Supplemental Information. The structural basis of R Spondin recognition by LGR5 and RNF43

Size: px
Start display at page:

Download "Supplemental Information. The structural basis of R Spondin recognition by LGR5 and RNF43"

Transcription

1 Supplemental Information The structural basis of R Spondin recognition by LGR5 and RNF43 Po Han Chen 1, Xiaoyan Chen 1, Deyu Fang 2, Xiaolin He 1* 1 Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois *Correspondence to Xiaolin He, Ph.D. Phone: Fax: E mail: x he@northwestern.edu 1

2 Supplemental Materials and Methods Cell culture, cloning and BacMam expression Sf9 insect cells were maintained in SF900 II media (Invitrogen) supplemented with 8% (v/v) heat inactivated fetal bovine serum. N acetylglucosaminyltransferase I (GnTI) deficient Human Embryonic Kindey 293 (GnTI HEK293) cells were maintained in CDM4HEK293 media (HyClone) supplemented with 4% heat inactivated fetal bovine serum. The coding sequence for the whole ectodomain of human LGR5 (GenBank: AF ), RNF43 (GenBank: AB ), RSPO1 CRD (GenBank: DQ , E35 S133), and RSPO1 TSP (E35 G209) without signal peptide were PCR amplified and subcloned into the baculovirus mediated mammalian cell gene transduction (BacMam) vector pvlad627 containing a N terminal Gaussia luciferase signal peptide and a C terminal 7 histidine tag. The constructs and the BacVector 3000 baculovirus DNA (EMD Chemicals) were used to co transfect Sf9 cells in 6 well plates in the presence of the CellfectinII reagent (Invitrogen). After incubation of the transfected cells at 27 o C for 5 days, the resulting low titer virus stock was harvested, and was used to infect Sf9 cells at cell/ml for amplification. The amplified BacMam viruses were used to transduce HEK293 GnTI cells at a density of cells/ml. Rspo1 and LGR5 were co expressed for improved protein stability, and RNF43 was expressed separately. The cells were pelleted 72 hours later, and the supernatants were concentrated and buffer exchanged to HBS (10mM Hepes ph7.5, 150mM NaCl). The recombinant proteins were captured by the Talon affinity resin and eluted with 300 mm imidazole ph 7.5, glycan minimized with endoglycosidase F1 (Sigma), and treated with bovine carboxypeptidase A (Sigma) for His tag removal. RNF43 and RSPO1/LGR5 were mixed 2

3 with appropriate ratios, and proteins were further purified with size exclusion columns (Superdex 200, Amersham Biosciences) pre equilibrated and eluted with HBS. Crystallization and data collection Crystallization was performed in a 20 0 C incubator using the sitting drop vapor diffusion method. Crystals of the complex were obtained from drops composed of 0.5 µl reservoir solution and 0.5 µl protein solution (10mg/ml) equilibrated against 1 ml reservoir solution. The composition of the reservoir solution is 10% PEG4000, 0.2M ammonium sulfate, 7% Sucrose and 0.1 M TRIS ph 8.0. Prior to being flash frozen in liquid nitrogen, the crystals were transferred to a cryosolution consisting of the reservoir solution supplemented with 19% ethylene glycol. The heavy atom derivatives were prepared by quick soaking selected crystals in the cryo protectant containing 0.2 M NaI for ~60 seconds. X ray diffraction datasets were collected at 100K at the Life Science Collaborative Access Team (LS CAT) beamline 21 ID D, the Advanced Photon Source, Argonne, Illinois, USA. The data were processed with HKL3000 (Otwinowski and Minor, 1997), and the statistics are summarized in Table S1. Structure determination and refinement The structure was solved by single isomorphous replacement with anomalous scattering (SIRAS) using Iodine soaked crystals. Taking advantage of non crystallographic symmetry present in the crystal, the electron density map was furthered improved by density modification in CNS (Brunger, 2007; Brunger et al., 1998). Initial atomic models were built manually in O and COOT 3

4 (Emsley and Cowtan, 2004). The model of LGR5 was built with LRR repeats of FSHR (PDB 4AY9) as template. Due to lack of similar structures, RSPO1 was built initially by baton building when electron density map was improved to sufficient details, followed by manual adjustment of amino acids that agree best with sequence information (i.e., by locating the conserved disulfide bonds. Model of RNF43 were built by manually fitting in the aminopeptidase structure, followed by adjustment of amino acid side chains and backbones. The complex structure was refined using CNS1.3 and REFMAC5 (Collaborative Computational Project, 1994). Local NCS restraint is maintained throughout the refinement process. Isothermal Titration Calorimetry experiment Calorimetric titrations were implemented with a VP ITC calorimeter (MicroCal) at 30 C. Using gel filtration chromatography (Superdex 200, Amersham Biosciences), all proteins to be used in the titrations were buffer exchanged by into an identical lot of HBS buffer (10 mm Hepes ph7.5, 150 mm NaCl) to minimize the dilution effects of buffer heat during titration. Proteins eluted from the column were concentrated and concentrations measured by Nanophotometer P330 (IMPLEN): RNF mm, R CRD 0.07 mm, R TSP 0.02 mm, R CRD/LGR mm, and R TSP/LGR mm. The protein samples were degassed for 5 min before being loaded separately into the reaction chamber (all samples except for RNF43) and injection syringe. The proteins in the syringe (RNF43) were injected into the reaction chamber in 3µL pulses at 5 min intervals. The data were processed with MicroCal Origin 5.0 software. References 4

