Journal: Cell chemical biology

Article Title: Chemical Cross-Linking Enables Drafting ClpXP Proximity Maps and Taking Snapshots of In Situ Interaction Networks.

doi: 10.1016/j.chembiol.2018.10.007

Figure Lengend Snippet: Figure 3. Analysis of the ClpXP Interface (A) The largest proportion of cross-links (FDR < 0.01) detected in the ClpXP samples are intra-ClpX links distributed all over the sequence (involved lysines are highlighted in blue in one monomer). The IGF loop is colored red, the pore-2 loop yellow. (B) Cross-links are projected onto a ClpX hexameric model of L. monocytogenes derived from an E. coli crystal structure (PDB: 4I81). The histogram of median Ca-Ca distances displays most of the cross-links within the maximum distance constraint of 35 A˚ . Cross-links exceeding the maximum distance constraint are highlighted in red. (C) LmClpX-ClpP2 cross-linking contacts: one subunit of LmClpP2 (PDB: 4RYF, colored gray) and one subunit of LmClpX (colored beige) are positioned next to each other and inter-facial cross-links (involved lysine residues highlighted in green) are displayed from LmClpP2 to LmClpX. Notably, only intermolecular links between LmClpP2 and LmClpX, but not between LmClpP1 and LmClpX, were detected. The ClpX IGF loop is colored red and the pore-2 loop yellow. ClpP N terminus is colored pink, E helix cyan, and G helix beige. Further, a representative molecular dynamics (MD) snapshot of an oligomeric LmClpX-ClpP2 model (two trimers) is presented. Only cross-links with the shortest distances from LmClpX subunits to the central LmClpP2 subunit are shown. See also Figures S5 and S6 and Tables S2 and S3 and Data S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-ClpP antibody abcam Cat# ab124822; RRID: AB_10975619 anti-Lon antibody Thermo Fisher Scientific Cat# PA5-50823 anti-SaClpP antibody custom made N/A rabbit isotype control Cell Signaling Technology Cat# 3900 Bacterial and Virus Strains Escherichia coli K12 DSMZ Cat# 498 Escherichia coli Rosetta 2 (DE3) Merck Cat# 71400 Escherichia coli BL21 (DE3) Sigma Aldrich Cat# CMC0014 Chemicals, Peptides, and Recombinant Proteins Silica gel 40-63 mm VWR Cat# 84894.460 3,3’-thiodipropionic acid AlfaAesar Cat# A17220 N-hydroxysuccinimide Iris Biotech Cat# RL-1038 N,N’-dicyclohexylcarbodiimide Sigma Aldrich Cat# D80002 3-chloroperbenzoic acid Acros Organics Cat# 255790250 Lysyl Endopeptidase Wako Cat# 125-05061 Trypsin, Sequencing grade Promega Cat# V5111 BS3 cross-linker Creative Molecules Cat# 001SS Critical Commercial Assays Roti Quant Universal Roth Cat# 0120.1 Deposited Data raw files, MaxQuant and cross-link analysis to PRIDE This paper https://www.ebi.ac.uk/pride/archive/; PRIDE: PXD009224 Experimental Models: Cell Lines Human HepG2 cell line ECACC via Sigma Aldrich Cat# 85011430; RRID: CVCL_0027 Human K562 cell line ECACC via Sigma Aldrich Cat# 89121407; RRID: CVCL_0004 Recombinant DNA pET300 LmClpX Dahmen et al., 2015 N/A pET301 SaClpP Gersch et al., 2012 N/A pET301 SaClpX Gersch et al., 2015 N/A pET301 hClpP Gersch et al., 2016 N/A pET301 hClpX Gersch et al., 2016 N/A pETDuet1 LmClpP1/2 Dahmen et al., 2015 N/A Software and Algorithms MaxQuant software MPI Biochemistry Martinsried http://www.biochem.mpg.de/5111795/maxquant Perseus software MPI Biochemistry Martinsried http://www.biochem.mpg.de/5111810/perseus Protoeme Discoverer 2.2. with XlinkX software node Thermo Fisher Scientific http://www.thermofisher.com/order/catalog/ product/OPTON-30795 Kojak software Institute for System Biology http://www.kojak-ms.org/ Swiss Model Swiss Institute of Bioinformatics https://swissmodel.expasy.org/ XLinkAnalyzer Kosinski et al., 2015 http://www.beck.embl.de/XlinkAnalyzer.html UCSF Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ PyMOL by Schrödinger DeLano, 2002 https://pymol.org/2/ HDOCK Yan et al., 2017 http://hdock.phys.hust.edu.cn/ Amber16 software package Case et al., 2016 http://ambermd.org/ (Continued on next page) Cell Chemical Biology 26, 1–12.e1–e7, January 17, 2019 e1

