Nanoflow electrospinning serial femtosecond crystallography

Abstract

An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.

Figure 1

Nanoflow electrospinning protein microcrystal suspensions in vacuo for serial femtosecond crystallography at the LCLS Coherent X-ray Imaging endstation. An electrospun microjet (a) (scale bar 150 µm) of a thermolysin crystal suspension (b) (microscope image) flowing at 0.17 µl min⁻¹ is emitted in an electrospun microjet from a 50 µm internal diameter silica capillary positioned <1 mm from the X-ray-interaction point. An average of 2 mJ is delivered in each 40 fs pulse of 9.7 keV X-rays. Single-pulse diffraction patterns from single crystals were recorded on a Cornell-SLAC Pixel Array Detector (CSPAD). A virtual powder pattern from 1024 LCLS shots that produced ≥16 Bragg peaks each (c) showed diffraction beyond 4 Å resolution. Purple and yellow squares denote the portions of the CSPAD shown in the virtual powder pattern.

Figure 2

Tuning the sample flow rate of electrospun microjets into the nanoflow regime using capillary internal diameter (ID) and the pressure difference between the liquid reservoir and the vacuum chamber. The sample flow rate was measured for 30%(w/v) glycerol, 10%(w/v) PEG 2000, pH 6.5, 5 mM CaCl₂, 100 mM MES buffer solution emitted into vacuum from 50, 75 and 100 µm ID silica capillaries that were 114, 110 and 120 cm long, respectively. Linear fits are added to aid the eye. The range of flow rates for published SFX experiments is highlighted for comparison but note that the GDVN operates with higher backing pressure on the liquid than the values on the abscissa.