Fig 3c. The L,D transpeptidase ldt2 is required for covalent attachment of BpbA and BbpB to PG.

Stationary phase C. burnetii samples were frozen on a glow discharged Quantifoil™ R 2/2 grid using a Leica EM GP plunge freezer (Leica Microsystems, Wetzlar, Germany) set to a relative humidity of 100%. Grids were mounted in an autogrid assembly and cryo 2D images collected using a FEI Titan Krios 300kV FEG-TEM and a FEI Falcon II CMOS direct electron detection camera. Low dose images of 13e/A2 were captured at 6 µm defocus with a nominal magnification of 29 K which corresponds to a pixel size of 2.87 Å using a 70 µm objective aperture. OM vesiculation was quantified by imaging 50 bacterial cells per strain and is expressed as the percentage of bacteria with vesicles clearly attached to the bacterial OM.

将稳定期的伯氏考克斯体（Coxiella burnetii，C. burnetii）样品置于经辉光放电处理的Quantifoil™ R 2/2载网上，使用参数设置为100%相对湿度的Leica EM GP plunge式冷冻制样仪（徕卡显微系统公司，德国韦茨拉尔）完成冷冻固定。将载网安装至自动载网夹持组件中，采用FEI Titan Krios 300kV场发射枪透射电子显微镜（FEG-TEM）与FEI Falcon II CMOS直接电子探测相机采集冷冻二维图像。本次实验采集的低剂量图像剂量为13 e/Ų，欠焦量为6 μm，标称放大倍数为29000倍，对应像素尺寸为2.87 Å，成像过程中使用了70 μm的物镜光阑。细菌外膜囊泡化（outer membrane vesiculation, OM vesiculation）的量化方法为：对每个菌株的50个细菌细胞进行成像，并以囊泡清晰附着于细菌外膜的细菌占比作为量化结果。