Ultrastructure and complex polar architecture of the human pathogen Campylobacter jejuni

Campylobacter jejuni is one of the most successful food-borne human pathogens. Here we use electron cryotomography to explore the ultrastructure of C. jejuni cells in logarithmically growing cultures. This provides the first look at this pathogen in a near-native state at macromolecular resolution (~5 nm). We find a surprisingly complex polar architecture that includes ribosome exclusion zones, polyphosphate storage granules, extensive collar-shaped chemoreceptor arrays, and elaborate flagellar motors

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy

Cryo-electron microscopy of viruses

Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions. The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy. Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method

Uncharacterized bacterial structures revealed by electron cryotomography

Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening

Mycobacter smegmatis pneumonia

KP3wt M. smegmatis mc2_155.Janelia Titan<strong>Tilt Series Date:</strong> 2013-07-01</p> <strong>Data Taken By:</strong> Alasdair McDowall</p> <strong>Species / Specimen:</strong> Mycobacterium smegmatis</p> <strong>Strain:</strong> mc2_155</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 180.0 eV/Ų, defocus: -8.0 μm, magnification: 26000x. </p> <strong>Acquisition Software:</strong> UCSF Tomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor Janelia Titan</p> <strong>Collaborators and Roles:</strong> Koushik Paul / Eric BrownGenentech, Inc.1 DNA WaySouth San Francisco, CA 94080(650) 225-6248Fax: (650) 225-1107</p> <strong>Purification / Growth Conditions / Treatment:</strong> 7H9 media</p> <strong>Sample Preparation:</strong> 3ul + 10nm AuR2/2 quantifoils 4.5offset 1 blot 2s 1s drain</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-07-01-5</p>montage_0005.mrc, Tilt Series (Pixel Size 0.42 nm), 3.3 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-07-01-5/rawdata/montage_0005.mrc" > <i class="download icon"></i> Download </a></p> montage_0005_part121_5.rec, Reconstruction (Pixel Size 1.68 nm), 602.8 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-07-01-5/3dimage_27785/montage_0005_part121_5.rec" > <i class="download icon"></i> Download </a></p&gt

Mycobacter smegmatis pneumonia

type VII secretion system in Mycobacterium smegmatis Wild type Mycobacterium smegmatis strain is expected to have T7SS, while mutant to be defective.KP48<strong>Tilt Series Date:</strong> 2013-06-10</p> <strong>Data Taken By:</strong> Alasdair McDowall</p> <strong>Species / Specimen:</strong> Mycobacterium smegmatis</p> <strong>Strain:</strong> 3877</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 180.0 eV/Ų, defocus: -8.0 μm, magnification: 22500x. </p> <strong>Acquisition Software:</strong> UCSF Tomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> Raptor</p> <strong>Collaborators and Roles:</strong> Koushik Paul / Eric BrownGenentech, Inc.1 DNA WaySouth San Francisco, CA 94080(650) 225-6248Fax: (650) 225-1107</p> <strong>Purification / Growth Conditions / Treatment:</strong> KP48 M. smegmatis Δ3877 (ΔeccD1) 7H9 media </p> <strong>Sample Preparation:</strong> 3ul + 10nm AuR2/2 quantifoils 4.5offset 1 blot 2s 1s drain</p> amw2013-06-10-17_slicer7421.jpg: REZFiles available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-06-10-17</p>kp48_10015.mrc, Tilt Series (Pixel Size 0.48 nm), 3.4 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-06-10-17/rawdata/kp48_10015.mrc" > <i class="download icon"></i> Download </a></p> kp48_10015_full.rec, Reconstruction (Pixel Size 1.92 nm), 623.6 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-06-10-17/3dimage_27559/kp48_10015_full.rec" > <i class="download icon"></i> Download </a></p&gt

