Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: (A) Bar diagram of Dot1L and ubiquitin used in this study. The domains that are discussed in the present study are displayed. The line below the diagram shows visible in the structure (solid line) and disordered (dashed line) regions of the catalytic domain. (B) Cryo-EM reconstruction of Dot1L bound to H2BK120 ubiquitinated nucleosome displayed in two separate views related by ~ 90 degrees; (C) Structural model of Dot1L-H2BK120Ub nucleosome. The structure is color-coded (Dot1L catalytic domain is depicted in pink, ubiquitin in cyan, DNA in grey, histone H2A in pale yellow, histone H2B in red salmon, histone H3 in slate blue and histone H4 in pale green).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Cryo-EM Sample Prep

Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: (A) Cryo-EM density map focused on the interface between Dot1L and the nucleosome. (B) Overview of interactions in the complex. Secondary structure elements are also depicted here. Dot1L catalytic domain is depicted in pink and orange, ubiquitin in cyan, histone H4 in pale green; histone H3 in slate blue; histone H2A in pale yellow; histone H2B in red salmon and DNA in grey. (C) Primary and secondary structure of the catalytic domain of Dot1L. N-terminal ‘head’ portion of catalytic domain of Dot1L is depicted in orange and C-terminal ‘tail’ portion depicted in pink. Loop L-EF between the two portions of Dot1L is depicted in green. Triangles below the sequence show histone and Ub interactions that are ordered and clearly visible in the cryo-EM density. Bars below the sequence show interactions for which side-chain density is less clear. Colors of triangles and bars represent which histone (or ubiquitin) interact with which region of Dot1L. Dashed grey line below the sequence shows potential interactions of previously mapped ubiquitin interaction motif and lysine-rich region of Dot1. (D) 4.6Å cryo-EM Arctica reconstruction showing nucleosomes that have Dot1L bound to both sides of the nucleosome. At lower contour level this map also reveals density at the bottom of Dot1L catalytic domain that seems to interact with ubiquitin and nucleosomal DNA. This density could correspond to the DNA binding K-rich region and a putative ubiquitin-interacting motif (UIM) of Dot1L. (E) Distance between the SAH and H3K79 observed in the current cryo-EM structure.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Cryo-EM Sample Prep, Sequencing, Binding Assay

Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: (A) Cryo-EM density map showing Dot1L interacting with Ubiquitin. (B) Model showing the interface and highlighting the path of Ubiquitin C-terminus and H2B (the model is color-coded as in Figure 1). (C) Detailed view of interactions between Dot1L and Ubiquitin. (D) Cryo-EM density of the interface between Dot1L and Ubiquitin. (E) Multiple sequence alignment of the R anchor/β10 region and the αK helix of Dot1L showing the conservation of the hydrophobic cage. Residues mutated for biochemistry experiments are indicated with a star. (F) Endpoint methyltransferase assay for Dot1L mutants using unmodified nucleosome (WT Nuc) or ubiquitinated nucleosome (Ub Nuc). Numbers above the columns represent ratios between Dot1L activity on H2BK120Ub and unmodified nucleosome substrates. (G) Binding curves of Dot1L to unmodified nucleosome (WT Nuc) (Kd= 52 nM) or ubiquitinated nucleosome (Ub Nuc) (Kd= 50 nM) measured by EMSA. Nucleosome ubiquitination does not change the affinity of Dot1L for nucleosome substrates. (H) Representative HMT assay measuring activity of WT Dot1L and αK mutants. HMT assays were performed with an increasing amount of Dot1L (10, 20, or 40 nM) in the presence of unmodified or H2BK120Ub nucleosomes and reaction products were identified by WB. (I) Binding curve of Dot1L mutants F326A (top) and L322D (bottom) to unmodified nucleosome (WT Nuc) (Kd= 57 nM and 69 nM, respectively) or ubiquitinated nucleosome (Ub Nuc) (Kd= 46 nM and 59 nM, respectively) measured by EMSA. Mutations F326A and L322D do not change the affinity of Dot1L for nucleosome substrates. Each data point and error bar represent the mean ± s.d. from 3 independent experiments. The standard errors of dissociation constants (Kd) are indicated. The apparent Kd values are summarized in Table S4.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Cryo-EM Sample Prep, Sequencing, Activity Assay, Binding Assay, HMT Assay

Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: (A) Top, Overview of the interactions between Dot1L R-anchor loop and acidic patch as well as general architecture of this region (the model is color-coded as in Figure 1). Bottom, Multiple sequence alignment of the R-anchor loop showing the high degree of conservation (residue identity shown in green), with the exception of yeast. Residues mutated for biochemistry experiments are indicated with a star. (B) Top, Detailed view of the interactions between Dot1L with the acidic patch residues in H2A/H2B. Bottom, cryo-EM density map of the region. (C) Top, Representative HMT assay measuring Dot1L and Dot1L R278E/R282E mutant activity. HMT assays were performed similarly to Fig. 3H using ubiquitinated nucleosome (H2BK120Ub Nuc). The Dot1L mutations result in significantly reduced HMT H3K79me3 activity and lower HMT H3K79me2 activity on Ub-nucleosome. Bottom, binding of Dot1L (Kd= 52 nM) or Dot1L R278E/R282E mutant (Kd= 50 nM) to ubiquitinated nucleosome (Ub Nuc) measured by EMSA. Binding curves are presented as mean ± s.d. (n = 3 for each data point). The standard errors of dissociation constants (Kd) are indicated.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Sequencing, Cryo-EM Sample Prep, HMT Assay, Mutagenesis, Activity Assay, Binding Assay

Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: (A) Cryo-EM reconstruction at 4.9 Å of Dot1L bound to unmodified nucleosome. (B) Fitted model of Dot1L-H2BK120Ub complex (without Ub) into the map of Dot1L bound to unmodified nucleosome. (C) Close up of R-anchor interaction with the acidic patch that can be observed in this structure. (D) Crosslinking mass spectrometry diagram of the complex of Dot1L with the unmodified nucleosome showing the interacting regions between Dot1L and the histones in solution (as represented with connecting lines). (E) Representative HMT assay measuring the activity of Dot1L and Dot1L R278E/R282E mutant (R-anchor loop mutations) on unmodified nucleosomes. HMT assays were performed similarly to Fig. 3H. (F) Binding of Dot1L (Kd= 73 nM) or Dot1L R278/282E (Kd= 48 nM) mutant to unmodified nucleosome, measured by EMSA. (G) Binding of full-length yeast Dot1 to unmodified (Kd= 21 nM) and ubiquitinylated nucleosomes (Kd=22 nM) measured by EMSA. (H) Binding of catalytic domain (158-582) of yeast Dot1 (Dot1Δ) to unmodified (Kd= 84 nM) and ubiquitinylated nucleosomes (Kd=38 nM) measured by EMSA. For each EMSA, data points and error bars represent the mean ± s.d. from 3 independent experiments. (I) Shift and angular rotation necessary for Dot1L to adopt conformation necessary for catalysis.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Cryo-EM Sample Prep, Mass Spectrometry, HMT Assay, Activity Assay, Mutagenesis, Binding Assay

Journal: Molecular cell

Article Title: Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination

doi: 10.1016/j.molcel.2019.03.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-H3K79Me2 Abcam Cat#Ab3594 anti-H3K79Me3 Abcam Cat#Ab2621 anti-Histone H4 Abcam Cat#Ab7311 Bacterial and Virus Strains E. coli One Shot™ BL21(DE3) Thermofisher Cat#C6000-03 SoluBL21 Amsbio Cat#C700200 Chemicals, Peptides, and Recombinant Proteins S -(5′-Adenosyl)- L -methionine (SAM) Sigma Cat#A7007 bis(sulfosuccinimidyl)suberate (BS3) Thermo Scientific Cat#21580 S-(5′-Adenosyl)-L-homocysteine (SAH) Sigma Cat#A9384 Critical Commercial Assays MTase-Glo Methyltransferase Assay Promega Cat#V7601 Q5® Site-Directed Mutagenesis Kit NEB Cat#E0554S Gibson Assembly® Master Mix NEB Cat#E2611S Deposited Data Dot1L bound to the H2B-Ubiquitinated nucleosome This paper PDB-6O96 Dot1L bound to the H2B-Ubiquitinated nucleosome, 3.5 Å map This paper EMDB: EMD-0652 Dot1L bound to the H2B-Ubiquitinated nucleosome, 4.6 Å map This paper EMDB: EMD-0653 Dot1L bound to the H2B-Ubiquitinated nucleosome, 5.2 Å map This paper EMDB: EMD-0654 Dot1L bound to the unmodified nucleosome This paper EMDB: EMD-0655 Raw EMSA gels images and Western Blots images This paper; and Mendeley Data doi: 10.17632/59wbz6gbcp.1 Recombinant DNA pET28-MHL-DOT1L (1-420) Addgene 40736 pET28-MHL-DOT1L (1-420), R278/278 This paper N/A pET28-MHL-DOT1L (1-420), F326A This paper N/A pET28-MHL-DOT1L (1-420), L322D This paper N/A pET28-MHL-DOT1L (1-420), I290D This paper N/A pET15b-His-3C-Dot1 (158-582) This paper N/A pET3a xlH2A Luger et al., 1997 N/A pET3a xlH2B Luger et al., 1997 N/A pET3a xlH3 Luger et al., 1997 N/A pET3a xlH4 Luger et al., 1997 N/A pET3a xLH2BK120C This paper N/A pET-His-UB G76C Long et al., 2014 N/A pUC57-601 Armache et al., 2011 Genscript Software and Algorithms MotionCor2 v1.2.1 Zheng et al., 2017 http://msg.ucsf.edu/em/software/motioncor2.html Gautomatch https://www.mrc-lmb.cam.ac.uk/kzhang/ https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ cisTEM Grant et al., 2018 https://cistem.org/ Relion 3.0 Zivanov et al., 2018 https://github.com/3dem/relion PHENIX Adams et al., 2010 https://www.phenix-online.org/ CryoSPARC Punjani et al., 2017 https://cryosparc.com/ pLink2 http://pfind.ict.ac.cn/software/pLink/ http://pfind.ict.ac.cn/software/pLink/ Coot Emsley et al., 2004 https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ Chimera Pettersen et al., 2004 https://www.cgl.ucsf.edu/chimera/ Prism 7 Ivashchenko et al., 2017 https://www.graphpad.com/scientific-software/prism/ ImageQuant 5.2v Molecular Dynamics.

Techniques: Recombinant, Mutagenesis, Western Blot, Software