DHVI Macromolecular X-ray Crystallography

Technology / Instruments / Resources Mission

Services

• Support high-throughput crystallography with modern instrumentation and robotics.

• Open access for anyone at Duke wanting to pursue structural biology aims.

• Protein production• Crystallization trials• Automated imaging of

crystallization experiments• X-ray diffraction and

data collection• Expert staff support

Rigaku MicroMax-007• Micro focus rotating anode x-ray generator• VariMax HR (high resolution) optics • RAXIS IV++ image plate detector• Inverse phi axis for easy crystal mounting• X-stream 2000 cryogenic system• Plexiglass enclosure

Recent Projects / Publications

Reservations / Service Requests The shared instruments are available for use by

properly trained users at their discretion.

• Martinez D, Vandergrift N, et al. (2017) Maternal binding and neutralizing IgG responses targeting the C terminal region of the V3 loop are predictive of reduced peripartum HIV-1 transmission risk. Journal of Virology 91(9):e02422-16• Saunders K, Nicely NI, et al. (2017) Vaccine elicitation of high mannose-

dependent neutralizing antibodies against the V3-glycan broadly neutralizing epitope in nonhuman primates. Cell Reports 18(9):2175-2188• Shwab E, Juvvadi P, et al. (2017) A Novel Phosphoregulatory Switch

Controls the Activity and Function of the Major Catalytic Subunit of Protein Kinase A in Aspergillus fumigatus. mBio 8(1):e02319-16• Williams LD, Ofek G, et al. (2017) Potent and Broad HIV Neutralizing

Antibodies in Memory B Cells and Plasma. Science Immunology 2(7):eaal2200• Nicely NI, Wiehe K, et al. (2015) Structural analysis of the unmutated

ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee. EBioMedicine 2(7): 713-22• Deis LN, Wu Q, et al. (2015) Suppression of conformational

heterogeneity at a protein–protein interface. PNAS USA 112(29):9028-33• Santra S, Tomaras GD, et al. (2015) Human Non-neutralizing HIV-1

Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques. PLoS Pathog. 11(8):e1005042.• Burg JM, Makhoul AT, et al. (2015) A rationally-designed chimeric

KDM1A/KDM1B histone demethylase tower domain deletion mutant retaining enzymatic activity. FEBS Lett. S0014-5793(15)00653-5.

Leadership / Experience Director: Nathan Nicely, PhD • Assistant Professor in Medicine / DHVI• Ten years at Duke• Experience in diverse topics including immunology, enzymology, pathogenic metabolism, oncology

Contact Us

Levine Science Research Center room A06

crystallography@duke.edu

xray.dhvi.duke.edu

X-ray diffraction

Minstrel HT UV• Every drop dual imaged in white and UV light• Fully automated operation with a manual mode• Inspection schedule of choice

Crystallization experiment setups• Phoenix robot dispenses 96-well screens all at once• Oryx4 robot dispenses proteins and protein-reagent mixtures• Drop volumes of 100-500 nl• Handles seeds

Robots

Crystal imaging

Protein crystalsFluoresce under UV.

Salt crystalsDo not illuminate.

Crystals in precipitateRevealed by UV.

Instrument ThroughputPhoenix Dispenses one screen in ~3 minutes.

Oryx Dispenses one plate in ~20 minutes; typically ~100-500 nl drop volumes

Gallery incubators House ~200 plates per Gallery

Minstrel HT UV Images one 96x3 plate in ~30 min007/Varimax HR Runs 24/7

HIV antibody DH522.2. A) The structure of DH522.2 (red, pink) in complex with a constituent of the HIV Envelope trimer (grey) shows why the antibody does not broadly neutralize: B) it is unable to access its binding interface (blue) on the fully assembled trimeric structure (white).

A

B

Effects of phosphorylation events on Protein Kinase A function. A) The structure of the protein kinase A catalytic subunit threaded with the Aspergillus fumigatus ortholog sequence (white trace) and bound to inhibitory peptide (green) shows the location of key Thr mutations where phosphorylation modifications abrogate enzyme function. B) Other inappropriate phosphorylation sites negatively impact interaction of the catalytic subunit with the regulatory subunit (violet).

A B