a comparison of membrane-containing bacteriophage and archaeal virus structures by cryo-em

A comparison of membrane-containing bacteriophage and archaeal virus structures by cryo-EM

Sarah Butcher

ERICE 2006

Enveloped virus session

PRD1 irido adeno

phi6 BTV

herpes

Lineage 1Lineage 1

Lineage 2Lineage 2

Lineage 3Lineage 3

Membrane-containing viruses: antipasta misto

Membrane required for host cell entry e.g. fusion with endosomes.Lipids derived from the host.

Bacteriophage:Cystoviridae – 6 and 8

- fuse with Gram-negative host inner and outer membranes, releasing polymerase complex into cytoplasm

Tectiviridae – PRD1 and Bam35- fuse, releasing DNA + terminal proteins into cytoplasm

Archaeal:SH1 - not known

Cryo-em studies

Relatively unstable viruses, so rapid preparation an advantage Membrane structure preserved in native state Possibility to look at mutants, protease treated, chemically

disrupted Resolution typically 8-12 Å, but varies within the reconstruction

depending on the order. Capsid proteins mainly peripheral membrane proteins Integral membrane proteins occasionally revealed Image reconstruction generally relies on icosahedral symmetry for

orientation determination and averaging. Hence symmetry

mismatches will not be resolved.

OMCMPG

Pseudomonas syringae

6 entry

3D reconstruction of 6 and 8 virion

Jäälinoja et al. (in preparation)

Structure of the 6 nucleocapsid

Three-dimensional reconstruction

from cryo-EM data

applying icosahedral

symmetry

Two protein shells

Hexameric packaging

motor at the vertices

(symmetry-mismatch)

Bacteriophage PRD1

PRD1 cryo-EM

25 Å

14 Å

12 ÅButcher et al. 1995 EMBO J.San Martin et al. 2001 StructureSan Martin et al. 2002 NSB

Major capsid

protein is a

peripheral membrane protein

R=36%R=36%

R=34%R=34%

R=31%R=31%

Benson et al, 1999 Cell; San Martin et al. 2001 Structure

EM/Xray combination

Benson et al, 1999 Cell; San Martin et al. 2001 Structure; San Martin et al. 2002 NSB

Bam35

Infects Gram-positive host Bacillus thuringiensis Very limited sequence similarity to bacteriophage PRD1 Address structural similarity using cryo-EM and X-ray

data from PRD1

7.3 Å icosahedral reconstruction of bacteriophage Bam35

Reconstructions calculated

from both full virion and

particle lacking DNA

Laurinmäki et al. (2005) Structure

Modelling with homologous proteins

Laurinmäki et al. (2005) Structure

Bam35 membrane proteins

Laurinmäki et al. (2005) Structure

Zhang et al. 2003, NSB; Laurinmäki et al. 2005, Structure

Archaeal virus SH1

100Å

800Å

Jäälinoja et al. (in preparation)

Density distribution and resolution

DNA

Membrane

Capsid

Spike

Jäälinoja et al. (in preparation)

Archaeal virus SH1

Jäälinoja et al. (in preparation)

Unusual capsomers

2- and 3–fold symmetric

capsomers on capsid surface.

Quasi-hexagonal capsomer

base.

Slightly skewed at 2f and

adjacent capsomers, not

elsewhere.

All with similar mass

Jäälinoja et al. (in preparation)

Structure at 5-fold axis

Very weak signal from peripheral domain.

Possible symmetry mismatch in external domains.

Transmembrane complex.Jäälinoja et al. (in preparation)

Vertex Reconstruction

Orientation angles and vertex positions derived from output of an icosahedral reconstruction.

Vertices are compared and classified revealing differences in composition and orientation.

Subsets of vertices can be used for reconstruction and any desired symmetry can be applied.

SH1 spike reconstruction

Briggs et al. (2005) JSB; Huiskonen et al. (submitted); Jäälinoja et al. (in preparation)

Cryo-em studies – conclusions

Capsid proteins mainly peripheral membrane proteins, connected

by -helices Integral membrane proteins occasionally revealed, so far

apparently -helical Membranes are icosahedral, locally affected by membrane

proteins

Acknowledgements

Benita Koli

Dennis Bamford Hanna Kivelä

John Briggs Stephen Fuller