5 Brunger, A.T. (2007). Version 1.2 of the Crystallography and NMR system. Nature protocols 2, Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta crystallographica Section D, Biological crystallography 54, Collaborative Computational Project, N. (1994). The CCP4 suite: programs for protein crystallography. Acta crystallographica Section D, Biological crystallography 50, Emsley, P., and Cowtan, K. (2004). Coot: model building tools for molecular graphics. Acta Crystallogr D 60, Otwinowski, Z., and Minor, W. (1997). Processing of X ray diffraction data collected in oscillation mode. Method Enzymol 276,

6 Supplemental Table 1 crystals Human LGR5-Rspo1-RNF43 native I-soaked Data collection Space group P P Cell parameters / / /80.07/ Resolution range ( ) ( ) Unique reflections (5121) (952) Completeness (%) 97.8 (91.9) 99.2 (99.7) I/sigma(I) 19.5 (4.2) 18.6 (6.6) Redundancy 6.2 (4.8) 16.2 (12.8) R merge (%)* 6.1 (33.8) 8.0 (38.2) phasing Number of heavy atoms 8 R iso (%) 13.4 R ano (%) 4.8 FOM 0.53 Refinement Resolution range ( ) r.m.s.d bonds (Å) r.m.s.d angles ( ) 1.67 R free (%) 23.0 R work (%) 27.8 Number of protein atoms, averaged B factor (Å 2 ) Number of glycan atoms, averaged B factor (Å 2 ) Number of waters, averaged B factor (Å 2 ) 11019, , , 48.8 Ramachandran plot (%) (favored, allowed, outliers) 76.6, 12.5, 10.9 *R merge = Σ hkl I-<I> /Σ hkl I, where I is the intensity of unique reflection hkl, and <I> is the average over symmetry-related observation of unique reflection hkl. R work =Σ F obs -F calc /ΣF obs, where F obs and F calc are the observed and the calculated structure factors, respectively. R free is calculated using 5% of reflections set aside from refinement. 6

7 Supplemental Table 2. Selected interface residues* between Rspo1 CRD and LGR5 ECD Rspo1 CRD LGR5 ECD Asp85 R144 (LRR4) Arg87 Asp146 (LRR4), Asp171 (LRR5) F106 His166, Trp168 (LRR5) F110 Val213/214 (LRR7) Lys122 Val213 (LRR7), Glu237 (LRR8) *Interface residues are listed according to PISA analysis and visual inspection of electron density map. 7

8 Supplemental Table 3. Selected interface residues* between Rspo1 CRD and RNF43 PA Rspo1 CRD RNF43 PA Ser48 Glu110 Leu64 His86, Leu88, Tyr89 Arg66 Asp97, Gln84 Asp68 Lys81, His183 Ile69 Leu82, Gln84, Lys181, Val176 Arg70 Gln84 Gln71 Gln84, His86, Asp97 *Interface residues are listed according to PISA analysis and visual inspection of electron density map. 8

9 Chen Supplemental Figure 1. Comparison between LGR5 RSPO1 and FSHR FSH. Direct view of the LRR concave face in both structures. 9

10 Supplemental Figure 2. Crystal packing of back to back dimer. The side view and top view are presented. The modeled Ni 2+ ion is shown as the gray sphere surrounded by His residues in sticks. Boxed region indicates site of RNF43 LGR5 packing in the crystal structure. 10

11 Supplemental Figure 3. Electron density surrounding the RSPO1 LGR5 interface. RSPO1 is shown in cyan and LGR5 in green. 2Fo Fc electron density map contoured at 2.0 σ. The interface consists of hydrophilic interactions (RSPO1 D85 and R87 with LGR5 R144 and D146) and hydrophobic interactions (right part of the figure, in particular RSPO1 F106 and F110 and LGR5 W168, H166, and V213). 11

12 Supplemental Figure 4. Electron density surrounding the RSPO1 β hairpin (L64 Q71) interaction with RNF43 β3. Orientation is the same as in Fig. 3. RSPO1 is shown in marine and RNF43 shown in hotpink. The 2Fo Fc map is contoured at 1.0 σ. 12

The Skap-hom Dimerization and PH Domains Comprise

The Skap-hom Dimerization and PH Domains Comprise Molecular Cell, Volume 32 Supplemental Data The Skap-hom Dimerization and PH Domains Comprise a 3 -Phosphoinositide-Gated Molecular Switch Kenneth D. Swanson, Yong Tang, Derek F. Ceccarelli, Florence Poy,

More information

Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the

Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the Supplementary information, Data S1 Methods Clones and protein preparation Six genes, Lsm1, Lsm2, Lsm3, Lsm5, Lsm6, and Lsm7, were amplified from the Saccharomyces cerevisiae genomic DNA by polymerase chain

More information

Proteins were extracted from cultured cells using a modified buffer, and immunoprecipitation and

Proteins were extracted from cultured cells using a modified buffer, and immunoprecipitation and Materials and Methods Immunoprecipitation and immunoblot analysis Proteins were extracted from cultured cells using a modified buffer, and immunoprecipitation and immunoblot analyses with corresponding

More information

Supporting Online Material. Av1 and Av2 were isolated and purified under anaerobic conditions according to

Supporting Online Material. Av1 and Av2 were isolated and purified under anaerobic conditions according to Supporting Online Material Materials and Methods Av1 and Av2 were isolated and purified under anaerobic conditions according to published protocols (S1). Crystals of nf-, pcp- and adp-av2:av1 complexes

More information

Appendix B Dansyl probe syntheses and characterization and D-8-Ad:P450cam structure determination

Appendix B Dansyl probe syntheses and characterization and D-8-Ad:P450cam structure determination 201 Appendix B Dansyl probe syntheses and characterization and D-8-Ad:P450cam structure determination Acknowlegements. The structure of the D-8-Ad:P450cam conjugate was determined by Anna-Maria A. Hays.

