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Dive into the research topics where Eric Koesema is active.

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Featured researches published by Eric Koesema.


Proteins | 2008

Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts

Heath E. Klock; Eric Koesema; Mark W. Knuth; Scott A. Lesley

Successful protein expression, purification, and crystallization for challenging targets typically requires evaluation of a multitude of expression constructs. Often many iterations of truncations and point mutations are required to identify a suitable derivative for recombinant expression. Making and characterizing these variants is a significant barrier to success. We have developed a rapid and efficient cloning process and combined it with a protein microscreening approach to characterize protein suitability for structural studies. The Polymerase Incomplete Primer Extension (PIPE) cloning method was used to rapidly clone 448 protein targets and then to generate 2143 truncations from 96 targets with minimal effort. Proteins were expressed, purified, and characterized via a microscreening protocol, which incorporates protein quantification, liquid chromatography mass spectrometry and analytical size exclusion chromatography (AnSEC) to evaluate suitability of the protein products for X‐ray crystallography. The results suggest that selecting expression constructs for crystal trials based primarily on expression solubility is insufficient. Instead, AnSEC scoring as a measure of protein polydispersity was found to be predictive of ultimate structure determination success and essential for identifying appropriate boundaries for truncation series. Overall structure determination success was increased by at least 38% by applying this combined PIPE cloning and microscreening approach to recalcitrant targets. Proteins 2008.


Proceedings of the National Academy of Sciences of the United States of America | 2010

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency

Rodolfo Gonzalez; Lori L. Jennings; Mark W. Knuth; Anthony P. Orth; Heath E. Klock; Weija Ou; Julie Feuerhelm; Mitchell V. Hull; Eric Koesema; Yuping Wang; Jia Zhang; Chunlei Wu; Charles Y. Cho; Andrew I. Su; Serge Batalov; Hong Chen; Kristen Johnson; Bryan A. Laffitte; Deborah G. Nguyen; Evan Y. Snyder; Peter G. Schultz; Jennifer L. Harris; Scott A. Lesley

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.


Protein Science | 2006

Crystal structure of the human TRPV2 channel ankyrin repeat domain

Clare J. McCleverty; Eric Koesema; Ardem Patapoutian; Scott A. Lesley; Andreas Kreusch

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein–protein recognition domain, is conserved in the N‐terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 Å resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.


Proteins | 2002

Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 Å resolution

Peter Kuhn; Scott A. Lesley; Irimpan I. Mathews; Jaume M. Canaves; Linda S. Brinen; Xiaoping Dai; Ashley M. Deacon; Marc André Elsliger; Said Eshaghi; Ross Floyd; Adam Godzik; Carina Grittini; Slawomir K. Grzechnik; Chittibabu Guda; Keith O. Hodgson; Lukasz Jaroszewski; Cathy Karlak; Heath E. Klock; Eric Koesema; John M. Kovarik; Andreas Kreusch; Daniel McMullan; Timothy M. McPhillips; Mark A. Miller; Mitchell D. Miller; Andrew T. Morse; Kin Moy; Jie Ouyang; Alyssa Robb; Kevin Rodrigues

Peter Kuhn, Scott A. Lesley, Irimpan I. Mathews, Jaume M. Canaves, Linda S. Brinen, Xiaoping Dai, Ashley M. Deacon, Marc A. Elsliger, Said Eshaghi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Chittibabu Guda, Keith O. Hodgson, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John M. Kovarik, Andreas T. Kreusch, Daniel McMullan, Timothy M. McPhillips, Mark A. Miller, Mitchell Miller, Andrew Morse, Kin Moy, Jie Ouyang, Alyssa Robb, Kevin Rodrigues, Thomas L. Selby, Glen Spraggon, Raymond C. Stevens, Susan S. Taylor, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The Genomics Institute of Novartis Foundation, San Diego, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, California The Scripps Research Institute, La Jolla, California


PLOS ONE | 2010

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus Pneumoniae Pilus.

Glen Spraggon; Eric Koesema; Maria Scarselli; Enrico Malito; Massimiliano Biagini; Nathalie Norais; Carla Emolo; Michèle A. Barocchi; Fabiola Giusti; Markus Hilleringmann; Rino Rappuoli; Scott A. Lesley; Antonello Covacci; Vega Masignani; Ilaria Ferlenghi

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility.


Proteins | 2005

Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold

Chris Rife; Robert Schwarzenbacher; Daniel McMullan; Polat Abdubek; Eileen Ambing; Herbert L. Axelrod; Tanya Biorac; Jaume M. Canaves; Hsiu-Ju Chiu; Ashley M. Deacon; Michael DiDonato; Marc-André Elsliger; Adam Godzik; Carina Grittini; Slawomir K. Grzechnik; Joanna Hale; Eric Hampton; Gye Won Han; Justin Haugen; Michael Hornsby; Lukasz Jaroszewski; Heath E. Klock; Eric Koesema; Andreas Kreusch; Peter Kuhn; Scott A. Lesley; Mitchell D. Miller; Kin Moy; Edward Nigoghossian; Jessica Paulsen

Chris Rife, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Herbert Axelrod, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Mitchell D. Miller, Kin Moy, Edward Nigoghossian, Jessica Paulsen, Kevin Quijano, Ron Reyes, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Aprilfawn White, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The University of California, San Diego, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The Scripps Research Institute, La Jolla, California