Techniques: Sequencing, Derivative Assay

Journal: Cell chemical biology

Article Title: Chemical Cross-Linking Enables Drafting ClpXP Proximity Maps and Taking Snapshots of In Situ Interaction Networks.

doi: 10.1016/j.chembiol.2018.10.007

Figure Lengend Snippet: Figure 5. In Situ Chemical Cross-Linking Enhances CoIP Coverage in E. coli Volcano plots representing t test results of anti-ClpP coIP compared with the isotype control coIP (n = 4). Cutoff values were defined as enrichment factor of log2 = 2 (4-fold enrichment) and log10 (p value) of 1.3 (solid lines). (A) Conventional coIP in E. coli compared with in situ cross-linking and pull-down (B). The major target ClpP, together with its two interacting chaperones ClpA and ClpX as well as the ATPase ClpB, are colored in blue, flagellum-related proteins in magenta, chemotaxis-related proteins in green, and previously identified substrates in orange. See also Tables S4 and S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-ClpP antibody abcam Cat# ab124822; RRID: AB_10975619 anti-Lon antibody Thermo Fisher Scientific Cat# PA5-50823 anti-SaClpP antibody custom made N/A rabbit isotype control Cell Signaling Technology Cat# 3900 Bacterial and Virus Strains Escherichia coli K12 DSMZ Cat# 498 Escherichia coli Rosetta 2 (DE3) Merck Cat# 71400 Escherichia coli BL21 (DE3) Sigma Aldrich Cat# CMC0014 Chemicals, Peptides, and Recombinant Proteins Silica gel 40-63 mm VWR Cat# 84894.460 3,3’-thiodipropionic acid AlfaAesar Cat# A17220 N-hydroxysuccinimide Iris Biotech Cat# RL-1038 N,N’-dicyclohexylcarbodiimide Sigma Aldrich Cat# D80002 3-chloroperbenzoic acid Acros Organics Cat# 255790250 Lysyl Endopeptidase Wako Cat# 125-05061 Trypsin, Sequencing grade Promega Cat# V5111 BS3 cross-linker Creative Molecules Cat# 001SS Critical Commercial Assays Roti Quant Universal Roth Cat# 0120.1 Deposited Data raw files, MaxQuant and cross-link analysis to PRIDE This paper https://www.ebi.ac.uk/pride/archive/; PRIDE: PXD009224 Experimental Models: Cell Lines Human HepG2 cell line ECACC via Sigma Aldrich Cat# 85011430; RRID: CVCL_0027 Human K562 cell line ECACC via Sigma Aldrich Cat# 89121407; RRID: CVCL_0004 Recombinant DNA pET300 LmClpX Dahmen et al., 2015 N/A pET301 SaClpP Gersch et al., 2012 N/A pET301 SaClpX Gersch et al., 2015 N/A pET301 hClpP Gersch et al., 2016 N/A pET301 hClpX Gersch et al., 2016 N/A pETDuet1 LmClpP1/2 Dahmen et al., 2015 N/A Software and Algorithms MaxQuant software MPI Biochemistry Martinsried http://www.biochem.mpg.de/5111795/maxquant Perseus software MPI Biochemistry Martinsried http://www.biochem.mpg.de/5111810/perseus Protoeme Discoverer 2.2. with XlinkX software node Thermo Fisher Scientific http://www.thermofisher.com/order/catalog/ product/OPTON-30795 Kojak software Institute for System Biology http://www.kojak-ms.org/ Swiss Model Swiss Institute of Bioinformatics https://swissmodel.expasy.org/ XLinkAnalyzer Kosinski et al., 2015 http://www.beck.embl.de/XlinkAnalyzer.html UCSF Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ PyMOL by Schrödinger DeLano, 2002 https://pymol.org/2/ HDOCK Yan et al., 2017 http://hdock.phys.hust.edu.cn/ Amber16 software package Case et al., 2016 http://ambermd.org/ (Continued on next page) Cell Chemical Biology 26, 1–12.e1–e7, January 17, 2019 e1

Techniques: In Situ, Control, Chemotaxis Assay