More information

Figure S1 Alpha-carbon backbones of components from FSH-FSHRHB complex. a, Stereo view of FSHRHB (red) with every 10 th residue marked.

Figure S1 Alpha-carbon backbones of components from FSH-FSHRHB complex. a, Stereo view of FSHRHB (red) with every 10 th residue marked. a 230 80 80 130 110 230 110 180 60 180 130 60 210 200160 40 210 200 160 40 90 20 90 20 250 150 150 190 100 250 50 30 100 220 140 220 190 140 50 30 240 240 120 70 120 70 170 170 b 70 70 60 60 20 20 50 50

More information

The YTH domain (residues ) of human YTHDF2 (NP_ ) was subcloned

The YTH domain (residues ) of human YTHDF2 (NP_ ) was subcloned Supplementary information, Data S1 Materials and Methods Protein Expression, Purification and Crystallization The YTH domain (residues 383-553) of human YTHDF2 (NP_057342.2) was subcloned into a modified

More information

Protein expression and purification from Hi5 insect cells is as described (1, 2).

Protein expression and purification from Hi5 insect cells is as described (1, 2). Materials & methods Protein expression and purification Protein expression and purification from Hi5 insect cells is as described (1, 2). The purified complex exhibited a 2:2:2 stoichiometry as determined

More information

Nature Structural & Molecular Biology: doi: /nsmb.1969

Nature Structural & Molecular Biology: doi: /nsmb.1969 Supplementary Methods Structure determination All the diffraction data sets were collected on BL-41XU (using ADSC Quantum 315 HE CCD detector) at SPring8 (Harima, Japan) or on BL5A (using ADSC Quantum

More information

Supplemental Information

Supplemental Information Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2014 Supplemental Information Experimental Procedures Cloning, expression, purification and mutagenesis

More information

Supporting Information

Supporting Information Supporting Information Slep et al. 10.1073/pnas.0801569105 SI Materials and Methods Mouse RGS16, residues 53 180, was subcloned into a modified pgex-2t vector (GE Healthcare) to yield a thrombin-cleavable

More information

Supporting Information

Supporting Information Supporting Information Nishimura et al. 10.1073/pnas.1003553107 SI Text SI Materials and Methods. Plasmid construction. The cdnas of human Gα q were amplified and subcloned into pcmv5. Mutants of Gα q

More information

Suppl. Figure 1: RCC1 sequence and sequence alignments. (a) Amino acid

Suppl. Figure 1: RCC1 sequence and sequence alignments. (a) Amino acid Supplementary Figures Suppl. Figure 1: RCC1 sequence and sequence alignments. (a) Amino acid sequence of Drosophila RCC1. Same colors are for Figure 1 with sequence of β-wedge that interacts with Ran in

More information

Introduction to Protein Purification

Introduction to Protein Purification Introduction to Protein Purification 1 Day 1) Introduction to Protein Purification. Input for Purification Protocol Development - Guidelines for Protein Purification Day 2) Sample Preparation before Chromatography

More information

Supporting Information

Supporting Information Supporting Information Xiao et al. 10.1073/pnas.1309211110 SI Methods Protein Expression and Purification. Bacmid was generated and recombinant baculovirus amplified using standard procedures (Bac-to-Bac;

More information

Purification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract

Purification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract Purification: Step 1 Lecture 11 Protein and Peptide Chemistry Cells: Break them open! Crude Extract Total contents of cell Margaret A. Daugherty Fall 2003 Big Problem: Crude extract is not the natural

More information

Purification: Step 1. Protein and Peptide Chemistry. Lecture 11. Big Problem: Crude extract is not the natural environment. Cells: Break them open!

Purification: Step 1. Protein and Peptide Chemistry. Lecture 11. Big Problem: Crude extract is not the natural environment. Cells: Break them open! Lecture 11 Protein and Peptide Chemistry Margaret A. Daugherty Fall 2003 Purification: Step 1 Cells: Break them open! Crude Extract Total contents of cell Big Problem: Crude extract is not the natural

More information

Figure S2, related to Figure 1. Stereo images of the CarD/RNAP complex and. electrostatic potential surface representation of the CarD/RNAP interface

Figure S2, related to Figure 1. Stereo images of the CarD/RNAP complex and. electrostatic potential surface representation of the CarD/RNAP interface Structure, Volume 21 Supplemental Information Structure of the Mtb CarD/RNAP -Lobes Complex Reveals the Molecular Basis of Interaction and Presents a Distinct DNA-Binding Domain for Mtb CarD Gulcin Gulten

More information

Structural basis of transferrin sequestration by transferrin-binding protein B

Structural basis of transferrin sequestration by transferrin-binding protein B Supplementary Information for Structural basis of transferrin sequestration by transferrin-binding protein B Charles Calmettes, Joenel Alcantara, Rong-Hua Yu, Anthony B. Schryvers and Trevor F. Moraes*

More information

Solutions to 7.02 Quiz II 10/27/05

Solutions to 7.02 Quiz II 10/27/05 Solutions to 7.02 Quiz II 10/27/05 Class Average = 83 Standard Deviation = 9 Range Grade % 87-100 A 43 74-86 B 39 55-73 C 17 > 54 D 1 Question 1 (56 points) While studying deep sea bacteria, you discover

More information

absorption spectra were measured on a Hewlett Packard 8452A diode array spectrophotometer.

absorption spectra were measured on a Hewlett Packard 8452A diode array spectrophotometer. S1 Experimental General: P450cam was expressed and purified as previously described. 1 Steady state UV-visible absorption spectra were measured on a Hewlett Packard 8452A diode array spectrophotometer.