Proteins | 2004

Crystal structure of a tandem cystathionine-β-synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 Å resolution

Mitchell D. Miller; Robert Schwarzenbacher; Frank von Delft; Polat Abdubek; Eileen Ambing; Tanya Biorac; Linda S. Brinen; Jaume M. Canaves; Jamison Cambell; Hsiu-Ju Chiu; Xiaoping Dai; Ashley M. Deacon; Mike DiDonato; Marc-André Elsliger; Said Eshagi; Ross Floyd; Adam Godzik; Carina Grittini; Slawomir K. Grzechnik; Eric Hampton; Lukasz Jaroszewski; Cathy Karlak; Heath E. Klock; Eric Koesema; John S. Kovarik; Andreas Kreusch; Peter Kuhn; Scott A. Lesley; Inna Levin; Daniel McMullan

Mitchell D. Miller, Robert Schwarzenbacher, Frank von Delft, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jaume M. Canaves, Jamison Cambell, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Said Eshagi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Andrew Morse, Kin Moy, Jie Ouyang, Rebecca Page, Kevin Quijano, Alyssa Robb, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park California Genomics Institute of the Novartis Research Foundation, San Diego, California San Diego Supercomputer Center, La Jolla, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California


PLOS ONE | 2010

RNAi Screen Indicates Widespread Biological Function for Human Natural Antisense Transcripts

Mohammad Ali Faghihi; Jannet Kocerha; Farzaneh Modarresi; Pär G. Engström; Alistair Morgan Chalk; Eric Koesema; Georges St. Laurent; Claes Wahlestedt

Natural antisense transcripts represent a class of regulatory RNA molecules, which are characterized by their complementary sequence to another RNA transcript. Extensive sequencing efforts suggest that natural antisense transcripts are prevalent throughout the mammalian genome; however, their biological significance has not been well defined. We performed a loss-of-function RNA interference (RNAi) screen, which targeted 797 evolutionary conserved antisense transcripts, and found evidence for a regulatory role for a number of natural antisense transcripts. Specifically, we found that natural antisense transcripts for CCPG1 and RAPGEF3 may functionally disrupt signaling pathways and corresponding biological phenotypes, such as cell viability, either independently or in parallel with the corresponding sense transcript. Our results show that the large-scale siRNA screen can be applied to evaluate natural antisense transcript modulation of fundamental cellular events.


Journal of Structural and Functional Genomics | 2005

Methods and Results for Semi-automated Cloning Using Integrated Robotics

Heath E. Klock; Aprilfawn White; Eric Koesema; Scott A. Lesley

The Joint Center for Structural Genomics (JCSG) has emphasized automation and parallel processing approaches. Here, we describe automated methods used across the cloning process with results from JCSG projects. The protocols for PCR, restriction digests and ligations, as well as for gel electrophoresis and microtiter plate assays have all been automated. The system has the capacity to routinely process 384 clones a week. This throughput can adequately supply our expression and purification pipeline with expression-ready clones, including novel targets and truncations. The utility of our system is demonstrated by our results from three diverse projects. In summary, 94% of the PCR amplicons generated to date have been successfully cloned and verified by sequencing (83% of the total attempted targets). Our results demonstrate the capabilities of this robotic platform to provide an avenue to high-throughput cloning which requires little manpower and is rapid and cost-effective while providing insights for method optimization.


Proteins | 2004

Crystal structure of a PIN (PilT N‐terminus) domain (AF0591) from Archaeoglobus fulgidus at 1.90 Å resolution

Inna Levin; Robert Schwarzenbacher; Rebecca Page; Polat Abdubek; Eileen Ambing; Tanya Biorac; Linda S. Brinen; Jamison Campbell; Jaume M. Canaves; Hsiu-Ju Chiu; Xiaoping Dai; Ashley M. Deacon; Mike DiDonato; Marc-André Elsliger; Ross Floyd; Adam Godzik; Carina Grittini; Slawomir K. Grzechnik; Eric Hampton; Lukasz Jaroszewski; Cathy Karlak; Heath E. Klock; Eric Koesema; John S. Kovarik; Andreas Kreusch; Peter Kuhn; Scott A. Lesley; Daniel McMullan; Timothy M. McPhillips; Mitchell D. Miller

Inna Levin, Robert Schwarzenbacher, Rebecca Page, Polat Abdubek, Eileen Ambing, Tanya Biorac, Linda S. Brinen, Jamison Campbell, Jaume M. Canaves, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Mike DiDonato, Marc-André Elsliger, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Eric Hampton, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Jie Ouyang, Kevin Quijano, Ron Reyes, Fred Rezezadeh, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Frank von Delft, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics, Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park California Genomics Institute of the Novartis Research Foundation, San Diego, California San Diego Supercomputer Center, La Jolla, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California

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Heath E. Klock

Genomics Institute of the Novartis Research Foundation

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Daniel McMullan

Genomics Institute of the Novartis Research Foundation

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Andreas Kreusch

Genomics Institute of the Novartis Research Foundation

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Mitchell D. Miller

SLAC National Accelerator Laboratory

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Peter Kuhn

University of Southern California

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Scott A. Lesley

Genomics Institute of the Novartis Research Foundation

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