Mar 10, 2025

Public workspaceREADDI protocol: Crystallisation of Dengue NS5 RdRp in I222

 Forked from READDI protocol: Crystallisation of CHIKV nsP3 macrodomain
DOI
dx.doi.org/10.17504/protocols.io.5qpvo9wn9v4o/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3READDI-AC;
  • 4Rutgers University
  • ASAP Discovery
Peter Marples
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo9wn9v4o/v1
External link: https://readdi-ac.org/
Protocol Citation: Jasmin Aschenbrenner, Manisha Saini, Ashima Chopra, Peter Marples, Francesc Xavier Ruiz, Eddy Arnold, Daren Fearon 2025. READDI protocol: Crystallisation of Dengue NS5 RdRp in I222. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo9wn9v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2025
Last Modified: March 10, 2025
Protocol Integer ID: 119264
Keywords: crystallisation, XChem, Diamond Light Source, i04-1, READDI, AViDD, Research complex at Harwell, Dengue, NS5, RdRp, Flavivirus, Dengue virus, virus
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171292
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Acknowledgements:
READDI-AC, https://readdi-ac.org
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK
Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot OX11 0FA, UK
Oxford Lab Technologies crystal shifter https://doi.org/10.1107/S2059798320014114
Abstract
The crystallisation protocol and buffer conditions used to obtain reproducible Dengue virus serotype 2 RNA-dependent RNA polymerase crystals in I222 suitable for XChem fragment screening.
Guidelines
N/A
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

1 M MES, pH 6.7 (Catalog # MD2-013-PH)
5 M Magnesium chloride hexahydrate (Catalog # MD2-100-47)
50 % (w/v) PEG 4000 (Catalog # MD2-100-11)

Purified protein (12 mg/mL) in 20 mM HEPES, pH 7.0, 300 mM NaCl, 5 mM TCEP.
Safety warnings
Follow all handling warnings for the chemicals used in the crystalllisation screen composition.
Ethics statement
N/A
Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
https://www.sptlabtech.com/products/liquid-handling/mosquito-hv/
LINK
P100 8 multi-channel pipette
SwissCI 3 lens plate

Crystallisation experiment
Crystallisation experiment
5d 0h 27m 40s
5d 0h 27m 40s
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 5000 dilution SampleSample seeds
17m 40s
Protein and buffer requirements:
Amount30 µL Concentration12 mg/mL SampleSample
Amount2.9 mL Crystallisation screen
Amount14.4 µL Dengue NS5 seeds , dilution 1:5000

Crystallisation screen composition:
Concentration0.1 Molarity (M) MES Ph6.7
Concentration0.35 Molarity (M) MgCl2
Concentration10 % (w/v) PEG 4000

Stock solutions used:
Concentration1 Molarity (M) MES Ph6.7
Concentration5 Molarity (M) MgCl2
Concentration50 % (w/v) PEG 4000
Note
The crystallisation screen can be stored in a Duran bottle or aliquoted into 96 deep-well blocks for easy dispensing into SwissCI 3 lens plates.

For long-term storage keep the crystallisation screen in the fridge at 4°C.

Dispense Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount100 nL Dengue NS5 protein Concentration12 mg/mL to each lens using the SPT mosquito.
Dispense Amount150 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 nL Dengue NS5 Seeds to each lens using the SPT mosquito.

Drop ratio: 2:3:1 ratio (100 nl Protein: 150 nl Crystallisation solution: 50 nl Seeds)
Final drop volume: 300 nl
10m
Incubate at Temperature20 °C for Duration120:00:00 in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections.
5d

Expected result
The crystals reach their maximum size after 3 to 5 d.

Crystals typically form as chunky, often layered, needles.

Morphology: long, thin or chunky needles
Size: ~400 μm in length
Appearance: needle clusters
Average resolution: 1.9 Å
Space group: I222
Unit cell: 83 Å, 117 Å, 149 Å
90°, 90°, 90°



An example of a drop containing DENV2 NS5 RdRp crystals.




Data collection at Synchrotron
Data collection at Synchrotron
Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 XE 9M
Beamline: I04-1
Wavelength: 0.9212 Å
Resolution (Å): 1.62
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 3.80e+12
Acknowledgements
READDI-AC, https://readdi-ac.org
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK
Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot OX11 0FA, UK
Oxford Lab Technologies crystal shifter https://doi.org/10.1107/S2059798320014114