More information

Protein analysis. Dr. Mamoun Ahram Summer semester, Resources This lecture Campbell and Farrell s Biochemistry, Chapters 5

Protein analysis. Dr. Mamoun Ahram Summer semester, Resources This lecture Campbell and Farrell s Biochemistry, Chapters 5 Protein analysis Dr. Mamoun Ahram Summer semester, 2015-2016 Resources This lecture Campbell and Farrell s Biochemistry, Chapters 5 Bases of protein separation Proteins can be purified on the basis Solubility

More information

PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%)

PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%) 1 AFFINITY HIS-TAG PURIFICATION PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%) DESCRIPTION Nickel NTA Magnetic Agarose Beads are products that allow rapid and easy small-scale purification of

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Molecular basis of RNA-dependent RNA polymerase II activity Elisabeth Lehmann, Florian Brueckner, and Patrick Cramer Gene Center Munich and Center for integrated Protein Science CiPS M, Department of Chemistry

More information

Supplementary information, Figure S1A ShHTL7 interacted with MAX2 but not another F-box protein COI1.

Supplementary information, Figure S1A ShHTL7 interacted with MAX2 but not another F-box protein COI1. GR24 (μm) 0 20 0 20 GST-ShHTL7 anti-gst His-MAX2 His-COI1 PVDF staining Supplementary information, Figure S1A ShHTL7 interacted with MAX2 but not another F-box protein COI1. Pull-down assays using GST-ShHTL7

More information

SUPPLEMENTAL DATA Experimental procedures

SUPPLEMENTAL DATA Experimental procedures SUPPLEMENTAL DATA Experimental procedures Cloning, protein production and purification. The DNA sequences corresponding to residues R10-S250 (DBD) and E258-T351 (IPT/TIG) in human EBF1 (gi:31415878), and

More information

Supplementary Information For. A genetically encoded tool for manipulation of NADP + /NADPH in living cells

Supplementary Information For. A genetically encoded tool for manipulation of NADP + /NADPH in living cells Supplementary Information For A genetically encoded tool for manipulation of NADP + /NADPH in living cells Valentin Cracan 1,2,3, Denis V. Titov 1,2,3, Hongying Shen 1,2,3, Zenon Grabarek 1* and Vamsi

More information

SERVA Ni-NTA Magnetic Beads

SERVA Ni-NTA Magnetic Beads INSTRUCTION MANUAL SERVA Ni-NTA Magnetic Beads Magnetic beads for Affinity Purification of His-Tag Fusion Proteins (Cat. No. 42179) SERVA Electrophoresis GmbH - Carl-Benz-Str. 7-69115 Heidelberg Phone

More information

Supporting Information

Supporting Information Title Structure of bacterial cellulose synthase subunit D Hu, Song-Qing; Gao, Yong-Gui; Tajima, Kenji; Sunagaw Author(s) Yoda, Takanori; Shimura, Daisuke; Satoh, Yasuharu; M CitationProceedings of the

More information

Applications involving the ViroCyt Virus Counter in the production of various recombinant proteins

Applications involving the ViroCyt Virus Counter in the production of various recombinant proteins Applications involving the ViroCyt Virus Counter in the production of various recombinant proteins Chris Kemp Kempbio, Inc. Frederick, MD USA chris.kemp@kempbioinc.com Presentation Summary Kempbio, Inc.

More information

Structure of the C-cadherin ectodomain and implications for the mechanism of cell adhesion

Structure of the C-cadherin ectodomain and implications for the mechanism of cell adhesion Structure of the C-cadherin ectodomain and implications for the mechanism of cell adhesion Titus J. Boggon, John Murray, Sophie Chappuis-Flament, Ellen Wong, Barry M. Gumbiner, and Lawrence Shapiro Ref.

More information

INSECT CELL/BACULOVIRUS PRODUCTION

INSECT CELL/BACULOVIRUS PRODUCTION INSECT CELL/BACULOVIRUS PRODUCTION PEF # GENE NAME TRANSFER VECTOR BEVS MOLECULAR WEIGHT 2015-XXXX XXXX pbac1 flashbacultra TM 36.0 kda EXPRESSION METHOD OVERVIEW: Insect cells Spodoptera frugiperda (Sf9)

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Structure of a tyrosyl-trna synthetase splicing factor bound to a group I intron RNA Paul J. Paukstelis 1, Jui-Hui Chen 2, Elaine Chase 2, Alan M. Lambowitz 1,*, and Barbara L. Golden 2,*,. 1 Institute

More information

Extracting Pure Proteins from Cells

Extracting Pure Proteins from Cells Extracting Pure Proteins from Cells 0 Purification techniques focus mainly on size & charge 0 The first step is homogenization (grinding, Potter Elvejhem homogenizer, sonication, freezing and thawing,

More information

OPPF-UK Standard Protocols: Insect Cell Purification

OPPF-UK Standard Protocols: Insect Cell Purification OPPF-UK Standard Protocols: Insect Cell Purification Last Updated 6 th October 2016 Joanne Nettleship joanne@strubi.ox.ac.uk OPPF-UK SOP: Insect Cell Purification Table of Contents Suggested Schedule...

More information

Supplemental Material Liu et al.

