2, with a maximum of 3 missed cleavages and a minimum peptide length of 5 with peptide masses ranging from 300 – 7,000 Da. Cross-linker modifications were defined for lysine residues and protein N-termini (+209.97 Da). Oxidation of methionine (+ 15.995 Da) was considered as a variable and carbamidomethylation of cysteine (+ 57.021 Da) as a static modification. Searches were conducted against a database containing the target protein sequences. The false discovery rate was controlled at 1% on cross-link spectrum match (CSM)- and cross-link level, with separate estimation for intra- and inter-links. Cross-links were exported and dynamic score thresholds were applied based on the maximum decoy CSM scores, and identifications were further filtered post-search by score or minimum CSM count. |
| Keywords: | Micos, Cross-linking, Mitochondria, Xl-ms, Phox |
| Source: | PRIDE |
| Publisher: | EMBL-EBI |
| Date: | 4 August 2026 |
| External Fulltext: | View full text on external repository or document server |
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