Ecoli JM109 Metabolosome

Bifurcating tubules/fibres in the periphery of the cells, Pentameric structures, carboxysomes, metabolosomes, pdu enzymes, Periplasmic cone<strong>Tilt Series Date:</strong> 2009-07-09</p> <strong>Data Taken By:</strong> Alasdair McDowall</p> <strong>Species / Specimen:</strong> Escherichia coli</p> <strong>Strain:</strong> </p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (60.0°, 60.0°), step: °, constant angular increment, dosage: 0.0 eV/Ų, defocus: 10.0 μm, magnification: 0x. </p> <strong>Acquisition Software:</strong> Leginon</p> <strong>Upload Method:</strong> webload</p> <strong>Processing Software Used:</strong> IMOD </p> <strong>Collaborators and Roles:</strong> PI of visitor on short term fellowship Science foundation Ireland Eur 6,673Michael B PrenticeProfessor of Medical MicrobiologyDepartment of MicrobiologyUniversity College CorkCork, IrelandTel +353 214901420Fax +353 [email protected] Liang, PhDDepartment of MicrobiologyUniversity College CorkCork Ireland"Liang, Mingzhi" </p> <strong>Purification / Growth Conditions / Treatment:</strong> Single colony of E.coliJM109:pAR3114 was picked up and inoculate in NCE minimal medium supplemented with 1% (w/v) succinate, 5 g/litre of yeast extract, 50 mM 1,2-propanediol, 20 ?g/ml of tetracycline, 30 ?g/ml of cefsulodin. Cells were grown at 37oC with shaking for 24 hours. The culture at an OD600 of 0.5 was mixed with 10 nm colloidal gold and applied to holey carbon grids. </p> <strong>Sample Preparation:</strong> Vitrobot 100r/h 1 blot 1 sec drain, 10 nm colloidal gold and applied to 2/2 holey carbon grids.</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2009-07-09-20</p>032.rec, Reconstruction (Pixel Size 1.922 nm), 13.4 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2009-07-09-20/3dimage_6809/032.rec" > <i class="download icon"></i> Download </a></p&gt

#4-60 BaxH6 Lysogen Ecoli phage

Bax delta H6 bigger hole Bax mutantBaxH6 Lysogen Ecoli lamdaphageMC4100[lamdadelta (SR)]<strong>Tilt Series Date:</strong> 2013-07-12</p> <strong>Data Taken By:</strong> Alasdair McDowall</p> <strong>Species / Specimen:</strong> Escherichia coli</p> <strong>Strain:</strong> </p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 150.0 eV/Ų, defocus: -8.0 μm, magnification: 26000x. </p> <strong>Microscope:</strong> Janelia Titan</p> <strong>Acquisition Software:</strong> UCSFTomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> raptor failed</p> <strong>Collaborators and Roles:</strong> Xo Luo and Xiaming Pang [email protected]</p> <strong>Purification / Growth Conditions / Treatment:</strong> see Xiamings notes</p> <strong>Sample Preparation:</strong> Titan 26k 7/11/13 expo 0.9 150e- poor fudicials</p>Files available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-07-12-4</p>B1_4-600004.mrc, Tilt Series (Pixel Size 0.42 nm), 3.3 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2013-07-12-4/rawdata/B1_4-600004.mrc" > <i class="download icon"></i> Download </a></p&gt

Rhodopseudomonas palustris TIE-1

Raw data files of Rhodopseudomonas palustris TIE-1<strong>Tilt Series Date:</strong> 2012-05-09</p> <strong>Data Taken By:</strong> Alasdair McDowall</p> <strong>Species / Specimen:</strong> Rhodopseudomonas palustris</p> <strong>Strain:</strong> None</p> <strong>Tilt Series Settings:</strong> Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 100.0 eV/Ų, defocus: None μm, magnification: 22500x. </p> <strong>Acquisition Software:</strong> UCSFTomo</p> <strong>Upload Method:</strong> pipeline</p> <strong>Processing Software Used:</strong> raptor</p> <strong>Collaborators and Roles:</strong> Nicholas ShikumaDianne Newman</p> <strong>Purification / Growth Conditions / Treatment:</strong> wild type and shc mutantgrown by photoheterotrophic anaerobic exponential and stationary</p> <strong>Sample Preparation:</strong> vitrobot</p> amw2012-05-09-9_slicer4822.jpg: unknown amw2012-05-09-9_slicer4823.jpg: Granules amw2012-05-09-9_slicer4824.jpg: Granules amw2012-05-09-9_slicer4826.jpg: unknown vesicles amw2012-05-09-9_slicer4827.jpg: unknown vesiclesFiles available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2012-05-09-9</p>WT_expo0004.mrc, Tilt Series (Pixel Size 0.9 nm), 1.0 GB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2012-05-09-9/rawdata/WT_expo0004.mrc" > <i class="download icon"></i> Download </a></p> WT_expo0004_part122_4.rec, Reconstruction (Pixel Size 1.8 nm), 516.1 MB <a role="button" class="ui compact mini button" href="https://renc.osn.xsede.org/ini210004tommorrell/tomography_archive/amw2012-05-09-9/3dimage_18462/WT_expo0004_part122_4.rec" > <i class="download icon"></i> Download </a></p&gt