Supplemental Material Liu et al. Supplemental Material Liu et al. Supplemental Methods Protein expression, purification Recombinant etud11 (a.a 2344-2515) and Tud7-11 (a.a. 1617-2515) proteins (Tud7-11) were expressed in the Rosetta strain

More information

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Focus concept Purification of a novel seed storage protein allows sequence analysis and determination of the protein

More information

Supporting Information

Supporting Information Supporting Information Chan et al. 10.1073/pnas.0903849106 SI Text Protein Purification. PCSK9 proteins were expressed either transiently in 2936E cells (1), or stably in HepG2 cells. Conditioned culture

More information

A(+1) A( 7) RNA hairpin

A(+1) A( 7) RNA hairpin PP7 CP dimer A(+1) A( 7) RNA hairpin 5 3 Supplemental Figure 1. Sample electron density of RNA hairpin. Cartoon showing PP7 FG CP (wheat) bound to RNA hairpin (violet) showing 2Fo-Fc electron density (blue)

More information

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Last modified 29 September 2005

Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Last modified 29 September 2005 Case 7 A Storage Protein From Seeds of Brassica nigra is a Serine Protease Inhibitor Last modified 9 September 005 Focus concept Purification of a novel seed storage protein allows sequence analysis and

More information

Preparative Protein Chemistry

Preparative Protein Chemistry Biochemistry 412 Preparative Protein Chemistry 19 February 2008 The Three Eras of Protein Purification 1. The Classical (Pre-Recombinant DNA) Era (pre-1978) - Proteins purified from natural sources only

More information

Structural bases for N-glycan processing by mannosidephosphorylase

Structural bases for N-glycan processing by mannosidephosphorylase Supporting information Volume 71 (2015) Supporting information for article: Structural bases for N-glycan processing by mannosidephosphorylase Simon Ladevèze, Gianluca Cioci, Pierre Roblin, Lionel Mourey,

More information

SUPPLEMENTARY INFORMATION. Reengineering Protein Interfaces Yields Copper-Inducible Ferritin Cage Assembly

SUPPLEMENTARY INFORMATION. Reengineering Protein Interfaces Yields Copper-Inducible Ferritin Cage Assembly SUPPLEMENTARY INFORMATION Reengineering Protein Interfaces Yields Copper-Inducible Ferritin Cage Assembly Dustin J. E. Huard, Kathleen M. Kane and F. Akif Tezcan* Department of Chemistry and Biochemistry,

More information

Ali Yaghi. Tamara Wahbeh. Mamoun Ahram

Ali Yaghi. Tamara Wahbeh. Mamoun Ahram 28 Ali Yaghi Tamara Wahbeh Mamoun Ahram This sheet is a continuation of protein purification methods. Isoelectric focusing Separation of proteins based on Isoelectric points(charge),and it is a horizontal

More information

Supplementary Table 1: List of CH3 domain interface residues in the first chain (A) and

Supplementary Table 1: List of CH3 domain interface residues in the first chain (A) and Supplementary Tables Supplementary Table 1: List of CH3 domain interface residues in the first chain (A) and their side chain contacting residues in the second chain (B) a Interface Res. in Contacting

More information

Steps in solving a structure. Diffraction experiment. Obtaining well-diffracting crystals. Three dimensional crystals

Steps in solving a structure. Diffraction experiment. Obtaining well-diffracting crystals. Three dimensional crystals Protein structure from X-ray diffraction Diffraction images: ciprocal space Protein, chemical structure: IALEFGPSLKMNE Conformation, 3D-structure: CRYST1 221.200 73.600 80.900 90.00 90.00 90.00 P 21 21

More information

Ni-NTA Agarose. User Manual. 320 Harbor Way South San Francisco, CA Phone: 1 (888) MCLAB-88 Fax: 1 (650)

Ni-NTA Agarose. User Manual. 320 Harbor Way South San Francisco, CA Phone: 1 (888) MCLAB-88 Fax: 1 (650) Ni-NTA Agarose User Manual 320 Harbor Way South San Francisco, CA 94080 Phone: 1 (888) MCLAB-88 Fax: 1 (650) 871-8796 www. Contents Introduction -----------------------------------------------------------------------

More information

Product. Ni-NTA His Bind Resin. Ni-NTA His Bind Superflow. His Bind Resin. His Bind Magnetic Agarose Beads. His Bind Column. His Bind Quick Resin

Product. Ni-NTA His Bind Resin. Ni-NTA His Bind Superflow. His Bind Resin. His Bind Magnetic Agarose Beads. His Bind Column. His Bind Quick Resin Novagen offers a large variety of affinity supports and kits for the purification of recombinant proteins containing popular peptide fusion tags, including His Tag, GST Tag, S Tag and T7 Tag sequences.

More information

Michael A. DiMattia, Norman R. Watts, Stephen J. Stahl, Jonathan M. Grimes, Alasdair C. Steven, David I. Stuart, and Paul T.

Michael A. DiMattia, Norman R. Watts, Stephen J. Stahl, Jonathan M. Grimes, Alasdair C. Steven, David I. Stuart, and Paul T. Structure, Volume 21 Supplemental Information Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein Michael A. DiMattia, Norman R. Watts, Stephen J. Stahl, Jonathan

More information

AFFINITY HIS-TAG PURIFICATION

AFFINITY HIS-TAG PURIFICATION DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous suspension containing 30 vol % ethanol. INSTRUCTIONS The resins are adapted

More information

Lecture 7: Affinity Chromatography-II

Lecture 7: Affinity Chromatography-II Lecture 7: Affinity Chromatography-II We have studied basics of affinity purification during last lecture. The current lecture is continuation of last lecture and we will cover following: 1. Few specific

More information

Supplemental Material

Supplemental Material Supplemental Material Molecular basis for oncohistone H3 recognition by SETD2 methyltransferase Shuang Yang, 1, 2 Xiangdong Zheng, 1, 2, 3 Chao Lu, 4 Guo-Min Li, 2 C. David Allis, 4 1, 2, 3, 5* and Haitao

More information

Purification and crystallization of the bimagrumab Fab. The light- (residues 1 to 216, C216A

Purification and crystallization of the bimagrumab Fab. The light- (residues 1 to 216, C216A SI Methods Purification and crystallization of the bimagrumab Fab. The light- (residues 1 to 216, C216A variant) and heavy-chain (residues 1 to 219, R212K variant) of the Bimagrumab Fab were cloned on

More information

BACTERIAL PRODUCTION EXPRESSION METHOD OVERVIEW: PEF # GENE NAME EXPRESSION VECTOR MOLECULAR WEIGHT kda (full-length) 34.

