Dare to spin – well diffracting protein nanocrystals through on-vortex crystallisation

Presented During:

07/30/2022: 5:30 PM - 7:30 PM
Portland Marriott Downtown Waterfront  


2022: 72nd ACA Annual Meeting

Session Type:


Presenting Author :

Gerhard Hofer  
Stockholm University

Additional Author(s):

Laura Calmanovici Pacoste  
Stockholm University
Lei Wang  
Stockholm University
Hongyi Xu  
Department of Materials and Environmental Chemistry
Xiaodong Zou  
Stockholm University

Abstract Body:

Protein crystallisation has been extensively studied for X-ray and neutron diffraction.
The hunt for optimised conditions yielding large single crystals has become the
staple of protein crystallography labs around the world. Recently new advances in
synchrotron beam lines and free electron X-ray lasers have opened the world of
smaller (<40 μm a side) crystals being used for structure determination.
Electron diffraction (3D ED / microED) brings altogether new challenges to the field.
Not only can it utilise even smaller crystals, it even requires one dimension to be sub
1 μm to allow the electron beam to pass through. Additionally, it benefits from the
other dimensions being significantly larger, since that allows for more protein to
participate in the diffraction measurement - which in turn improves signal strength
while reducing beam damage effects. Such ideal plate shaped crystals are often
created by focused ion beam milling of larger crystals, but this approach is time
consuming and requires specialised machinery.
We have found that leaving behind the careful and slow techniques of X-ray protein
crystallography and instead employing rapid crystallisation on a vortex mixer can
offer surprisingly good control over crystal size in the range required for 3D ED. The
optional addition of metal or PTFE beads to create shear forces capable of
fragmenting larger crystals provides a feedback loop that seeds new crystal growth
should the seed count be insufficient at first.
With an optimized protocol taking only minutes, we have succeeded in creating
solutions containing countless well diffracting crystal plates of a urate oxidase
(UOX), a ribonucleotide reductase R2 and two arginine kinases amongst others.
Diffraction data sets to 2 Å are commonly obtainable from these plates with both the
diffraction quality and resolution improving with decreasing plate thickness. In the
case of UOX the achievable resolution changed from 3 Å to 1.4 Å when switching to
the new method.

Additional Information - Poster (2022)

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1.1.4 MicroED of small and macromolecules

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