BACTERIAL PRODUCTION EXPRESSION METHOD OVERVIEW: PEF # GENE NAME EXPRESSION VECTOR MOLECULAR WEIGHT kda (full-length) 34. BACTERIAL PRODUCTION PEF # GENE NAME EXPRESSION VECTOR MOLECULAR WEIGHT 2015-XXXX XXXX pet-32a 50.9 kda (full-length) 34.0 kda (cleaved) EXPRESSION METHOD OVERVIEW: Plasmid DNA was transformed into BL21

More information

Innovation Moléculaire et Thérapeutique, Université François Rabelais, Tours, France *For correspondence:

Innovation Moléculaire et Thérapeutique, Université François Rabelais, Tours, France *For correspondence: Expression and Purification of the Eukaryotic MBP-MOS1 Transposase from sf21 Insect Cells Jérôme Jaillet, Audrey Dussaussois-Montagne, Sylvaine Renault and Corinne Augé-Gouillou * Innovation Moléculaire

More information

Virtual bond representation

Virtual bond representation Today s subjects: Virtual bond representation Coordination number Contact maps Sidechain packing: is it an instrumental way of selecting and consolidating a fold? ASA of proteins Interatomic distances

More information

INSTRUCTIONS The resins are adapted to work mainly in native conditions like denaturing.

INSTRUCTIONS The resins are adapted to work mainly in native conditions like denaturing. 1 AFFINITY HIS-TAG PURIFICATION PROCEDURE FOR USE Nickel NTA Agarose Beads DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous

More information

MBMB451A Section1 Fall 2008 KEY These questions may have more than one correct answer

MBMB451A Section1 Fall 2008 KEY These questions may have more than one correct answer MBMB451A Section1 Fall 2008 KEY These questions may have more than one correct answer 1. In a double stranded molecule of DNA, the ratio of purines : pyrimidines is (a) variable (b) determined by the base

More information

SERVA IMAC Ni-IDA Test Kit Agarose for Affinity Purification of His-Tag Fusion Proteins

SERVA IMAC Ni-IDA Test Kit Agarose for Affinity Purification of His-Tag Fusion Proteins INSTRUCTION MANUAL SERVA IMAC Ni-IDA Test Kit Agarose for Affinity Purification of His-Tag Fusion Proteins (Cat. No.42164, 42165) SERVA Electrophoresis GmbH - Carl-Benz-Str. 7-69115 Heidelberg Phone +49-6221-138400,

More information

Supporting Information. Noncovalent insertion of ferrocenes into the protein shell of apo-ferritin

Supporting Information. Noncovalent insertion of ferrocenes into the protein shell of apo-ferritin Supporting Information Noncovalent insertion of ferrocenes into the protein shell of apo-ferritin Jochen Niemeyer, Satoshi Abe, Tatsuo Hikage, Takafumi Ueno, Gerhard Erker, Yoshihito Watanabe Department

More information

Ligand immobilization using thiol-disulphide exchange

Ligand immobilization using thiol-disulphide exchange A P P L I C A T I T E 9 Ligand immobilization using thiol-disulphide exchange Abstract This Application ote describes an immobilization procedure based on thioldisulphide exchange, providing a valuable

More information

Zwitterion Chromatography ZIC

Zwitterion Chromatography ZIC Zwitterion Chromatography ZIC A novel technique, with unique selectivity, suitable for preparative scale separations? PhD Einar Pontén What is Zwitterion Chromatography? Our definition: Liquid chromatography

More information

Purification, Optimization, and Growth of New Delhi metallo-β-lactamase-1 protein crystals mixed with NZ218 inhibitor

Purification, Optimization, and Growth of New Delhi metallo-β-lactamase-1 protein crystals mixed with NZ218 inhibitor Augustana College Augustana Digital Commons Celebration of Learning Purification, Optimization, and Growth of New Delhi metallo-β-lactamase-1 protein crystals mixed with NZ218 inhibitor Brandon M. Wills

More information

Supplementary Materials for

Supplementary Materials for advances.sciencemag.org/cgi/content/full/2/11/e1601625/dc1 Supplementary Materials for A molecular mechanism of chaperone-client recognition This PDF file includes: Lichun He, Timothy Sharpe, Adam Mazur,

More information

Nickel-NTA Agarose Suspension

Nickel-NTA Agarose Suspension Nickel-NTA Agarose Suspension Agarose beads for purification of His-tagged proteins Product No. A9735 Description Nickel-NTA Agarose Suspension is an agarose-based affinity chromatography resin allowing

More information

Description of Changes and Corrections for PDB File Format Version 4.0. Provisional Document April 12, 2011

Description of Changes and Corrections for PDB File Format Version 4.0. Provisional Document April 12, 2011 Description of Changes and Corrections for PDB File Format Version 4.0 Provisional Document April 12, 2011 The wwpdb has reviewed the PDB archive and created a new set of corrected files that will be released

More information

SUPPLEMENTARY MATERIAL

SUPPLEMENTARY MATERIAL SUPPLEMENTARY MATERIAL Purification and biochemical characterization of acid phosphatase-i from seeds of Nelumbo nucifera Sanaullah Khan a*, Shahnaz Asmat c, Sajida Batool a, Mushtaq Ahmed b a Department

More information

X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction

X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction Tomohisa Shimasaki 1, Hiromi Yoshida 2, Shigehiro Kamitori 2 & Koji

More information

ProteIndex TM Co-NTA Agarose 6 Fast Flow

ProteIndex TM Co-NTA Agarose 6 Fast Flow Store product at 2 C 8 C. Do not freeze. The product is shipped at ambient temperature. ProteIndex TM Co-NTA Agarose 6 Fast Flow Cat. No. Package Size 11-0231-010 ProteIndex Co-NTA Agarose 6 Fast Flow,

More information

SUPPORTING INFORMATION

SUPPORTING INFORMATION SUPPORTING INFORMATION Text S1: EvoDesign profile construction Table S1 lists the pair-wise structural alignments generated by the TM-align program [1], between the XIAP scaffold protein (PDB ID: 2OPY)

More information

Supplementary materials for Structure of an open clamp type II topoisomerase-dna complex provides a mechanism for DNA capture and transport

Supplementary materials for Structure of an open clamp type II topoisomerase-dna complex provides a mechanism for DNA capture and transport Supplementary materials for Structure of an open clamp type II topoisomerase-dna complex provides a mechanism for DNA capture and transport Ivan Laponogov 1,2, Dennis A. Veselkov 1, Isabelle M-T. Crevel

More information

AVB Sepharose High Performance

AVB Sepharose High Performance GE Healthcare Life Sciences Data file 28-927-54 AB Custom designed media AVB Sepharose High Performance AVB Sepharose High Performance is an affinity chromatography medium (resin) designed for the purification

More information

Supplemental Figure 1

Supplemental Figure 1 Supplemental Figure 1 Supplemental Fig. 1. (A) The binding avidity between Fz4-FcH6 and MBP-Norrin dimers measured by biolayer interferometry. 5 µg/ml Fz4-Fc protein was loaded onto anti human Fc capture

More information

OPPF-UK Standard Protocols: Mammalian Expression

OPPF-UK Standard Protocols: Mammalian Expression OPPF-UK Standard Protocols: Mammalian Expression Joanne Nettleship joanne@strubi.ox.ac.uk Table of Contents 1. Materials... 3 2. Cell Maintenance... 4 3. 24-Well Transient Expression Screen... 5 4. DNA

More information

Supplemental Information. Structural Basis of Zika Virus-Specific. Antibody Protection

Supplemental Information. Structural Basis of Zika Virus-Specific. Antibody Protection Cell, Volume 166 Supplemental Information Structural Basis of Zika Virus-Specific Antibody Protection Haiyan Zhao, Estefania Fernandez, Kimberly A. Dowd, Scott D. Speer, Derek J. Platt, Matthew J. Gorman,

More information

Index 1. Product Description 2. Purification Procedure 3. Troubleshooting 4. Ordering Information

Index 1. Product Description 2. Purification Procedure 3. Troubleshooting 4. Ordering Information High Affinity Ni-Charged Resin Cat. No. L00223 Technical Manual No. TM0217 Version 07132010 Index 1. Product Description 2. Purification Procedure 3. Troubleshooting 4. Ordering Information 1. Product

More information

AFFINITY HIS-TAG PURIFICATION

AFFINITY HIS-TAG PURIFICATION DESCRIPTION Resins are products that allow batch or column purifications. This product is supplied as a suspension in 50% aqueous suspension containing 30 vol % ethanol. INSTRUCTIONS The resins are adapted

More information

BIOC 463A Expt. 4: Column Chromatographic Methods Column Chromatography

BIOC 463A Expt. 4: Column Chromatographic Methods Column Chromatography Column Chromatography Chromatography is the process use to separate molecules based on SOME physical property of the molecule: Mass (i.e. size) Charge Affinity for ligands or substrates Hydrophobic interactions

More information

Supporting Information

Supporting Information Supporting Information Crystal Structure of a Mycoestrogen-Detoxifying Lactonase from Rhinocladiella mackenziei: Molecular Insight into ZHD Substrate Selectivity Yingying Zheng, 1, Wenting Liu, 1,2, Chun-Chi

More information

AminTRAP HIS Prepacked Column

AminTRAP HIS Prepacked Column INDEX Ordering Information... 3 Intended Use... 3 Product Description... 3 Purification Procedure... 4 Sample Preparation... 5 Sample Purification... 6 Analysis... 6 Regeneration Procedure... 6 Use and

More information

Supplemental Materials and Methods:

Supplemental Materials and Methods: Supplemental Materials and Methods: Cloning: Oligonucleotides used in the subcloning steps are listed in Supplemental Table 1. Human FANCI (isoform 1, KIAA1794) was subcloned from pcmv6-xl4 [FANCI] in

More information

Supporting Information Contents

Supporting Information Contents Supporting Information Choy Theng Loh, Kiyoshi Ozawa, Kellie L. Tuck, Nicholas Barlow, Thomas Huber, Gottfried Otting, and Bim Graham Lanthanide tags for site-specific ligation to an unnatural amino acid

More information

Supplementary Online Material. An Expanded Eukaryotic Genetic Code 2QH, UK. * To whom correspondence should be addressed.

Supplementary Online Material. An Expanded Eukaryotic Genetic Code 2QH, UK. * To whom correspondence should be addressed. Supplementary Online Material An Expanded Eukaryotic Genetic Code Jason W. Chin 1, T. Ashton Cropp, J. Christopher Anderson, Mridul Mukherji, Zhiwen Zhang and Peter G. Schultz* Department of Chemistry

More information

Supporting Information

Supporting Information Supporting Information Supplementary Figure-1 Results of Phage Display Screening Against TNT The sequences seen in Supplementary Figure-1 represent the phages sequenced after the third and fourth rounds

More information

Nickel Chelating Resin Spin Columns

Nickel Chelating Resin Spin Columns 326PR-02 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Nickel Chelating Resin Spin Columns A Ni-IDA IMAC resin for 6X-His Tagged Protein

More information

Protein Purification Products. Complete Solutions for All of Your Protein Purification Applications

Protein Purification Products. Complete Solutions for All of Your Protein Purification Applications Protein Purification Products Complete Solutions for All of Your Protein Purification Applications FLAG-Tagged Protein Products EXPRESS with the pcmv-dykddddk Vector Set Fuse your protein of interest to

More information

The Study of NF-κB Peptide Mimics and How Proteins Bind DNA

The Study of NF-κB Peptide Mimics and How Proteins Bind DNA Georgia Southern University Digital Commons@Georgia Southern University Honors Program Theses Student Research Papers 2016 The Study of NF-κB Peptide Mimics and How Proteins Bind DNA Allee M. Murray Georgia

More information

Supplementary Figure 1. Electron microscopy of gb-698glyco/1g2 Fab complex. a)

Supplementary Figure 1. Electron microscopy of gb-698glyco/1g2 Fab complex. a) Supplementary Figure 1. Electron microscopy of gb-698glyco/1g2 Fab complex. a) Representative images of 2D class averages of gb-698glyc bound to 1G2 Fab. Top views of the complex were underrepresented

More information

Cobalt Chelating Resin

Cobalt Chelating Resin 078PR-05 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Cobalt Chelating Resin A Co-IDA IMAC resin for 6X-His Tagged Protein Purification

More information

TransIT -Lenti Transfection Reagent

TransIT -Lenti Transfection Reagent Quick Reference Protocol, SDS and Certificate of Analysis available at mirusbio.com/6600 INTRODUCTION Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family capable of infecting

More information

High-Affinity Ni-NTA Resin

High-Affinity Ni-NTA Resin High-Affinity Ni-NTA Resin Technical Manual No. 0217 Version 20070418 I Description.... 1 II Key Features... 1 III His-Tagged Fusion Protein Purification Procedure.. 1 IV Resin Regeneration. 4 V Troubleshooting...

More information

The Transfection Collection TCF/LEF Transient Pack Wnt / -catenin Signaling Pathway Catalog #: 79273

The Transfection Collection TCF/LEF Transient Pack Wnt / -catenin Signaling Pathway Catalog #: 79273 Data Sheet The Transfection Collection TCF/LEF Transient Pack Wnt / -catenin Signaling Pathway Catalog #: 79273 Background The Wnt / -catenin signaling pathway controls a large and diverse set of cell

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Table 1. Crystallographic statistics CRM1-SNUPN complex Space group P6 4 22 a=b=250.4, c=190.4 Data collection statistics: CRM1-selenomethionine SNUPN MAD data Peak Inflection Remote Native

More information

HELIX TM HT-96 MD1-69

HELIX TM HT-96 MD1-69 HELIX TM HT-96 MD1-69 Crystallize a diverse range of nucleic acid topologies and molecular weights. This screen targets all types of DNA/RNA, triplexes, quadruplexes, pseudoknots, i-motifs, and large molecular

More information

蛋白質體學. Proteomics Amino acids, Peptides and Proteins 陳威戎 & 21

蛋白質體學. Proteomics Amino acids, Peptides and Proteins 陳威戎 & 21 蛋白質體學 Proteomics 2015 Amino acids, Peptides and Proteins 陳威戎 2015. 09. 14 & 21 Outline 1. Amino Acids 2. Peptides and Proteins 3. Covalent Structure of Proteins Amino Acids Proteins are polymers of amino

More information

Data Sheet Quick PCR Cloning Kit

Data Sheet Quick PCR Cloning Kit Data Sheet Quick PCR Cloning Kit 6044 Cornerstone Ct. West, Ste. E DESCRIPTION: The Quick PCR Cloning Kit is a simple and highly efficient method to insert any gene or DNA fragment into a vector, without

More information

Separation of Monoclonal Antibodies Using TSKgel HPLC Columns

Separation of Monoclonal Antibodies Using TSKgel HPLC Columns ANALYSIS S e p a r a t i o n R e p o r t N o. 7 4 Separation of Monoclonal Antibodies Using TSKgel HPLC Columns Table of Contents 1. Introduction 1 2. Separation Mode and Purification Method 1 3. Applications

More information

TECHNICAL BULLETIN. HIS-Select HF Nickel Affinity Gel. Catalog Number H0537 Storage Temperature 2 8 C

TECHNICAL BULLETIN. HIS-Select HF Nickel Affinity Gel. Catalog Number H0537 Storage Temperature 2 8 C HIS-Select HF Nickel Affinity Gel Catalog Number H0537 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select High Flow (HF) is an immobilized metal-ion affinity chromatography (IMAC)

More information

High-Affinity Ni-NTA Resin

High-Affinity Ni-NTA Resin High-Affinity Ni-NTA Resin Technical Manual No. 0237 Version 20070418 I Description.. 1 II Key Features... 1 III His-Tagged Fusion Protein Purification Procedure 1 IV Resin Regeneration. 4 V Troubleshooting...

More information

Structural bioinformatics

Structural bioinformatics Structural bioinformatics Why structures? The representation of the molecules in 3D is more informative New properties of the molecules are revealed, which can not be detected by sequences Eran Eyal Plant

More information